ABSTRACTS PART 2:

Abstracts PhD Programme in Biosciences

e00035 Immune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic cells

e00036 Aortic disease in Marfan syndrome is caused by overactivation of sGC-PRKG signaling by NO

e00037 Characterization of novel KCNA5 loss-of-function mutations in a Spanish cohort of pulmonary arterial hypertension patients

e00038 p38γ/δ hyperactivation alters Ca2+ handling and predisposes to cardiac hypertrophy and arrhythmias.

e00039 Colorectal cancer stem cell fusion with human monocytes: an explanation for metastasis.

e00040 Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia, a novel therapeutic anti-CCR7 antibody

e00041 Galectin-1 expression in CD8+ T lymphocytes controls inflammation in contact hypersensitivity

e00042 Studies for new applications of a monoclonal antibody antiCCR7. Validation as a therapy in onco-immunology.

e00043 Immunecheckpoints in sepsis: an approach to diagnosis and therapy

e00044Lamin A/C regulates epigenetic changes in CD4+ T-cells favoring Th1 commitment

e00045A novel NRF2-βTrCP Protein-Protein Interaction (PPI) inhibitor suppresses lipopolysaccharide-mediated inflammation through the activation of transcription factor NRF2

e00046WIP uses the NRF2/KEAP1 axis in glioblastoma cells to promote oxidant tolerance.

e00047METPlatform identifies brain metastasis vulnerabilities and predicts patient response to therapy

e00048  A global map of the impact of deletion of Post-Translational Modification sites in genetic diseases

e00049  Prioritizing variants of uncertain significance for reclassification using a rule-based algorithm in inherited retinal dystrophies

e00050  KV1.3 channel inhibition by a family of indolic compounds.

e00051 Disruption of liver homeostasis and systemic metabolism upon concomitant hepatic activation of growth factor and nutrient signaling

e00052Myeloid p38s modulate BAT function through hepatic FGF21 during obesity

e00053NEW GENE, NEW SKELETAL DYSPLASIA Identification and functional characterization of biallelic variants in PRKG2 as cause of a new Acromesomelic Dysplasia

e00054HIV reverse transcriptases defective in strand displacement activity

Abstracts PhD Programme in Neuroscience

e00055 The primate striatum: morphological and stereological study of neurons and interneurons in the MPTP non-human primate model 

e00056 Distribution of thyroid hormone transporters MCT8 and OATP1C1 in the human and monkey cerebral cortex.

e00057 Distribution of thyroid hormone transporters MCT8 and OATP1C1 in the human and monkey the basal ganglia and thalamus.

e00058 Anatomical study of the striatal and cortical projections arising from the posterior intralaminar thalamic nucleus neurons in the mouse

e00059 Diversity and organization of the thalamocortical projections from the ventroposterior complex of the thalamus

e00060Distribution of the noradrenaline innervation and Alpha adrenoceptors in human higher-order thalamic nuclei

Abstracts PhD Other Programmes

e00061Effect of a workplace program to promote physical activity on metabolic syndrome risk factors during the COVID-19 pandemic

Abstracts CIVIS Alliance Universities

e00062On the improvement of aortic anastomosis

e00063Methylglyoxal stress induces a major epigenetic deregulation leading to a pro-migratory phenotype in breast cancer and significant clinical relevance

e00064Combinations of BH3 mimetics with the TKI nilotinib synergistically induce apoptosis in blast phase chronic myeloid leukaemia cells

e00065Heterotypic cell-cell communication regulates glandular stem cell multipotency

e00066High-Sensitivity Detection and Genotyping of Yellow Fever Virus

e00067Chemerin regulates normal angiogenesis and hypoxia-driven neovascularization

e00068 Identification of an immune profile able to improve IMDC stratification in mRCC patients

Abstracts University of Malaya

e00069Investigation of capillary leak syndrome induced by the eastern (Daboia siamensis) and western (Daboia russelii) Russell’s viper venoms.

e00070Gut microbiota dysbiosis in survivors of childhood acute lymphoblastic leukemia and the association with immune dysregulation and metabolic derangement.

e00071 Real-time Impedance Monitoring of Biofilm Formation from Dual Flagellar Systems in Aeromonas dhakensis.

e00072The Burden of Multidrug-Resistant Tuberculosis in Malaysian TB Patients

e00073The effects of attitude, self-efficacy and perceived usefulness on the actual use of personal radiation dosimeter among multiethnic Malaysian radiology workers

e00074Profiling of the Malayan blue coral snake (Calliophis bivirgata flaviceps) venom through an integrated -omics approach

e00075Medication safety: Evaluating and enhancing implementation of risk minimisation measures in Malaysia.

e00076Biosimilars in Malaysia: Regulation and factors associated with confidence to promote their use among hospital pharmacists.

 

ABSTRACTS PhD Programme in Molecular Biosciences

e00035
Immune synapse instructs epigenomic and transcriptomic
functional reprogramming in dendritic cells

Ana Alcaraz-Serna1,2†, Eugenio Bustos-Morán1,2†, Irene Fernández-Delgado1,2†, Diego Calzada-Fraile2†, Daniel Torralba2,
Ester Marina-Zárate2, Erika Lorenzo-Vivas2, Enrique Vázquez2, Juliana Barreto de Alburquerque3, Nora Ruef3, Manuel
José Gómez2, Fátima Sánchez-Cabo2, Ana Dopazo2, Jens V. Stein3, Almudena Ramiro2, Francisco Sánchez-Madrid1,2,4*.

*Corresponding author: Francisco Sánchez-Madrid1,2,4,Madrid, Spain. E-mail: fsmadrid@salud.madrid.org

Details of affiliation

1Immunology Department, Instituto de Investigación Sanitaria Hospital Universitario La Princesa, Universidad Autónoma de Madrid,
28006 Madrid, Spain.
2Vascular Pathophysiology Department, Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain.
3Department of Oncology, Microbiology, and Immunology, University of Fribourg, 1700 Fribourg, Switzerland.
4Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), 28029 Spain.
†These authors contributed equally to this work.

Funding

This study was supported by grant SAF2017-82886-R from the Spanish Ministry of Economy and Competitiveness (MINECO), grant
S2017/BMD-3671-INFLAMUNE-CM from the Comunidad de Madrid, a grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud”
(XIX Concurso-2018), a grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (BIOMEDICINA-018), the Fundació Marató TV3
(grant 122/C/2015), “la Caixa” Banking Foundation (HR17-00016), BIOIMID (PIE13/041) from Instituto de Salud Carlos III, CIBER Cardiovascular
(CB16/11/00272), and Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional
FEDER). I.F.-D. is supported by a Fellowship from the Spanish Ministry of Science, Innovation, and Universities (FPU15/02539). The Centro Nacional
de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC
Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).

Competing Interests:

The authors declare that they have no competing interests

Keywords: immune synapse, postsynaptic dendritic cells, epigenomic reprogramming.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00035

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ana Alcaraz-Serna, Eugenio Bustos-Morán, Irene Fernández-Delgado, Diego Calzada-Fraile, Daniel Torralba2, Ester Marina-Zárate, Erika
Lorenzo-Vivas, Enrique Vázquez, Juliana Barreto de Alburquerque, Nora Ruef, Manuel José Gómez, Fátima Sánchez-Cabo, Ana Dopazo, Jens V. Stein,
Almudena Ramiro, Francisco Sánchez-Madrid, et al. Immune synapse instructs epigenomic and transcriptomic functional reprogramming in dendritic
cells. IBJ Plus 2021 (S4)e000035:10.24217/2531-0151.21v1s4.00035

Edited: Madrid, España.

Abstract

Introduction: Understanding the fate of dendritic cells (DCs) after productive immune synapses (postsynaptic
DCs) with T cells during antigen presentation has been largely neglected in favor of deciphering the nuances
of T cell activation and memory generation. We have previously described that T cells prime DCs through the
transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to
DCs against pathogen infection. In this study, we have analyzed the epigenetic and functional changes induced in
CD11c+ bone marrow–derived DCs (BMDCs) upon being instructed by antigen cognate interaction with T cells.

Material and Methods: For that, DCs were isolated by cell sorting or CD11c+ positive selection after a productive
or non-productive immune synapse with CD4+ Naïve T cells. Then, we assessed the transcriptomic or epigenomic
signature of nonsynaptic and postsynaptic DCs by RNA-seq or ATAC-seq, respectively.

Results: Here, we describe that postsynaptic DCs switch their transcriptomic signature, correlating with epigenomic
changes including DNA accessibility and histone methylation. We observed the upregulation of several
genes such as Ccr7, Tlr3, Fscn1, Cd40, Isg15, Ifit1, and Cxcl10. These leads towards a more mature, migratory, and
inflammatory phenotype, that also occurs in a CD8+ T cell immune synapse. We focus on the chemokine receptor
Ccr7 as a proof-of-concept gene that is increased in postsynaptic DCs. Consistent with our epigenomic observations,
postsynaptic DCs migrate more efficiently toward CCL19 in vitro and display enhanced homing to draining
lymph nodes in vivo.

Conclusions: This work describes a previously unknown DC population whose transcriptomics, epigenomics, and
migratory capacity change in response to their cognate contact with T cells.

e00036
Aortic disease in Marfan syndrome is caused by
overactivation of sGC-PRKG signaling by NO

Andrea de la Fuente-Alonso1,2,#, Marta Toral1,2,#, Miguel R. Campanero2,3,4,* and Juan Miguel Redondo1,2,*..

*Corresponding author: Juan Miguel Redondo, E-mail: jmredondo@cnic.es  and Miguel R. Campanero,E-mail: mcampanero@cbm.csic.es

Details of affiliation

1Gene regulation in cardiovascular remodeling and inflammation group, Centro Nacional de Investigaciones Cardiovasculares (CNIC),
Madrid 28029, Spain.
2Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain.
3Department of Cancer Biology, Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones
Científicas–Universidad Autónoma de Madrid, Madrid 28029, Spain.
4Present address: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas–Universidad
Autónoma de Madrid, Madrid 28049, Spain.
#These authors contributed equally to this work.

Funding

The project leading to these results has received funding from “La Caixa” Banking Foundation HR18-00068 and HR17-00247; Spanish
Ministerio de Ciencia e Innovación grants RTI2018-099246-B-I00 (MICIU/AEI/FEDER, UE), SAF2015-636333R (MINECO/FEDER, UE), SAF2017-
88881R (MINECO/AEI/FEDER, UE), BIO2015-67580-P and PGC2018-097019-B-I00; Comunidad de Madrid through the European Social Fund
(ESF)-financed program AORTASANA-CM (B2017/BMD-3676); Instituto de Salud Carlos III (CIBER-CV CB16/11/00264 and CB16/11/00277; PRB3-
IPT17/0019-ProteoRed; and PI17/00381, with cofinancing from the European Regional Development Fund); Fundacio La Marato TV3 (20151330, and
122/C/2015); The Marfan Foundation USA Faculty grant 2017 MRF/1701; and Spanish Ministerio de Ciencia e Innovación fellowships FPU (17/05866)
and Sara Borrell (CD18/00028).

Competing Interests:

The authors declare that they have no competing interests

Keywords: Nitric Oxide, NO-donors, sGC-PRKG signaling, Marfan Syndrome, aortic aneurysm, therapy, biomarker.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00036

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Andrea de la Fuente-Alonso, Marta Toral,Miguel R. Campanero, and Juan Miguel Redondo, et al. Aortic disease in Marfan syndrome is caused
by overactivation of sGC-PRKG signaling by NO. IBJ Plus 2021 (S4)e000036:10.24217/2531-0151.21v1s4.00036

Edited: Madrid, España.

Abstract

Introduction: Thoracic aortic aneurysm, as occurs in Marfan Syndrome (MFS), is generally asymptomatic until
dissection (TAAD), requiring surgical intervention as the only available treatment. Nitric Oxide Synthase 2 (NOS2)
expression is induced in Marfan patients and in a mouse model of MFS, and TAAD is reversed in the mouse model
by pharmacological NOS2 inhibitors, raising the possibility that Nitric Oxide (NO) is crucial for MFS-associated TAAD.
However, the mechanisms by which NOS2 contributes to TAAD in MFS remain unclear. Our objective is to elucidate
if the NO-sGC-PRKG signaling pathway is implicated in MFS aortopathy and identify novel targets that could improve
MFS diagnosis, treatment and/or prognosis.

Material and methods: This study combines different in vivo and in vitro approaches: MFS established disease was
studied in a genetic MFS mouse model; NO-donors were administered to assess their capacity to induce aortopathy
in wild-type mice, whereas pharmacological inhibitors and lentivirus encoding shRNA specific of sGC-PRKG pathway
components were administered to study TAAD reversion in MFS mice. Primary cultures of vascular smooth muscle
cells were isolated from mouse aortas for in vitro studies. Additionally, blood and aortic tissue samples from healthy
donors and Marfan patients were studied. In vivo ultrasound imaging was used to evaluate TAAD in our different
mouse models. Aortic tissue and plasma protein nitration levels were determined by proteomics analysis. Confocal
imaging, histological studies, circulating cGMP determination, RT-PCR and western blotting were combined to test
our hypothesis.

Results: Here, we show that NO signaling dysregulates actin cytoskeleton dynamics in MFS vascular smooth muscle
cells and that NO-donors induce MFS-like aortopathy in wild-type mice, indicating that a marked increase in
NO suffices to induce TAAD. Levels of nitrated proteins are higher in plasma from Marfan patients and mice and in
aortic tissue from MFS mice than in control samples, indicating elevated circulating and tissue NO. Soluble guanylate
cyclase (sGC) and cGMP-dependent protein kinase (PRKG) are both activated in Marfan patients and mice and
in wild-type mice treated with NO-donors, as shown by increased plasma cGMP and pVASP-S239 staining in aortic
tissue. MFS aortopathy in mice is reverted by pharmacological inhibition of sGC and PRKG and lentiviral-mediated
Prkg1 silencing.

Conclusions: The NO-sGC-PRKG signaling pathway mediates aortopathy in a mouse model of MFS and is activated
in MFS mice and Marfan patients. These findings identify potential biomarkers for monitoring Marfan Syndrome in
patients and urge evaluation of PRKG and sGC as therapeutic targets.

e00037
Characterization of novel KCNA5 loss-of-function mutations
in a Spanish cohort of pulmonary arterial
hypertension patients

Alba Vera-Zambrano1, Mauro Lago-Docampo2, Jair Tenorio3, Pilar Escribano4, Juan Felipe Franco-Gonzalez5,
Francisco Pérez-Vizcaíno6, Diana Valverde2, Teresa González1, Angel Cogolludo6.

*Corresponding author: Alba Vera-Zambrano, Biochemistry Department, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain. Instituto de
Investigaciones Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain. E-mail: albavera@iib.uam.es

Details of affiliation

1Biochemistry Department, School of Medicine, Universidad Autónoma de Madrid, Madrid, Spain. Instituto de Investigaciones
Biomédicas Alberto Sols (CSIC-UAM), Madrid, Spain.
2CINBIO, Universidad de Vigo, Vigo, Spain.
3Instituto de Genética Médica y Molecular (INGEMM), Hospital Universitario La Paz-IdiPaz, Universidad Autónoma de Madrid,
Madrid, Spain. Centro de Investigación en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain.
4Unidad multidisciplinar de Hipertensión Pulmonar, Hospital 12 de Octubre, Madrid, Spain.
5Structural and Chemical Biology Department, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain.
6Departamento de Farmacología y Toxicología, Facultad de Medicina, Universidad Complutense de Madrid (UCM), Madrid, Spain.

Funding

Supported by Fundación contra la Hipertensión Pulmonar

Competing Interests:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

Keywords: KCNA5, Kv1.5, pulmonary arterial hypertension, PAH, mutations

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00037

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Alba Vera-Zambrano, Mauro Lago-Docampo, Jair Tenorio, Pilar Escribano, Juan Felipe Franco-Gonzalez, Francisco Pérez-Vizcaíno, Diana
Valverde, Teresa González, Angel Cogolludo, et al. Manuscript´s full TitleCharacterization of novel KCNA5 loss-of-function mutations in a Spanish
cohort of pulmonary arterial hypertension patients. I IBJ Plus 2021 (S4)e0037:10.24217/2531-0151.21v1s4.00037.

Edited: Madrid, España.

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is a rare and debilitating cardiopulmonary disorder characterized
by persistent vasoconstriction and pulmonary vascular remodelling. Among the different mechanisms
contributing to the disease, K+ channels dysfunction in the pulmonary artery smooth muscle cells (PASMC) is a
common feature in most forms of PAH. Thus, reduced K+ channel activity leads to PASMC membrane depolarization
and activation of voltage-gated L-type Ca2+ channels, resulting in increased vasoconstriction and proliferation.
It is remarkable that different mutations in genes encoding K+ channels (KCNK3, KCNJ8, and ABCC8/9) have
been described in heritable PAH. Likewise, reduced expression and/or activity of Kv1.5 channels has been reported
in human or experimental PAH and single nucleotide polymorphisms in its KCNA5 gene have been found in
PAH patients, which suggest that Kv1.5 channel dysfunction may be a risk factor for PAH.

Material and Methods: In the present study, we aimed to characterize the functional consequences of 7 KCNA5
variants found in a Spanish cohort of PAH patients. For this purpose, potassium currents were recorded by whole-
cell patch-clamp in HEK293 cells transfected with WT or mutant Kv1.5 cDNA, and flow cytometry, western blot
and immunocitochemistry techniques were used for measuring protein expression.

Results: We found that two of the KCNA5 variants, R184P and G384R, generated Kv1.5 channels with an important
loss-of-function. These mutations decreased the current amplitude by 80% and 42%, respectively (n=10-21,
p<0.05) and altered the gating of the channel. For R184P, we also detected a significant decrease in Kv1.5 protein
expression.

Conclusions: Our preliminary data indicates that some KCNA5 mutations present in PAH patients have critical
consequences for channel function. This strongly suggests loss-of-function KCNA5 mutations as a risk factor for
PAH and opens a new field of research directed to design and develop pharmacological tools to restore Kv1.5
channel function.

e00038
p38γ/δ hyperactivation alters Ca2+ handling and predisposes
to cardiac hypertrophy and arrhythmias.

Rafael Romero-Becerra1#, Bárbara González-Terán1,2#, Juan Antonio Lopez1,4, Daniela Ponce-Balbuena3, Andrew
Allan3, Roberto Ramos-Mondragón3, Elisa Manieri1, Laura Sanz1, Ivana Nikolic1, Valle Montalvo-Romeral1, Ayelén M.
Santamans1, Maria Elena Rodríguez1, Luis Leiva-Vega1, Víctor Bondía1, Guadalupe Guerrero-Serna3, Eric N. Jimenez-
Vazquez3, David Filgueiras-Rama1,4,5, Luis Jesús Jiménez-Borreguero1, Deepak Srivastava2,6,7,8, Jesús Vázquez1,4, José
Jalife1,3,4* & Guadalupe Sabio1*.

*Corresponding author: Guadalupe Sabio & José Jalife
Centro Nacional de Investigaciones Cardiovasculares (CNIC).
Calle de Melchor Fernández Almagro, 3, 28029, Madrid, Spain.
gsabio@cnic.es, jose.jalife@cnic.es

Details of affiliation

1Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
2 Gladstone Institutes, San Francisco, CA, USA.
3Center for Arrhythmia Research, Department of Internal Medicine, University of Michigan, Ann Arbor, MI
4CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain
5Hospital Clínico Universitario San Carlos, Madrid, Spain.
6Department of Pediatrics, UCSF School of Medicine, San Francisco, CA, USA.
7Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, CA, USA.
8Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.
# Equal contribution

Funding

This work was funded by a CNIC Intramural Project Severo Ochoa (Expediente 12-2016 IGP) to GS and JJ. GS is an investigator of the Ramón
y Cajal Program. BGT was a fellow of FPI Severo Ochoa CNIC Program (SVP‐2013‐067639) and is an American Heart Association Postdoctoral Fellow
(18POST34080175). R.R.B is a fellow of the FPU Program (FPU17/03847). The CNIC is supported by the Ministerio de Economía y Competitividad and
the Pro‐CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). D.S. is supported by NIH/NHLBI P01 HL098707,
P01 HL146366, R01 HL057181, R01 HL127240, and by the Roddenberry Foundation, the L.K. Whittier Foundation, and the Younger Family Fund.

Competing Interests:

D.S. is scientific co-founder, shareholder and director of Tenaya Therapeutics.

Keywords: MKK6, MKK3, p38 MAPK, cardiac hypertrophy, mTOR, ryanodine receptor, arrhythmia

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00038

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rafael Romero-Becerra, Bárbara González-Terán, Juan Antonio Lopez, Daniela Ponce-Balbuena, Andrew Allan, Roberto Ramos-Mondragón,
Elisa Manieri, Laura Sanz, Ivana Nikolic, Valle Montalvo-Romeral, Ayelén M. Santamans, Maria Elena Rodríguez, Luis Leiva-Vega1, Víctor Bondía1,
Guadalupe Guerrero-Serna3, Eric N. Jimenez-Vazquez3, David Filgueiras-Rama1,4,5, Luis Jesús Jiménez-Borreguero1, Deepak Srivastava, Jesús
Vázquez, José Jalife & Guadalupe Sabio, et al. p38γ/δ hyperactivation alters Ca2+ handling and predisposes to cardiac hypertrophy and arrhythmias.
IBJ Plus 2021 (S4)e0038:10.24217/2531-0151.21v1s4.00038.

Edited: Madrid, España.

Abstract

Introduction: Cardiac hypertrophy is a stereotyped response to a variety of intrinsic and extrinsic stimuli that can result
in maladaptive states associated with increased risks for arrhythmias and sudden death. Although several signal
transduction cascades have been identified as critical regulators of cardiac hypertrophy, the molecular mechanisms
underlying altered electrical activity of the hypertrophic heart remain limited.

Objective: To explore the role of the MKK3/6-p38γ/δ stress signaling pathway in the control of cardiac hypertrophy
and predisposition to stress-induced ventricular arrhythmia.

Methods and Results: We leveraged several constitutive and tissue-specific knockout mouse models and subjected
them to echocardiography, heart histology and molecular biology analysis to definitively show that MKK6 deficiency
results in cardiac MKK3-p38γ/δ hyperactivation and mTOR-mediated cardiac hypertrophic growth in the absence of
obvious ventricular pathophysiological changes. An intensive swim exercise protocol and ex-vivo cardiac β-adrenergic
stimulation coupled with electrophysiology assays demonstrating action potential duration prolongation in adult
ventricular myocytes showed that hypertrophic MKK6-deficient hearts were susceptible to stress-induced malignant
arrythmias and sudden death. Co-immunoprecipitation and unbiased heart phosphoproteomics uncovered a
central role for p38γ/δ in regulating a multi-kinase protein module that tunes ryanodine receptor 2 (RyR2)-mediated
intracellular calcium handling. Thus, hyperactive p38γ/δ signaling results in RyR2 hyperphosphorylation leading to
ectopic activity and stress-induced arrhythmias in live mice.

Conclusions: Our work highlights a fundamental role for MKK3/6-p38γ/δ signaling in ion channel function and
cardiac calcium handling and highlights how its dysregulation can predispose to stress-induced arrhythmias and premature
death. More broadly, these findings implicate the MKK3/6-p38γ/δ pathway as a nodal regulator of cardiac
homeostasis and adaptation to physiologic and pathologic stressors.

e00039
Colorectal cancer stem cell fusion with human monocytes:
an explanation for metastasis.

Karla Montalbán-Hernández1,2,$, Ramón Cantero-Cid1,2,3,$, Elvira Marín1,2, José Carlos Casalvilla1,2, Elisa Pulido Lucas
1,2, José Avendaño Ortiz1,2,4, Roberto Lozano-Rodríguez1,2 , Verónica Terrón-Arcos1,2 , Jaime Valentín1,2, Luis Augusto
Aguirre1,2,*, Eduardo López-Collazo1,2,4*.

*Corresponding author: Eduardo López-Collazo. Innate Immunity Group, IdiPAZ, La Paz University Hospital, Madrid, Spain.
E-mail: elopezc@salud.madrid.org

Details of affiliation

1The Innate Immune Response Group IdiPAZ, La Paz University Hospital, Madrid, Spain.
2Tumour Immunology Lab, IdiPAZ, La Paz University Hospital, Madrid, Spain.
3 Surgery Department, La Paz University Hospital, Madrid, Spain.
4Centre for Biomedical Research Network of Respiratory Diseases (CIBERES), Madrid, Spain.
$ co-authors

Funding

This work was supported by Foundation for the Hospital La Paz Institute for Health Research (FIBULP).

Competing Interests:

All authors: No reported conflicts of interest.

Keywords: Colorectal cancer, cell fusion, hybrids, monocytes, SigleC5.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00039

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Karla Montalbán-Hernández, Ramón Cantero-Cid, Elvira Marin, Jose Carlos Casalvilla, Elisa Pulido Lucas, Jose Avendaño Ortiz, Roberto
Lozano, Veronica Terrón , Jaime Valentin, Luis Augusto Aguirre and Eduardo López-Collazo. Colorectal cancer stem cell fusion with human
monocytes: an explanation for metastasis. IBJ Plus 2021 (S4)e0038:10.24217/2531-0151.21v1s4.00039.

Edited: Madrid, España.

Abstract

Introduction: Cell fusion is a physiological mechanism which occurs during processes such as muscle and bone
differentiation or embryogenesis. Pathologically, various authors have described fusion between leukocytes and
cancer cells yielding new hybrid entities with enhanced tumorigenic abilities. Among these, high migration and
immune evasion can be highlighted.

Material and Methods: To study monocyte-cancer cell fusion in colorectal cancer, the SW620 cell line was
dedifferentiated to obtain a stem cell phenotype and co-cultured in vitro with human monocytes isolated from
peripheral blood mononuclear cells (PBMCs). Tumour Hybrid cells (THCs) were established through flow cytometry
with the surface marker EpCAM for the cancer cell line, and CD14 for the human monocytes. Additionally,
immunofluorescence was also performed to study the presence of these THCs in primary tumour tissue samples.

Results: Characterisation of THCs demonstrated a clear EpCAM+CD14+CD45+ signature, demonstrating how these
THCs are not circulating tumour cells (CTCs). These hybrid entities had increased migratory abilities in vitro when
compared to its parental cancer cell line, illustrating their strong involvement in metastatic spread. M2 polarised
monocytes-tumour cells cocultures were found to yield a significantly higher percentage of THCs than M1
monocytes, showing how this event would be favoured in the tumour microenvironment. Moreover, an elevated
expression of SigleC5 on THCs was also found. This fact could explain the immune evasion of these hybrids, as T
cell proliferation against sorting-isolated THCs became restored in presence of a SigleC5 blocking antibody. Additionally,
these hybrid entities were also found in primary tumour samples from colorectal cancer patients and
statistical analyses showed how the higher the number of THCs found, the higher the probability of later developing
metastasis.

Conclusion: Cancer cell fusion in colorectal cancer is a poorly studied phenomenon which could aid in the selection
of new targets, disease biomarkers or immunotherapies. These EpCAM+CD14+ tumour hybrid cells have
been found both in vitro and ex vivo in colorectal cancer patients samples, indicating a new cell signature which
appears to be involved in the development of metastasis. The next steps would involve finding specific targets on
these cells, such as the novel SigleC5, which could serve as therapies to prevent disease progression.

e00040
Effect of ibrutinib on CCR7 expression and functionality in chronic lymphocytic leukemia, a novel therapeutic anti-CCR7 antibody.

Tamara Mateu-Albero1, Raquel Juárez-Sánchez1,2, Javier Loscertales3, Wim Mol4,5, Fernando Terrón2,4, Cecilia MuñozCalleja1,6, Carlos Cuesta-Mateos1,2,4.

*Corresponding author: Tamara Mateu-Albero Tamara Mateu-Albero, Immunology Department, Madrid, Spain. E-mail: tamara.mateu@estudiante.uam.es

Details of affiliation

1Immunology Department, Hospital Universitario de La Princesa, Madrid, Spain 2IMMED S.L., Immunological and Medicin Products, Madrid, Spain 3Hematology Department, Hospital Universitario de La Princesa, Madrid, Spain 4Catapult Therapeutics, Lelystad, The Netherlands 5Pepscan, Lelystad, The Netherlands 6Medicine Faculty, Universidad Autónoma de Madrid, Madrid, Spain

Funding

The authors declare that no fundings

Competing Interests:

The authors declare that no competing interest exit. 

Keywords: CCR-7, CLL, antibody, immunotherapy, migration, ibrutinib.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00040

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mateu-Albero, T. Juárez-Sánchez, R. Loscertales, J. Mol, W. Terrón, F. Muñoz-Calleja, C. Cuesta-Mateos, C. Effect of ibrutinib on CCR7 expressionn and functionality in chronic lymphocytic leukemia, a novel therapeutic anti-CCR7 antibody. IBJ Plus 2021 (S4)e0040:10.24217/25310151.21v1s4.00040.

Edited: Madrid, España.

Abstract

Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by a clonal expansion of CD5+CD23+CD19+ B cells that accumulates in the peripheral blood, bone marrow and lymph nodes. In the last years a BTK inhibitor (BTKi), Ibrutinib, was approved in the treatment of CLL. However, a growing number of patients discontinue ibrutinib and this likely to be increase in the up-coming years. In the last years, growing evidence suggests that clinical benefit of ibrutinib in CLL is not only a result of its activity on BTK but also to compelling off-target effects on a number of dysregulated pathways in CLL. This regard, pathological B cells of CLL patient over-express CCR7, a chemokine receptor that, upon binding to CCL19 and CCL21 drives migration of lymphocytes to lymph nodes. In this study we aimed to analyze the impact of ibrutinib on surface CCR7 (sCCR7) expression and functionality, as well as on the therapeutic activity of a novel anti-CCR7 mAb.

Material and Methods: Immunophenotyping was used to determine the modulation of sCCR7 expression by ibrutinib in primary CLL samples donated by patients and in vitro assays. CCR7 functionality was tested by means of chemotaxis assays towards CCL19 and CCL21. Anti-CCR7 activity on ibrutinib-treated patients was determined in chemotaxis assays as well as in antibody-dependent cell-mediated cytotoxicity (ADCC) assays.

Results: Our results demonstrated that ibrutinib exposure did not induce a complete loss of sCCR7, as both ibrutinib-treated and refractory/relapsed patients had almost 100% of sCCR7 expression. Despite sCCR7 slightly reduced (in terms of RMFI) after ibrutinib treatment, this down-modulation did not impair the sCCR7 functionality as demonstrated in CCR7-mediated chemotactic assays using CLL cells with a previous in vivo or in vitro exposure to the BTKi. Finally, sCCR7 down-modulation mediated by ibrutinib did not affect blocking or killing activities of the novel anti-CCR7.

Conclusions: Together, these results demonstrated that ibrutinib did not affected either CCR7 expression or functionality and validate the therapeutic utility of anti-CCR7 mAb as a next line single agent therapy for CLL patients who failed to ibrutinib treatment, as well as for combination therapy.

e00041
Galectin-1 expression in CD8+ T lymphocytes controls inflammation in contact hypersensitivity.

Raquel Castillo-González1-3#, Danay Cibrian1-4#, Nieves Fernández-Gallego2,3, Marta Ramírez-Huesca3, María Laura Saiz1,2, María N. Navarro5, Manuel Fresno5, Hortensia de la Fuente1-4 and Francisco Sánchez-Madrid1-4*.

*Corresponding author: Francisco Sánchez-Madrid, Immunology Service, Hospital de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria del Hospital Universitario de La Princesa, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. E-mail: fsmadrid@salud.madrid.org

Details of affiliation

1 Immunology Service, Hospital de la Princesa, Universidad Autónoma de Madrid, Instituto de Investigación Sanitaria del Hospital Universitario de La Princesa, Madrid, Spain. 2 Universidad Autónoma de Madrid. Madrid, Spain. 3 Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. 4 CIBER de Enfermedades Cardiovasculares, Carlos III Health Institute, Madrid, Spain 5 Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain # These authors contribute equally

Funding

Supported by grant SAF2017-82886-R from the Spanish Ministry of Economy and Competitiveness (MINECO), grant S2017/BMD-3671INFLAMUNE-CM from the Comunidad de Madrid, a grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud” (XIX Concurso-2018) and a grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (BIOMEDICINA-2018), the Fundació Marató TV3 (grant 122/C/2015) and “La Caixa” Banking Foundation (HR17-00016).

Competing Interests:

The authors declare no conflict of interest. 

Keywords: Galectin-1; contact hypersensitivity; CD8 T cells

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00041

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Raquel Castillo-González, Danay Cibrian, Nieves Fernández-Gallego, Marta Ramírez-Huesca, María Laura Saiz, María N. Navarro, Manuel Fresno, Hortensia de la Fuente and Francisco Sánchez-Madrid, et al. Galectin-1 expression in CD8+ T lymphocytes controls inflammation in contact hypersensitivity. IBJ Plus 2021 (S4)e004100:10.24217/2531-0151.21v1s4.00041.

Edited: Madrid, España.

Abstract

Introduction: Allergic contact dermatitis (ACD), also known as contact hypersensitivity (CHS), is a frequent T-cell mediated inflammatory skin disease characterized by red, itchy, swollen and cracked skin. It is caused by the direct contact with an allergen and/or hapten like oxazolone (OXZ). This pathology depends on the activation of specific T cells and their cytokine and chemokine secretion. ACD presents two phases: (i) sensitization, in which the clonal expansion of specific T cells occurs, and (ii) elicitation, in which the activation and recruitment of specific T cells at the inflammation site take place after re-exposure to the hapten. Galectins are β-galactoside-binding animal lectins expressed in many tissues and organs. Galectin-1 (Gal-1) is highly expressed in several types of immune cells. The role of endogenous Gal-1 in CHS model is not known. Material and Methods: To develop the model of CHS the shaved abdomen of mice was treated with OXZ 3% at day 1. At day five, the second challenge with OXZ 1% was applied in one side of right ear. We used double reporter mice that express IL17-GFP and Foxp3-RFP proteins, backcrossed with Gal-1+/+ or Gal-1-/- mice. In addition, Rag1-/- mice were backcrossed with Gal-1+/+ and Gal-1-/- mice.

Results: We found that Gal-1-/- mice display more sustained and prolonged skin inflammation than Gal-1+/+ mice after OXZ treatment. Gal-1-/- mice have increased CD8+ T cells and neutrophilic infiltration in the skin. After the sensitization phase, Gal-1-depleted mice showed increased frequency of central memory CD8+ T cells and IFNγ secretion by CD8+ T cells. The absence of Gal-1 does not affect migration of transferred CD4+ and CD8+ T cells from the blood to the lymph nodes or to the skin. Depletion of CD4+ T lymphocytes as well as adoptive transfer experiments demonstrated that endogenous expression of Gal-1 on CD8+ T lymphocytes exerts a major role in the control of contact hypersensitivity model.

Conclusion: These data underscore the protective role of endogenous Gal-1 in CD8+ but not CD4+ T cells in the development of allergic contact dermatitis.

e00042
Studies for new applications of a monoclonal antibody antiCCR7. Validation as a therapy in onco-immunology.

Raquel Juárez-Sánchez1,3, Carlos Cuesta-Mateos1,2,3, Tamara Mateu-Albero3, Fernando Terrón1,2, Cecilia MuñozCalleja3,6. 

*Corresponding author: Raquel Juarez Sanchez, Madrid,Spain e-mail:rajusan@hotmail.es

Details of affiliation

(1) IMMED S.L., Immunological and Medicinal Products, Madrid, Spain (2) Catapult Therapeutics, Lelystad, The Netherlands (3) Immunology Department, Hospital Universitario de La Princesa, IIS-IP, Madrid, Spain (4) Hematology Department, Hospital Universitario de La Princesa, IIS-IP, Madrid, Spain (5) Pepscan, Lelystad, The Netherlands (6) Medicine Faculty, Universidad Autónoma de Madrid, Madrid, Spain

Funding

Fundación La Caixa, Torres Quevedo Fellowship for industrial PhD

Competing Interests:

The authors declare that no competing interests exist.

Keywords: CCR7, CLL, therapy

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00042

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Raquel Juárez-Sánchez, Carlos Cuesta-Mateos, Tamara Mateu-Albero, Fernando Terrón1,2, Cecilia Muñoz-Calleja, et al. Studies for new applications of a monoclonal antibody anti-CCR7. Validation as a therapy in onco-immunology. IBJ Plus 2021 (S4)e0042:10.24217/25310151.21v1s4.00042

Edited: Madrid, España.

Abstract

Introduction: Chemokines are a class of cytokines that are expressed on the cell surface or secreted into the cell mesenchyme. They are expressed by several types of cells from immune system, our chemokine of interest is CCR7, a potent leukocyte chemotactic receptor that together with its ligands CCL19 and CCL21, is responsible for the correct homing and trafficking of dendritic cells and lymphocytes to secondary lymphoid tissues. We studied the expression of CCR7 in chronic lymphocytic leukemia (CLL) and the function of a novel antibody anti-CCR7. CCR7 surface over-expression has been consistently reported in nearly all CLL patients. CCR7 over-expression is critical for CLL cell trafficking, firm arrest, and extravasation through high endothelial venules (HEVs) and CCR7 also guides CLL cells within the LN parenchyma. We performed the experiments with a novel humanized IgG1 anti-hCCR7 blocking antibody, specifically aimed for cancer therapy.

Material and Methods: Patients included in this study were diagnosed for CLL according to WHO consensus criteria. PBMC from patients were isolated from fresh samples. Migration assay and ADCC were performed to test the capacity of anti-CCR7 antibody to inhibit cell migration and capacity of cytotoxicity in tumor cells. Cynomolgus monkeys were used to establish a former pharmacodynamics and toxicological profile at different dose levels. In two separate studies we evaluated by flow cytometry the effect anti-CCR7 antibody on immune cells.

Results: The generated anti-CCR7 binds to CCR7 expressed in CLL cells and neutralizes target-induced signaling, chemotaxis, homing and survival. The antibody neutralizes CCR7-mediated CLL cells migration in response to CCL19 or CCL21 and induce a strong ADCC on target CLL cells.

Conclusion: In CLL, CCR7 is consistently found overexpressed and has been correlated with bulky lymphadenopathy and aggressive disease. Anti-CCR7 adds on the inhibition of migration of malignant cells to the LNs or other SLO, preventing therefore their escape to survival niche and provides additional cell killing through ADCC against accumulated CLL cells in bloodstream. We validate the utility of a novel generated antibody anti-CCR7 as a novel therapy for these patient

e00043
Immunecheckpoints in sepsis: an approach to diagnosis and therapy.

Roberto Lozano-Rodríguez1,2, José Avendaño-Ortiz1,2, Verónica Terrón-Arcos1,2, Carolina Rubio1,2, Karla Montalbán-
Hernández1,2, Jaime Valentin1,2, José Carlos Casalvilla1,2, Elisa Pulido-Lucas1,2, , Luis Augusto Aguirre1,2,
Eduardo López-Collazo1,2,3*. 

*Corresponding author: Eduardo López-Collazo. Innate Immunity Group, IdiPAZ, La Paz University Hospital, Madrid, Spain.
E-mail: elopezc@salud.madrid.org

Details of affiliation

1The Innate Immune Response Group IdiPAZ, La Paz University Hospital, Madrid, Spain.
2Tumour Inmunology Lab, IdiPAZ, La Paz University Hospital, Madrid, Spain.
3Centre for Biomedical Research Network of Respiratory Diseases (CIBERES), Madrid, Spain.

Funding

This work was supported by grants from Instituto de Salud Carlos III (ISCIII) and “Fondos FEDER” to Eduardo López Collazo (PI 18/00148).

Competing Interests:

All authors: No reported conflicts of interest.

Keywords: Immunecheckpoint, monocytes, RNAseq, sepsis.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00043

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Roberto Lozano-Rodríguez, José Avendaño-Ortiz, Verónica Terrón-Arcos, Carolina Rubio, Karla Montalbán-Hernández, José Carlos Casalvilla,
Elisa Pulido-Lucas, Jaime Valentín, Luis Augusto Aguirre, Eduardo López-Collazo. Immunecheckpoints in sepsis: an approach to diagnosis and therapy.
IBJ Plus 2021 (S4)e0043:10.24217/2531-0151.21v1s4.00043

Edited: Madrid, España.

Abstract

Introduction: Sepsis is defined as a dysregulated inflammatory response against an infection which, beyond antibiotics
and fluid restoration, still without effective treatment. However, deaths from sepsis reflect a host immunosuppression,
defined as endotoxin tolerance (ET), which implies a high risk to nosocomial infection. Due to therapeutic
limitations and the growing interest in that phase, immunecheckpoints (ICs) appear to be a good candidates as
therapeutic targets. The ICs ligand expression in monocytes/macrophages during the immunosuppressive phase in
sepsis could regulate the immune response by inducing the T cell exhaustion.

Methodology: To study the expression of these ICs in the extracellular membrane on monocytes in a sepsis context,
RNA sequencing (RNAseq) analysis was performed in an in vitro model of ET that simulates the two phases of sepsis
(inflammatory and immunosuppressive) on purified monocytes of healthy volunteers (HVs). It was used an enrichment
method based in a sequential permeabilization cellular to obtain messenger RNA (mRNA) encoding cytosolic
and soluble/extracellular membrane proteins. This enrichment method was validated by real-time quantitative
polymerase chain reactions (RT-qPCR). The focus was placed on those ICs that present the V-set type domain, since
the most of the ICs already described present this domain.

Results: RT-qPCRs of cytosolic and soluble/membrane genes demonstrated that sequential permeabilization was
effective. Moreover, RTqPCRs of genes widely studied in ET, such as TNFα and PD-L1, confirmed ET in vitro model it
turned out as expected. Next, RNAseq was performed using the mRNA encoding soluble/extracellular membrane
and a principal component analysis (PCA) shown that each condition of the ET model were grouped and fine separated
between them. Additionally, an unsupervised heatmap analysis showed that each condition of the ET model
were also grouped and separated in 5 clusters of genes. Finally, we identified genes differently expressed containing
the V-type domain, such as PD-L1 previously described in sepsis, confirming the model and the methodology used.

Conclusion: Sequential permeabilization methodology was effective and the endotoxin tolerance in vitro model
showed results as we expected. RNAseq data showed by PCA analysis that all conditions of the model were grouped
and fine separated between them, confirming that ET model was rigorously performed. Finally, we identified genes
differentially expressed containing the V-set type domain. Next steps would involve finding the differential expression
of the identified genes in monocytes of septic patients and perform ex vivo experiments to validate them as
therapeutic targets.

e00044
Lamin A/C regulates epigenetic changes in CD4+ T-cells
favoring Th1 commitment.

Beatriz Herrero Fernandez 1,2; Raquel Gómez Bris 1,2; Raquel Toribio-Fernandez 3; Virginia Zorita García 3; Ángela Sáez
1,4; Silvia Arribas 2*; José María Gonzalez Granado 1,2,3,5.*. 

*Corresponding author: SM Arribas. E-mail: silvia.arribas@uam.es
JM Gonzalez-Granado. E-mail: jmgonzalez.imas12@h12o.es

Details of affiliation

1 LamImSys Lab. Instituto de Investigación Hospital 12 de Octubre (imas12), 28041 Madrid, Spain.
2 Departamento de Fisiología. Facultad de Medicina. Universidad Autónoma de Madrid (UAM), 28029 Madrid, Spain.
3 Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029 Madrid, Spain.
4 Facultad de Ciencias Experimentales. Universidad Francisco de Vitoria (UFV), 28223 Pozuelo de Alarcón (Madrid), Spain
5 CIBER de Enfermedades Cardiovasculares (CIBER-CV), 28029 Madrid, Spain.

Funding

ISCIII (PI17/01395, PI20/00306, SNS I3), MICIU (FPU18/00895, FPU19/01774, RTI2018-097504-B-100).

Competing Interests:

No conflict of interest.

Keywords: Lamin A/C, T CD4 cell, epigenetics, T-bet, Foxp3.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00044

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Beatriz Herrero Fernandez; Raquel Gómez Bris; Raquel Toribio-Fernandez ; Virginia Zorita García ; Ángela Sáez; Silvia Arribas ;
José María Gonzalez Granado, et al. Lamin A/C regulates epigenetic changes in CD4+ T-cells favoring Th1 commitment. I IBJ Plus 2021 (S4)
e0044:10.24217/2531-0151.21v1s4.00044.

Edited: Madrid, España.

Abstract

Nuclear envelope protein lamin A/C in CD4+ T cells enhances T cell activation and differentiation towards T
helper 1 (Th1) phenotype while reduces Treg polarization. Since epigenetic regulation is a key determinant of
the Th fate, our objective is to analyze the capacity of lamin A/C to modulate epigenetic changes of the master
transcription factors of Th polarization. To achieve this, we analyzed post-translational histone modifications
on the promoters of the genes encoding these transcription factors by ChIP-qPCR in in vitro activated WT and
Lmna−/− CD4+ T cells (isolated from Lmnafl/fl or CD4-Cre+/- Lmnafl/fl mice). In addition, we performed in vitro
transduction with EZH1 and EED (two epigenetic-modifying enzymes which are components of the polycomb)
RNAi retroviruses in in vitro Th1 and Treg differentiated WT and Lmna−/− CD4+ T cells. ChIP-qPCR analysis of the
Foxp3 promoter revealed no differences for the studied modifications (H3K4me3, H3K27me3 and H3K4me1) while
Lmna−/− T cells had significantly fewer H3K4me1 marks on the Tbx1 (T-bet) promoter than WT cells. Regarding
the RNAi experiments, we observed that EZH1, but not EED, downregulation not only abolished Tbx21 mRNA
differences between WT and Lmna−/− CD4+ T cells, but also eliminated the differences in T-bet-regulated Ifng
(IFNγ) mRNA.
Our findings suggest that lamin A/C contributes to the regulation of T-bet expression during Th1 commitment,
at least in part through an epigenetic mechanism. In contrast, lamin A/C-dependent FOXP3 regulation does not
involve the same epigenetic changes than Tbet. For FOXP3, regulation might occur by the direct interaction of
lamin A/C with transcription factors.
In conclusion, knowledge of the mechanisms that define differentiation towards a specific Th or Treg phenotype
may be of interest for some diseases, such as inflammatory bowel disease, where modified cells could be
used as therapy.

e00045
A novel NRF2-βTrCP Protein-Protein Interaction (PPI) inhibitor suppresses lipopolysaccharide-mediated inflammation through the activation of transcription factor NRF2.

Raquel Fernández-Ginés 1, Ana I. Rojo 1, José Antonio Encinar2, and Antonio Cuadrado 1. 

*Corresponding author: Instituto de investigaciones Biomédicas “Albertos Sols”. Department of Biochemistry, Faculty of Medicine, Autonomous University of
Madrid (UAM), Madrid, Spain. E-mail: Rfgines@iib.uam.es

Details of affiliation

1 Instituto de Investigación Sanitaria La Paz (IdiPaz). Centro de Investigación Biomédica en Red sobre Enfermedades
Neurodegenerativas (Ciberned). Instituto de investigaciones Biomédicas “Albertos Sols”. Department of Biochemistry, Faculty of
Medicine, Autonomous University of Madrid (UAM), Madrid, Spain.
2 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, SpainInstituto de Biología Molecular y Celular
(IBMC), Alicante, Spain.

Funding

This study was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Grant SAF2016-76520-R) and The Autonomous
Community of Madrid (grant B2017/BMD-3827). R.F.G was recipient of an FPI contract of the Spanish Ministry of Economy and Competitiveness.

Competing Interests:

No conflict of interest.

Keywords: NRF2, β-TrCP, KEAP1, Protein-Protein Interaction (PPI) Inhibitor, Inflammation, LPS.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00045

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Raquel Fernández-Ginés, Ana I. Rojo, José Antonio Encinar, and Antonio Cuadrado, et al. A novel NRF2-βTrCP Protein-Protein Interaction
(PPI) inhibitor suppresses lipopolysaccharide-mediated inflammation through the activation of transcription factor NRF2. IBJ Plus 2021 (S4)
e0045:10.24217/2531-0151.21v1s4.00045

Edited: Madrid, España.

Abstract

Chronic diseases, such as neurodegenerative and metabolic disorders, are characterized by long-term mild
inflammation. Corticosteroids and non-steroidal anti-inflammatory drugs are not appropriate for the continuous
administration in these diseases because they exhibit many undesired effects. Therefore, it is necessary to find
other compounds that can exert an anti-inflammatory and cytoprotective function. A new strategy to control
inflammation is the activation of transcription factor NRF2, nowadays considered as a master regulator of cellular
homeostasis. From a clinical perspective, NRF2 activation produces a beneficial therapeutic effect in most chronic
diseases characterized by low-grade oxidative stress and inflammation. Intensive research has been focused on
the identification of small electrophilic molecules that inhibit the E3 ubiquitin ligase adapter KEAP1, which is the
canonical mechanism for the ubiquitin-proteasome degradation of NRF2. However, these electrophiles exhibit
many unspecific activities and are hardly advancing towards their clinical use. Interestingly, a completely unexplored
alternative is the pharmacological modulation of the E3 ubiquitin ligase β-TrCP, also involved in its proteasomal
degradation. Here we report the development of a Protein-Protein Interaction (PPI) inhibitor of NRF2-β-Tr-
CP that offers an alternative to KEAP1 for NRF2 activation. This small molecule increases NRF2 levels and induces
the expression of NRF2-regulated genes such as Hmox1, in control and in KEAP1-defficient fibroblasts, but not in
β-TrCP-knock-down cells. Moreover, the compound attenuates the production of pro-inflammatory markers in
cultured macrophages and in liver of mice submitted to the endotoxin lipopolysaccharide. These findings suggest
that this compound could be used as an alternative to conventional anti-inflammatory therapies.

e00046
WIP uses the NRF2/KEAP1 axis in glioblastoma cells to
promote oxidant tolerance.

Diego Lastra1,2,3,4,*, Natalia Robledinos-Antón1,2,3,4, Antonio Cuadrado1,2,3,4 and Maribel Escoll1,2,3,4. 

*Corresponding author: Diego Lastra1,2,3,4,*, Madrid, Spain . E-mail: diegolastra@hotmail

Details of affiliation

1 Department of Biochemistry, Faculty of Medicine, Autonomous University of Madrid (UAM), Madrid, Spain
2 Instituto de Investigaciones Biomédicas “Alberto Sols” UAM-CSIC, Madrid, Spain.
3 Instituto de Investigación Sanitaria La Paz (IdiPaz), Madrid, Spain.
4 Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain.

Funding

This work was supported by PID2019-110061RB-I00, RTI2018-096303-B-C31 and SAF2017-82436R of the Spanish Ministry of Economy and
Competiveness; and by the P_37_732/2016 grant (REDBRAIN) financed by the European Regional Development Fund, Competitiveness Operational
Program 2014–2020, and Comunidad Autónoma de Madrid (grant B2017/BMD-3827). M.E. was recipient of a postdoctoral contract Juan de la
Cierva; D.L. and N.R.-A. enjoied a FPU contract of MINECO.

Competing Interests:

The authors declare no conflict of interest.

Keywords: antioxidants; cytoskeleton; oxidative stress; redox.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00046

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Diego Lastra, Natalia Robledinos-Antón, Antonio Cuadrado and Maribel Escoll, et al. WIP uses the NRF2/KEAP1 axis in glioblastoma cells to
promote oxidant tolerance. IBJ Plus 2021 (S4)e0046:10.24217/2531-0151.21v1s4.00046.

Edited: Madrid, España.

Abstract

Introduction: High proliferation and metabolic rates are characteristic traits of tumor cells, which eventually produce
exacerbated levels of reactive oxygen species (ROS) that tumor cells must control to maintain proliferation.
Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) is a scaffold multifunctional protein that essentially
controls Actin polymerization, podosome and invadopodia formation, etc. These functions support the
pro-tumoral role of WIP, endowing cancer cells with anchorage-independent growth and higher motility. WIP is
also able to promote cell survival and proliferation through independent poorly understood mechanisms. In this
study, we have focused on a possible relation between WIP and redox homeostasis in glioblastomas.

Material and Methods: U-373 MG and U-87 MG were grown under adherent conditions, and infected with
lentiviral vectors shRNA control, shWIP, shKEAP1, NRF2WT, NRF2ΔETGE, and NRF26SA. Protein and mRNA levels
were analyzed through western blotting and qRT-PCR, respectively. ROS levels were detected in a FACScan flow
cytometer (Becton-Dickinson) with hydroethidine (HE) probe. Immunofluorescence and image analysis were also
carried out using the Fiji Software.

Results: We show that the absence of WIP induced an increase of ROS levels, which correlated with a reduction
of the levels of NRF2 (Nuclear factor (erythroid-derived 2)-like 2), master regulator of redox homeostasis. We
demonstrate that WIP stabilizes NRF2 through the inhibition of E3 ligase adapter KEAP1, main NRF2 postranslational
repressor, thus helping to maintain redox homeostasis. What is more, the overexpression of NRF2ΔETGE mutant,
resistant to KEAP1 targeted degradation, in WIP depleted cells, restored normal ROS levels. Finally, we show
that the mechanism underlying high KEAP1 (Kelch-like ECH-associated protein 1) activity in WIP-depleted cells
consists in a WIP-dependent Actin cytoskeleton reorganization, which probably modifies the binding between
KEAP1 and F-Actin.

Conclusions: Together, our results show a novel role of WIP in cancer development, through NRF2 activity regulation
and the maintenance of oxidant tolerance in cancer cells, which could be addressed as novel target for the
development of antitumoral therapies.

e00047
METPlatform identifies brain metastasis vulnerabilities and
predicts patient response to therapy.

Lucía Zhu1, Tobias Weiss2, Riccardo Soffietti3, Juan Manuel Sepúlveda4, Joaquín Pastor5, Manuel Valiente1*. 

*Corresponding author: Manuel Valiente, Brain Metastasis Group, CNIO, Madrid, Spain. E-mail: mvaliente@cnio.es

Details of affiliation

1Brain Metastasis Group, CNIO, Melchor Fernández Almagro, 3, 28029, Madrid, Spain.
2Department of Neurology, University Hospital Zurich, Rämistrasse 100, 8091, Zurich, Switzerland.
3Neuro-Oncology Department, University and City of Health and Science University Hospital of Turin, Via Giuseppe Verdi, 8, 10124,
Turin, Italy.
4Neuro-Oncology Unit, Hospital Universitario Doce de Octubre, Avenida de Córdoba, s/n, 28041, Madrid, Spain.
5Experimental Therapeutics Programme, CNIO, Melchor Fernández Almagro, 3, 28029, Madrid, Spain.

Funding

This work was supported by MINECO (SAF2017-89643-R, SAF2014-57243-R, SAF2015-62547-ERC) (M.V.), Fundación FERO (IX FERO Grant
for Research in Oncology) (M.V.), Fundació La Marató de TV3 (141) (M.V.), Beug Foundation (Prize for Metastasis Research 2017) (M.V.), Fundación
Ramón Areces (CIVP19S8163) (M.V.), Worldwide Cancer Research (19-0177) (M.V.), H2020-FETOPEN (828972) (M.V.), Cancer Research Institute
(Clinic and Laboratory Integration Program CRI Award 2018 (54545)) (M.V.), AECC (Coordinated Translational Groups 2017 (GCTRA16015SEOA) (M.V.),
LAB AECC 2019 (LABAE19002VALI) (M.V.)), ERC CoG (864759) (M.V.), Sophien-Stiftung zur Förderung der klinischen Krebsorschung (T.W.), Stiftung
für angewandte Krebsforschung (T.W.), Forschungskredit of the University of Zurich (FK-18-054) (T.W.), Betty and David Koetser Foundation for Brain
Research (T.W.), La Caixa-Severo Ochoa International PhD Program Fellowship (LCF/BQ/SO16/52270014) (L.Z.). The contribution of the Experimental
Therapeutics Programme was supported by core funding from the Spanish National Cancer Research Center (CNIO). CNIO is supported by the ISCIII,
the Ministerio de Ciencia e Innovación, and is a Severo Ochoa Center of Excellence (SEV-2015-0510). M.V. is a Ramón y Cajal Investigator (RYC-2013-
13365) and EMBO YIP (4053).

Competing Interests:

Authors declare no competing interests.

Keywords: brain metastasis, drug-screen, organotypic cultures.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00047

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Lucía Zhu, Tobias Weiss, Riccardo Soffietti, Juan Manuel Sepúlveda, Joaquín Pastor, Manuel Valiente, et al. METPlatform identifies brain
metastasis vulnerabilities and predicts patient response to therapy. IBJ Plus 2021 (S4)e0047:10.24217/2531-0151.21v1s4.00047

Edited: Madrid, España.

Abstract

Introduction: The diagnosis of brain metastasis involves high morbidity and mortality and remains an unmet clinical
need in spite of being the most common tumor in the brain. Exclusion of these cancer patients from clinical trials is a
major cause of their limited therapeutic options.

Material and methods: We report a novel drug-screening platform (METPlatform) based on organotypic cultures
which allows identifying effective anti-metastasis agents in the presence of the organ microenvironment. We have
applied this approach to clinically relevant stages of brain metastasis using both experimental models and human
tumor tissue (by performing patient-derived organotypic cultures – PDOCs –). We have also used METPlatform to
perform unbiased proteomics of brain metastases in situ to identify potential novel mediators of this disease and
explore resistance mechanisms to targeted therapy. Finally, we have exploited METPlatform as “avatars” to predict
response to therapy in patients with primary brain tumors.

Results and conclusions: We identified heat shock protein 90 (HSP90) as a promising therapeutic target for brain
metastasis. DEBIO-0932, a blood-brain barrier permeable HSP90 inhibitor, shows high potency against mouse and
human brain metastases from different primary origin and oncogenomic profile at clinically relevant stages of the
disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis of brain
metastases treated with the chaperone inhibitor revealed non-canonical clients of HSP90 as potential novel mediators
of brain metastasis and actionable mechanisms of resistance driven by autophagy. Combined therapy using
HSP90 and autophagy inhibitors showed synergistic effects compared to sublethal concentrations of each monotherapy,
demonstrating the potential of METPlatform to design and test rationale combination therapies to target metastasis
more effectively. Finally, we show that PDOCs from glioblastoma predict the response of the corresponding
patient to standard of care, thus proving the potential of this strategy for improving personalized care in cancer. In
conclusion, our work validates METPlatform as a potent resource for metastasis research integrating drug-screening
and unbiased omic approaches that is fully compatible with human samples and questions the rationale of excluding
patients with brain metastasis from clinical trials. We envision that METPlatform will be established as a clinically
relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.

e00048
A global map of the impact of deletion of Post-Translational
Modification sites in genetic diseases.

Perceval Vellosillo1,2, Pablo Mínguez1,2*. 

*Corresponding author: Pablo Mínguez. E-mail: pablo.minguez@quironsalud.es

Details of affiliation

1Department of Genetics, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz University Hospital, Universidad Autónoma de
Madrid (IIS-FJD, UAM), Madrid 28040, Spain. 2Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid
28040, Spain

Funding

Instituto de Salud Carlos III (ISCIII) Fondos FEDER by means of the Miguel Servet Program (CP16/00116), Instituto de Salud Carlos III (ISCIII)
(PI18/00579) and the Ramon Areces Foundation.

Competing Interests:

The authors declare that they have no competing interests.

Keywords: Protein post-translational modifications; Genetic diseases; Rare diseases; Proteomics; Genomics; Systems Biology.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00048

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Perceval Vellosillo Pablo Mínguez, et al. A global map of the impact of deletion of Post-Translational Modification sites in genetic diseases.
IBJ Plus 2021 (S4)e0048:10.24217/2531-0151.21v1s4.00048.

Edited: Madrid, España.

Abstract

Introduction: Protein post-translational modifications (PTMs) define protein functional sites. More than 200 PTM
types are described in eukaryotes, having diverse species conservation levels, coverage in the proteome, number of
high-throughput experiments and functional roles. The knowledge accumulated about every type also differs, from the
highly studied phosphorylation or ubiquitination to others like neddylation or malonylation. From a clinical perspective,
a number of diseases have been associated to deregulated PTM sites and missense rare variants are globally enriched
in PTMs. We hypothesize that some genetic diseases may be caused by the deregulation of particular functions
produced by the removal of a specific PTM type by genomic variants.

Material and Methods: We collected >320,000 human PTMs of 59 types experimentally validated and cross them with
>4M missense DNA variants annotated with pathogenic predictions and disease/phenotype associations. Statistical
methods were performed to extract significant associations between PTM types and genetic diseases that were further
evaluated using a confidence score based on PTMs position permutations.

Results: We report >1.74M PTM-variant concurrences in >16,500 proteins that an enrichment analysis distributed in
217 pairwise significant associations between 18 PTM types and 150 genetic diseases. Around 23% of these associations
are already described in the literature, 34% have partial evidences based on single variants, related diseases or regulatory evidences, and 43% are novel. Removal of acetylation presents almost 30% of the associations, still low studied PTM types like S-glutathionylation shows 14 connections. A network of 133 PTM types and phenotypes associations is also discussed.

Conclusions: Our results shows an unexpected impact of PTM removal producing genetic diseases and phenotypes that is PTM type specific. We describe for the first time a general scenario of PTM types and genetic diseases direct associations, that provides new capacities to understand and diagnose these disorders. Using pathogenicity predictions we identified 156 potential PTM sites to produce particular diseases if genomic variants remove them.
Acetylation or Carboxylation shown higher percentages of sites with significant pathogenicity. Several examples of proteins with clinical relevance are examined providing new potential sites associated to specific diseases.

Figure. PTM type-disease predicted associations represented as links between PTM types and disease families. PTM types are
arranged in clusters based on the disease families they share and disease families are colored according to their connections
to clusters.

e00049
Prioritizing variants of uncertain significance for
reclassification using a rule-based algorithm in inherited
retinal dystrophies.

Ionut-Florin Iancu1,2, Almudena Avila-Fernandez1,2, Ana Arteche1, María José Trujillo-Tiebas1,2, Rosa Riveiro-Alvarez1,2,
Berta Almoguera1,2, Inmaculada Martin-Merida1,2, Marta Del Pozo-Valero1,2, Irene Perea-Romero1,2, Marta Corton1,2,
Pablo Minguez1,2 *, Carmen Ayuso1,2 *. 

*Corresponding author: Carmen Ayuso. Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospital,
Universidad Autónoma de Madrid (IIS-FJD, UAM), Madrid, Spain.
E-mail: cayuso@fjd.es

Details of affiliation

1 Department of Genetics, Instituto de Investigación Sanitaria–Fundación Jiménez Díaz University Hospital, Universidad Autónoma
de Madrid (IIS-FJD, UAM), Madrid, Spain.
2 Center for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain.

Funding

This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including
CIBERER (06/07/0036), IIS-FJD Biobank PT13/0010/0012, and FIS (PI16/00425 and PI19/00321); and from the regional government of Madrid,
RAREGenomics-CM (CAM, B2017/BMD-3721), all partially supported by FEDER (European Regional Development Fund). University Chair UAM-IISFJD
of Genomic Medicine and the Ramon Areces Foundation (CIVP18A3862) also supported our work.

Competing Interests:

The authors declare that they have no competing interests.

Keywords: Variants of uncertain significance, Inherited retinal diseases, variant reclassification, rule-based algorithm

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00049

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Iancu, IF., Avila-Fernandez, A., Arteche, A., et al. Prioritizing variants of uncertain significance for reclassification using a rule-based algorithm
in inherited retinal dystrophies. IBJ Plus 2021 (S4)e0049:10.24217/2531-0151.21v1s4.00049.

Edited: Madrid, España.

Abstract

Introduction: Inherited retinal dystrophies (IRD) are a highly heterogeneous group of rare diseases with a molecular
diagnostic rate of >50%. Reclassification of variants of uncertain significance (VUS) poses a challenge for
IRD diagnosis.

Methods: We collected 668 IRD cases analyzed by our geneticists using two different clinical exome sequencing
tests: TruSightOne Sequencing Panel kit and Clinical Exome Solution-Sequencing Panel kit. We identified 114
unsolved cases pending reclassification of 125 VUS and studied their genomic, functional, and laboratory-specific
features, comparing them to pathogenic and likely pathogenic variants from the same cohort (N=390).

Results: While the clinical exome used did not show differences in diagnostic rate, the more IRD-experienced
geneticist reported more VUS (p = 4.07e-04). Significantly fewer VUS were reported in recessive cases (p = 2.14e-
04) compared to other inheritance patterns, and of all the genes analyzed, ABCA4 and IMPG2 had the lowest
and highest VUS frequencies, respectively (p = 3.89e-04, p = 6.93e-03). Moreover, fewer frameshift and stop-gain
variants were found to be informed VUS (p = 6.73e-08 and p = 2.93e-06). Last, we applied five pathogenicity
predictors and found there is a significant proof of deleteriousness when all score for pathogenicity in missense
variants. Together, these results provided input for a set of rules that correctly reclassified ~70% of VUS as pathogenic
in three different validation datasets. We applied the algorithm to the initial set of VUS and selected 49
(out of 117) VUS fulfilling at least one of the rules. We performed a reassessment of the cases with the selected
VUS and were able to compile new evidence in 13 variants from unsolved cases, 5 of them being reclassified to
likely pathogenic. The remaining prioritized VUS will be subject of a close follow-up hoping for a prompt conclusive
diagnosis.

Conclusion: In summary, we present a strategy to assist VUS reclassification by prioritizing those VUS more likely
to be causative. Our prioritization strategy comes from an exhaustive study of laboratory, cohort, and variant
features than can be performed elsewhere.

e00050
KV1.3 channel inhibition by a family of indolic compounds.

Baena-Nuevo M1,2*, Reyes A1,2, Vera-Zambrano A1,2, Zapata JM2,3, Perez-Chacon G2,3 and Gonzalez T1,2,3. 

*Corresponding author: María Baena-Nuevo, Biochemistry Department, UAM and Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC-UAM, Madrid,
Spain. E-mail: mbnuevo@iib.uam.es

Details of affiliation

1Biochemistry Department, UAM, Madrid, Spain
2Instituto de Investigaciones Biomédicas “Alberto Sols”, CSIC-UAM, Madrid, Spain
3Instituto de Investigación Hospital Universitario La Paz (IdiPaz), Madrid, Spain

Funding

This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) from the Spanish Ministry of Health, including
CIBERER (06/07/0036), IIS-FJD Biobank PT13/0010/0012, and FIS (PI16/00425 and PI19/00321); and from the regional government of Madrid,
RAREGenomics-CM (CAM, B2017/BMD-3721), all partially supported by FEDER (European Regional Development Fund). University Chair UAM-IISFJD
of Genomic Medicine and the Ramon Areces Foundation (CIVP18A3862) also supported our work.

Competing Interests:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

Keywords: Kv1.3, indole-3-carbinol (I3C), 3,3’-diindolylmethane (DIM), indolic compounds.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00050

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Baena-Nuevo M, Reyes A, Vera-Zambrano A, Zapata JM, Perez-Chacon G, and Gonzalez T, et al. Baena-Nuevo M1,2*, Reyes A1,2, Vera-
Zambrano A1,2, Zapata JM2,3, Perez-Chacon G2,3 and Gonzalez T1,2,3. IBJ Plus 2021 (S4)e0050:10.24217/2531-0151.21v1s4.0050.

Edited: Madrid, España.

Abstract

Introduction: KV1.3 potassium channels are involved in B and T cell function, cellular cycle regulation and cell
proliferation. Therefore, they have been identified as therapeutic targets against autoimmune diseases, like
arthritis rheumatoid, and some cancers. Indole-3-carbinol (I3C) and its main metabolite (DIM) induce tumour
regression, cell death in B cells from patients with chronic lymphocytic leukemia and inhibits Kv1.3 currents from
these cells. Here, we analyse the inhibitory action of I3C and its derivatives on Kv1.3 channels to determine the
molecular requirements of these compounds to bind to the channel.

Material and Methods: we assessed the effect of the indolic compounds on KV1.3 currents recorded in HEK-293
cells transfected with KV1.3-pEYFP-C1 and selected by flow cytometry. Currents were registered by whole-cell
patch-clamp. Statistical significance was determined by t-Student test.

Results: I3C and (6-Methyl-1H-indol-3yl)methanol (6Metil), both at 50 μM, inhibited KV1.3 current amplitude
recorded at +40 mV by 41.2±7.7 and 29.1±3.7 %, respectively. The other derivatives, 3-(2-hydroxythyl)-indole
(32HEI), indole-3-carboxylic acid (I3CA) and 2,3-dihydroindoline (indoline), at 50 μM, and DIM at 1.25 μM, did
not inhibit the KV1.3 current.

Conclusions: From the compounds tested, only I3C and 6Metil were able to inhibit the KV1.3 current. These compounds
have an ethyl group in position 3 which allow their dimerization. In contrast, I3CA, 32HEI and indoline
cannot form dimers. DIM, which is the dimeric form of I3C, did not inhibit the current due to the low concentration
tested (its low solubility prevented us to test relevant concentrations). In conclusion, an ethyl group in position
3 of the indolic compound seems to be necessary to inhibit the Kv1.3 current, either for the binding of the
compound to the Kv1.3 protein or for the dimerization of the compound previous to the binding to the channel.

e00051
Disruption of liver homeostasis and systemic metabolism
upon concomitant hepatic activation of growth factor and
nutrient signaling.

Ana Belén Plata-Gómez1, Celia de la Calle Arregui1, Alba Sanz1, Eduardo Caleiras2, Alejo Efeyan1. 

*Corresponding author: Alejo Efeyan, Metabolism and Cell Signaling Group, Molecular Oncology Programme, Spanish National
Cancer Research Centre (CNIO), Madrid, Spain. E-mail: aefeyan@cnio.es

Details of affiliation

1 Metabolism and Cell Signaling Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO),
Madrid, Spain
2 Histopathology Unit, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Funding

Research was supported by the RETOS projects Programme of Spanish Ministry of Science, Innovation and Universities, Spanish State
Research Agency, cofunded by the European Regional Development Fund, EU-H2020 Programme, Excellence Network Grant from MICIU/AEI, FERO
Grant for Research in Oncology; A.B.P.-G. is recipient of Ayudas de contratos predoctorales para la formacion de doctores from MICIU/AEI.

Competing Interests:

The authors declare no competing interests.

Keywords: mTORC1, liver, metabolism, zonation

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00051

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Plata-Gómez AB, et al. Disruption of liver homeostasis and systemic metabolism upon concomitant hepatic activation of growth factor and
nutrient signaling. IBJ Plus 2021 (S4)e0051:10.24217/2531-0151.21v1s4.00051.

Edited: Madrid, España.

Abstract

The mechanistic target of rapamycin complex I (mTORC1) coordinates cell growth and metabolism integrating
two major regulatory inputs: nutrients, which activate mTORC1 pathway through the family of Rag GTPases, and
growth factors, which inhibit the Tuberous Sclerosis Complex 1 (TSC1) to allow mTORC1 activation. Although progress
has been made in the understanding of mTORC1 pathway, we still have a poor comprehension about how
the regulatory inputs of mTORC1 can cooperate each other. Based on that we have engineered a mouse model
with liver specific loss of TSC1 that carries a replacement of a single nucleotide in the coding sequence of RagA
that translates as a RagA constitutively bound to GTP (RagAGTP/Δ). Our results show that while chronic activation
of mTORC1 by one of these branches of the pathway hardly leads to severe alterations in the liver, the Li-TSC1KO
RagAGTP/Δ model elicited several features of hepatic damage. Particularly, we have observed increased concentration
of liver injury markers in serum, histological alterations and loss of hepatic zonation together with perturbations
in glucose homeostasis. Moreover, Li-TSC1KO RagAGTP/Δ mice show reduced lifespan due to the development
of heterogeneous liver tumors. The exacerbation of the hepatic phenotype with the chronic activation of
mTORC1 by its two major inputs suggest that both nutrients and growth factors synergize to activate mTORC1
pathway in the liver. The strong phenotype of Li-TSC1KO RagAGTP/Δ mice is vulnerable to pharmacological inhibition
of mTORC1, thereby rapamycin administration rescues hepatic damage, corrects defects in glucose metabolism
and leads to an extension in survival. We are currently trying to determine the molecular mechanisms involved
in the development of this aberrant liver phenotype to understand the relevance of the cooperation between
mTORC1 inputs.

e00052
Myeloid p38s modulate BAT function through hepatic FGF21
during obesity.

Maria Crespo1 , Ivana Nikolic1 , Alfonso Mora1 , Aránzazu Pintor1 , Maria Elena Rodríguez 1, Luis Leiva-Vega 1,
Magdalena Leiva1* , Guadalupe Sabio1*. 

*Corresponding author: Guadalupe Sabio, Centro Nacional de Investigaciones Cardiovasculares Carlos III (F.S.P), Madrid, Spain.
E-mail: guadalupe.sabio@cnic.es ; Magdalena Leiva, Centro Nacional de Investigaciones Cardiovasculares Carlos III (F.S.P), Madrid,
Spain. E-mail: magdalena.leiva@cnic.es

Details of affiliation

1Centro Nacional de Investigaciones Cardiovasculares Carlos III (F.S.P)

Funding

FPI Ministerio de Economía y Competitividad BES-2017-079711, MINECO-FEDER SAF2016-79126-R, PROYE19047SABI, AECC
INVES20026LEIV, MINECO-FEDER-SAF2015-74112-JIN.

Competing Interests:

No competing interests declared

Keywords: p38, macrophages, FGF21, liver, BAT, obesity.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00052

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Maria Crespo, et al. Myeloid p38s modulate BAT function through hepatic FGF21 during obesity. IBJ Plus 2021 (S4)e0052:10.24217/2531-
0151.21v1s4.00052.

Edited: Madrid, España.

Abstract

Introduction: For years, macrophages were placed as key drivers of obesity associated inflammation through
the release of pro-inflammatory cytokines and recruitment of other immune cells into tissues. However, now we
know that they play important role maintaining tissue homeostasis and are key modulators of brown adipose
tissue (BAT) thermogenesis and liver metabolism. In fact, local resident macrophages regulate BAT activation
affecting energy expenditure. p38MAPKs constitute one of the main signaling pathways activated in macrophages
upon inflammation. However, while myeloid p38 was well studied during acute inflammation, it is poorly
understood the role of myeloid p38 in shaping macrophage response during obesity and metabolic disorders.
Here, using a conditional knock-out mice for the main upstream activators of p38s, MKK3 and MKK6, in myeloid
cells (MKK3/6Lyzs-KO), we described myeloid p38 pathway in liver control BAT thermogenesis axis during obesity.

Material and methods: To induce obesity, we fed Lyzs-Cre and MKK3/6Lyzs-KO mice a high-fat diet (HFD) for 10
weeks. We analyzed them in metabolic cages, evaluated their response to insulin and glucose and determined
BAT temperature. After sacrificing the mice, we analyzed myeloid infiltration and morphological and functional
changes in both, BAT and in the liver. In vitro, we characterized the response of bone-marrow derived macrophages
(BMDM) from Lyzs-Cre and MKK3/6Lyzs-KO mice and evaluated the crosstalk between BMDM and hepatocytes.

Results: We found that after HFD, MKK3/6Lyzs-KO mice were more susceptible to obesity and diabetes due to an
impaired BAT thermogenic function that resulted in decreased energy expenditure. Drastic alterations in the liver
macrophage pool of MKK3/6Lyzs-KO mice correlated with decreased hepatic and circulating FGF21 levels. Mechanistically,
we found that BMDM lacking MKK3/6 were more pro-inflammatory both in vitro and in vivo after HFD
challenge and that BMDM after immune activation directly inhibits the expression of Fgf21 in hepatocytes. As a
consequence of the increased pro-inflammatory phenotype of macrophages lacking MKK3/6, hepatic FGF21 content
was lower in MKK3/6Lyzs-KO mice comparing to controls after HFD, resulting in the downregulation of hepatic
FGF21-BAT thermogenesis axis and decreased BAT function in knock-out mice.

Conclusions: In this study we described liver macrophages as new modulators of hepatic FGF21 expression being
myeloid p38 pathway crucial for this macrophage-hepatocyte crosstalk during obesity and affecting whole-body
metabolism by regulating hepatic FGF21-BAT axis.

e00053
NEW GENE, NEW SKELETAL DYSPLASIA
Identification and functional characterization of biallelic
variants in PRKG2 as cause of a
new Acromesomelic Dysplasia.

Díaz-González Francisca1,2, Wadhwa Saruchi3, Kapoor Seema4, Nishimura Gen5, Offiah Amaka C6, Faruq Mohammed3,
Heath Karen E1,2.. 

*Corresponding author: Francisca Díaz González INGEMM, Hospital Universitario La Paz, Madrid 28046, Spain; fra.diaz@estudiante.uam.es

Details of affiliation

1 1Institute of Medical and Molecular Genetics (INGEMM), IdiPAZ, Hospital Universitario La Paz, UAM, Madrid, Spain
2 Skeletal Dysplasia Multidisciplinary Unit (UMDE) and ERN- BOND, Hospital Universitario La Paz, Madrid, Spain
3 Genomics and Molecular Medicine Division, CSIR—Institute of Genomics and Integrative Biology, New Delhi, India.
4Dept. of Pediatrics, Maulana Azad Medical College and Lok Nayak Hospital, New Delhi, India
5 Center for Intractable Disease, Saitama Medical University Hospital, Saitama, Japan
6 Dept. of Radiology and ERN- BOND, Sheffield Children’s Hospital NHS Foundation Trust, Sheffield, UK

Funding

This work was supported in part by the following grants: SAF201784646- R from MINECO (to KEH), Council of Scientific & Industrial
Research (CSIR), India (to MF) and Indian Council of Medical Research (to MF). FG- D was supported by an FPU studentship from the Spanish
Ministry of Science, Innovation and Universities

Competing Interests:

The authors declare no competing interests.

Keywords: PRKG2, Acromesomelic Dysplasia, skeletal dysplasia

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00053

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Díaz-González F, et al. NEW GENE, NEW SKELETAL DYSPLASIA
Identification and functional characterization of biallelic variants in PRKG2 as cause of a new Acromesomelic Dysplasia
. IBJ Plus 2021 (S4)e0053:10.24217/2531-0151.21v1s4.00053.

Edited: Madrid, España.

Abstract

Background: C- type natriuretic peptide (CNP), its endogenous receptor, natriuretic peptide receptor- B (NPR- B), as
well as its downstream mediator, cyclic guanosine monophosphate (cGMP) dependent protein kinase II (cGKII), have
been shown to play a pivotal role in chondrogenic differentiation and endochondral bone growth. The binding of
CNP to NPR-B leads to synthesis of intracellular cGMP that subsequently activates cGKII. In humans, homozygous or
compound heterozygous variants in NPR2, encoding NPR-B, cause acromesomelic dysplasia, type Maroteaux (AMDM
[MIM 602875]), a rare autosomal recessive skeletal dysplasia characterised by severe disproportionate short stature,
acromesomelic shortening of the extremities, and other skeletal anomalies. Moreover, heterozygous loss of function
variants in NPR2, and NPPC, encoding CNP, cause milder phenotypes. In contrast, no variants in cGKII, encoded by the
protein kinase cGMP- dependent type II gene (PRKG2), have been reported in humans to date, although its role in
longitudinal growth has been clearly demonstrated in several animal models which presented with postnatal dwarfism
and shortened limbs as consequence of an elongated and abnormal growth plate.

Methods: Whole exome sequencing (WES) was performed in two girls with severe disproportionate short stature due
to acromesomelia of the limbs, moderate brachydactyly, variable platyspondyly and progressively increasing metaphyseal
alterations of the long bones. Functional studies were undertaken for the identified variants to demonstrate its
pathogenesis.

Results: Two homozygous PRKG2 variants, a nonsense (NM_006259.2: c.1705C>T: p.(Arg569*)) and a frameshift
(c.491dup: p.(Asn164Lysfs*2)) were identified. Both mutant transcripts are exposed to nonsense- mediated decay
thus reducing PRKG2 expression. The truncated mutant cGKII proteins, partially or completely lack the kinase domain,
thus they downregulate the downstream mitogen activation protein kinase signalling pathway (MAPK) by failing to
phosphorylate c- Raf 1 at Ser43 and subsequently unable to reduce ERK1/2 activation in response to fibroblast growth
factor 2. Moreover, they also downregulate COL10A1 and upregulate COL2A1 expression (markers of chondrocyte
differentiation and hypertrophy respectively), through modulation of SOX9 transcription factor activity.

Conclusion: In conclusion, we have clinically and molecularly described the first loss of function variants in PRKG2 in
two patient associated with a new acromesomelic dysplasia, acromesomelic dysplasia, PRKG2 type (AMDP)
These data has been recently published in the Journal of medical genetics (JMG). doi:10.1136/jmedgenet-2020-107177

e00054
HIV reverse transcriptases defective in strand
displacement activity.

Samara Martín-Alonso1, Mar Álvarez1, María Nevot2, Miguel Ángel Martínez2 & Luis Menéndez-Arias1*. 

*Corresponding author: lmenendez@cbm.csic.es
Samara Martín-Alonso, Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad
Autónoma de Madrid), c/ Nicolás Cabrera 1, Campus Cantoblanco-UAM, 28049 Madrid, Spain: samara.martin@cbm.csic.es

Details of affiliation

1Centro de Biología Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Científicas & Universidad Autónoma de
Madrid), c/ Nicolás Cabrera 1, Campus de Cantoblanco-UAM, Madrid, Spain
2Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain

Funding

This work was supported by a predoctoral fellowship of the Spanish Ministry of Science and Innovation (BES-2017-079836), and grants
PID2019-104176RB-I00/AEI/10.13039/501100011033 to L.M.-A. and SAF2016-75277-R to M.A.M.

Competing Interests:

These authors declare no competing financial interest.

Keywords: strand displacement; HIV; reverse transcriptase (RT); thymidine analogue resistance-associated mutations (TAMs)

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00054

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Martín-Alonso, et al. HIV reverse transcriptases defective in strand displacement activity. IBJ Plus 2021 (S4)e0054:10.24217/2531-
0151.21v1s4.00054.

Edited: Madrid, España.

Abstract

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immunodeficiency syndrome (AIDS). HIV
reverse transcriptase (RT) converts the single-stranded viral genomic RNA into double-stranded DNA that integrates
in the host cell genome. HIV RT is a multifunctional enzyme with DNA-polymerase (RNA- and DNA-dependent) and
ribonuclease H (RNase H) activities. HIV-1 and HIV-2 RTs are asymmetric heterodimers. The large subunits (p66 and
p68 in HIV-1 and HIV-2, respectively) possess DNA-polymerase and RNase H domains. During DNA polymerization, RTs
displace non-templated RNA or DNA strands to generate a proviral DNA. This property, known as strand displacement,
is essential for completing the reverse transcription process.
We measured strand displacement activities of HIV-1 and HIV-2 RTs by using 54-nucleotide DNA or RNA templates (M54),
annealed to 5´-32P-labeled oligonucleotides of 17-22 nucleotides (3TRP-17, 3TRP-20, 3TRP-21 or 3TRP) that act as DNA primers. In addition, strand displacement reactions include a DNA oligonucleotide (4TRP) that needs to be displaced while the DNA strand is being synthesized. After screening a panel of >30 different purified HIV RTs, we identified a quadruple mutant HIV-2ROD RT with a pronounced defect in strand displacement activity when using either RNA or DNA templates. This HIV-2ROD RT contained four major thymidine analogue resistance-associated mutations (TAMs): M41L, D67N, K70R and S215Y, located at and around the fingers subdomain of the RT’s DNA-polymerase domain. In agreement with these findings, we found that recombinant HIV-2 containing the M41L/D67N/K70R/S215Y mutant RT showed reduced replication capacity in comparison with the wild-type virus, as determined in phenotypic assays using MT-4 cells.

Furthermore, we have identified HIV-1BH10 RTs containing RNase H-inactivating mutations that showed largely reduced
strand displacement activity when using RNA templates. Interestingly, experiments carried out with the strand displacement
complex M54rna/3TRP-17/4TRP revealed that the inactivation of the RNase H produced different primer extension
patterns, depending on whether the loss of activity was due to an inactivating mutation (e.g. E478Q) or an RNase H inhibitor
(thujaplicinol). The strand displacement defect was more pronounced in the presence of the inhibitor.
We conclude that both the HIV RT fingers subdomain and the RNase H domain play important roles in controlling
strand displacement activity during reverse transcription. These findings could be helpful for the design of novel defective
strand-displacement RTs which could be potentially useful in biotechnological applications, including the preparation
of RNA libraries for transcriptomics.

ABSTRACTS: PhD Programme in Neuroscience

e00055
The primate striatum: morphological and stereological
study of neurons and interneurons in the MPTP non-human
primate model.

Natalia López-González del Rey1,2*, Javier Blesa1,2, Carmen Cavada3, José A. Obeso1,2.. 

*Corresponding author:Natalia López-González del Rey, HM-CINAC, Móstoles, Spain. E-mail: natalia_lgr91@hotmail.com

Details of affiliation

1HM-CINAC, HM Puerta del Sur University Hospital, Móstoles, Madrid, Spain.
2CIBERNED (Center for Networked Biomedical Research on Neurodegenerative Diseases), Instituto Carlos III, Madrid, Spain.
3Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Spain.

Funding

Pre-doctoral student funded by grant S2017/BMD-3700 (NEUROMETAB-CM) from Comunidad de Madrid co-financed with the Structural
Funds of the European Union.

Competing Interests:

No competing interest.

Keywords: DARPP-32, Cholinergic, Nitrergic, Parvalbumin, Calretinin, Tyrosine Hydroxylase.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00055

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Natalia López-González del Rey, Javier Blesa, Carmen Cavada, José A. Obeso, et al. The primate striatum: morphological and stereological
study of neurons and interneurons in the MPTP non-human primate model. IBJ Plus 2021 (S4)e0055:10.24217/2531-0151.21v1s4.00055

Edited: Madrid, España.

Abstract

The striatum is the largest nucleus of the basal ganglia and is mainly composed of projection neurons, also called
medium spiny neurons (MSNs, DARPP-32+), and a small population of interneurons which modulate and control
the striatal circuitry. These interneurons are mainly characterized as GABAergic and are classified into several
subtypes based on their immunostaining for different markers such as parvalbumin (PV+), calretinin (CR+),
neuropeptide Y/somatostatin/nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH+), tyrosine
hydroxylase (TH+), and a single population of non-GABAergic cholinergic interneurons, which express the enzyme
choline acetyltransferase (ChAT+). There are highly selective and specific interactions between MSNs and interneuron
subtypes and among interneurons themselves that result in the formation of important functional striatal
networks. Indeed, striatal interneurons are crucial for the processing of different behaviors that are affected in
states of altered dopamine (DA) transmission, such as Parkinson’s disease (PD), Huntington’s disease (HD) or drug
addiction. Thus, targeting the different interneuron population with different methods could provide therapeutic
approaches for these diseases. Accurate stereological data on the absolute number of all neuronal subtypes
would help to determine if there is a species difference, or not, for the percentage of interneurons in the monkey
striatum versus human or rodents. In this work, we provide a morphological description and the anteroposterior
striatal staining pattern for each striatal population. We also have used unbiased stereological methods on
consecutive sections of the same animals to estimate the density of all these neurons to obtain an unbiased
general landscape of the proportion and distribution of interneurons in the control non-human primate striatum.
Furthermore, we also analyzed their density in MPTP-treated monkeys with different degrees of DA loss to assess
how DA depletion affects these striatal populations. We report a gradient of striatal neuronal subtypes, being the
most abundant the DARPP-32+ neurons, followed by CR+, PV+, NADPH+ and ChAT+ interneurons and, finally, the
least abundant are the TH+ interneurons. Although few in number, these TH+ interneurons are the only neuronal
subtype that increase in the MPTP monkey model, even in the pre-symptomatic group. The presented data is
important for our understanding of striatal circuits and how they adapt in DA deficient models and for determining
the validity of this model of human pathology for translational studies involving targeting specific striatal
neuronal populations

e00056
Distribution of thyroid hormone transporters MCT8 and
OATP1C1 in the human and monkey cerebral cortex.

Wang Yu1,2, Wang Ting1,2,3, Prensa Lucía1, Guadaño-Ferraz Ana2, Rausell Estrella1. 

*Corresponding author: Wang Yu, Department of Anatomy, Histology and Neuroscience. School of Medicine. Universidad Autónoma de Madrid. Spain.
E-mail: wangyu_493889962@yahoo.com

Details of affiliation

1Department of Anatomy, Histology and Neuroscience. School of Medicine. Universidad Autónoma de Madrid. Spain.
2Instituto de Investigaciones Biomédicas Alberto Sols”. CSIC-UAM. Madrid, Spain.
3Xi’an Lintong Shiyoucheng General Clinic, Xi’an, China.

Funding

funded by SAF2017-86342-R; Yu Wang has a predoctoral fellowship from CSC (China Scholarship Council).

Competing Interests:

No competing interest.

Keywords: MCT8, OATP1C1, cerebral cortex, monkey, human

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00056

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Wang Yu, Wang Ting , Prensa Lucía, Guadaño-Ferraz An2, Rausell Estrella, et al. Distribution of thyroid hormone transporters MCT8 and
OATP1C1 in the human and monkey cerebral cortex. IBJ Plus 2021 (S4)e0056:10.24217/2531-0151.21v1s4.00056.

Edited: Madrid, España.

Abstract

Monocarboxylate transporter 8 (MCT8) and organic anion-transporting polypeptide 1C1 (OATP1C1) are thyroid
hormone (TH) transmembrane transporters that play a crucial role in the availability of systemic THs for neural
cells allowing their appropriate development and function. The loss of the function mutations in human MCT8
lead to a dramatic syndrome with very important implications in the motor system. This study aims to analyze
the presence of these two proteins at the cellular level in the monkey and human brain.
The distribution of those two transporters in the cerebral cortex was analyzed by immunostaining in 30-50 μm
floating frozen sections taken from three cynomolgus monkeys and four adult humans, with either rabbit anti-
MCT8 (or rabbit anti- OATP1C1 antibodies. To analyze their expression within the cortical neuron population,
double labeling immunofluorescent studies were performed for both antibodies and RC3/Neurogranin or calbindin
as well as immunostaining for either of those transporters combined with NADPH-diaphorase histochemistry.
The distribution of the immunolabeling was scanned with light/fluorescence epi-illumination and plotted with
Neurolucida system (MBF Biosciences) into all-section maps.
OATP1C1 is expressed in the soma, membrane, basal and apical dendrites of large and medium-sized pyramidal
neurons in layers II, III, V, and VI in both the monkey and the human brain, as evidenced by the histological
analysis of single immunostaining, colocalization with RC3/Neurogranin, and non-colocalization with NADPH-diaphorase.
MCT8 distribution is similar in monkey and human brain, although the intensity of its signal is lower.
Interestingly we found small layer I neurons expressing OATP1C1, which polygonal shape, short and intricate
dendrites, and close location to the pial surface suggest that they are Cajal Retzius cells. OATP1C1 and MCT8
immunostained cells with very small soma and large processes compatible with astrocytes, were found in the
subcortical white matter in close relation to vessels. MCT8 is also expressed in the endothelial cells of the large,
medium, and small size vessels and capillaries throughout the monkey and human cortex, the pial surface, and
the subcortical white matter, while OATP1C1 is occasionally observed in the endothelium of large and medium-
sized vessels.
Our study provides the first evidence for the abundance of MCT8 and OATP1C1 TH transporters in the long and
short projection pyramidal cortical neurons and in the astrocyte-vessel complexes in adult human and non-human
primates, which suggests their important role in the efferent cortical motor system.

e00057
Distribution of thyroid hormone transporters MCT8 and
OATP1C1 in the human and monkey
the basal ganglia and thalamus..

Wang Ting1,2,3, Wang Yu1,2, Prensa Lucía1, Guadaño-Ferraz Ana2, Rausell Estrella1. 

*Corresponding author: Wang Yu, Department of Anatomy, Histology and Neuroscience, School of Medicine, Universidad Autónoma de Madrid, Madrid,
Spain.
E-mail: wangyu_493889962@yahoo.com

Details of affiliation

1Department of Anatomy, Histology and Neuroscience. School of Medicine. Universidad Autónoma de Madrid. Madrid, Spain.
2Instituto de Investigaciones Biomédicas Alberto Sols”. CSIC-UAM. Madrid, Spain.
3Xi’an Lintong Shiyoucheng General Clinic, Xi’an, China.

Funding

funded by SAF2017-86342-R; Yu wang has a predoctoral fellowship from CSC (China Scholarship Council).

Competing Interests:

No competing interest.

Keywords: MCT8, OATP1C1, the basal ganglia, human, monkey

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00057

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Wang Ting, Wang Y1,2, Prensa Lucía, Guadaño-Ferraz Ana, Rausell Estrella, et al. Distribution of thyroid hormone transporters MCT8 and
OATP1C1 in the human and monkey the basal ganglia and thalamus. IBJ Plus 2021 (S4)e0057:10.24217/2531-0151.21v1s4.00057.

Edited: Madrid, España.

Abstract

Thyroid hormone (TH) is essential for the proper brain development and function and requires TH transporters
across the plasma membrane to perform its biological effect, mainly by modulating gene expression. The purpose
of the present study was to investigate the presence of two highly specific TH transporters, monocarboxylate
transporter 8 (MCT8) and organic anion transporting polypeptide 1C1 (OATP1C1), at the cellular level in the adult
monkey and human basal ganglia and motor related structures.
In order to identify the distribution of the expression of both transporters within the local neuron population we
used specific antibodies against either MCT8 or OATP1C1 to perform immunohistochemistry (IHC), IHC combined
with NADPH-diaphorase histochemistry or double immunofluorescence (IF) for calbindin in 30-50 μm floating
frozen sections of the basal ganglia and thalamus obtained from three cynomolgus monkeys and four adult humans.
The immunolabeling was scanned with light/fluorescence epi-illumination and plotted with a Neurolucida
system (MicroBrightField Biosciences) into all-section maps.
MCT8 and OATP1C1 are expressed in multiple neurons with different morphologies in the caudate and the putamen
nuclei both in human and monkey; in cells with large soma, multiple dendrites, compatible with large aspiny
cholinergic neurons, as well as in small and medium multipolar neurons which are compatible with medium-sized
spiny neurons. OATP1C1 and MCT8 distribution is similar in the human and monkey basal ganglia although MCT8
signal is less abundant. In the globus pallidus, OATP1C1 and MCT8 are expressed in medium-large spindle-shaped
neuron that also showed colocalization with calbindin, suggesting they are GABAergic neurons. In the motor thalamus,
MCT8 is expressed in medium-sized spherical cells in human and monkey, while OATP1C1 is more widely
expressed in medium to large size irregular neurons. In addition, we have noticed that both transporters are also
strongly expressed in substantia nigra in the monkey and in nucleus basalis of Meynert in human and monkey.
MCT8 is expressed extensively in the endothelial cells of the large, medium, and small size vessels and capillaries
in the basal ganglia and thalamus, while OATP1C1 is occasionally observed.
Our study provides the first evidence for the abundance of MCT8 and OATP1C1 TH transporters in the basal
ganglia and thalamus neurons in the adult human and non-human primates, which suggests their important role
in the coordination motor system.

e00058
Anatomical study of the striatal and cortical projections
arising from the posterior intralaminar thalamic nucleus
neurons in the mouse.

Enrique Gonzalo Martín*, Lucía Prensa Sepúlveda, Francisco Clascá Cabré. 

*Corresponding author:Enrique Gonzalo Martín. E-mail: kikegomar@gmail.com

Details of affiliation

Universidad Autónoma de Madrid. School of Medicine. Anatomy, Histology and Neuroscience department.
Calle Arzobispo Morcillo, 4. 28019 Madrid (Spain).

Funding

Supported by: Human Brain Project (HBP)-SGA3, Grant No 945539, WP2; European Union’s Horizon 2020 (Grant Agreement No. 785907
HBP SGA2) and Ministerio de Economía y Competitividad / Fondo Europeo para el Desarrollo Regional (MINECO/FEDER) grant BFU2017-88549.

Competing Interests:

The authors declare no competing interests

Keywords: parafascicular, thalamus, striatum, cerebral cortex

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00058

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Gonzalo-Martín et al. Anatomical study of the striatal and cortical projections arising from the posterior intralaminar thalamic nucleus
neurons in the mouse. IBJ Plus 2021 (S4)e0058:10.24217/2531-0151.21v1s4.00058.

Edited: Madrid, España.

Abstract

Introduction. The basal ganglia are several deep cerebral nuclei involved in the regulation of movements, behaviors
and habits. The neural information enters the basal ganglia mostly via the striatum, a nucleus which can be
subdivided functionally into three domains: a) sensorimotor (SM), receiving information of sensory perception
and about body posture and movement; b) associative, related to more integrated cognitive information; and c)
limbic, related to visceral and emotional information. It is also histologically divided in the matrix and the striosomes,
two biochemically different compartments. Two main projections activate striatal neurons: the corticostriatal
pathway (CS), coming from the cerebral cortex, and the thalamostriatal pathway (TS), which arises from
the thalamus. In rodents, most TS axons arise from the intralaminar thalamic nuclei (IL), divided into two groups:
anterior (aIL) and posterior (pIL), and which also innervate the cerebral cortex. There appears to be a functional
relation between the TS axons and the thalamocortical (TC) axons arising from IL, but it is unclear to what point.
Our aim is to investigate this relation for the pIL, which in rodents is the parafascicular nucleus (Pf).

Materials & Methods. To achieve our goal, we are labelling, in adult C57BL/6 male mice, both small groups of
neurons (micropopulations) and single neurons within the Pf. Micropopulations are labelled by microiontophoretic
injection of the anterograde tracer BDA. Then, their overall patterns of striatal and cortical projection are
analyzed using light microscopy. Single neurons are labelled by in vivo transfection with pseudoviral GFP-encoding
vectors (Sindbis), and then their axons are 3-D reconstructed entirely with Neurolucida® software in order
to obtain quantitative data of its features. We use different (immuno)histochemical approaches to delineate
cerebral structures. Striatal striosomes are made evident by immunolabelling of mu-opioid receptor (MOR).

Results. Our results show that Pf axons reach both SM and non-SM striatal territories and somatosensory, motor,
frontal association, orbital and insular areas within the cerebral cortex, mainly. The TS projection is much denser
than the TC projection, and most TS axons reach the matrix. The TS and TC projections vary depending on the
position of the labelled neurons within the Pf, reaching cortical and striatal territories which seem to be functionally
related, at least in part.

Conclusion. Our findings suggest that TS and TC projections arising from pIL neurons are functionally related. The
cortex and the striatum seem to receive similar information, possibly to allow a more optimal elaboration of the
response to each behavioral context.

e00059
Diversity and organization of the thalamocortical projections
from the ventroposterior complex of the thalamus.

Mario Rubio-Teves1, Pablo J. Martín-Correa1, César Porrero1, Francisco Clascá1. 

Details of affiliation

1 Cellular Connectomics Laboratory, School of Medicine, Universidad Autónoma de Madrid

Funding

Competing Interests:

Keywords: VPM, thalamus, somatosensory system.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00059

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mario Rubio-Teves, Pablo J. Martín-Correa, César Porrero, Francisco Clascá., et al. Diversity and organization of the thalamocortical
projections from the ventroposterior complex of the thalamus. IBJ Plus 2021 (S4)e0059:10.24217/2531-0151.21v1s4.00059.

Edited: Madrid, España.

Abstract

Introduction: The somatic sensory system is the component of the nervous system that detects, encodes and allows
the perception of a wide array of physical stimuli such as touch, pain, temperature or the position and movement
of the body. To do so, information detected by sensory receptors in the skin is transmitted over a series of neurons
connecting the body surface (where the stimulus is applied) with the neocortex (where perception is finally produced).
In this pathway, the last station before reaching the cortex is the thalamus, a region of the forebrain where clusters of
cells with specific functions and connections are spatially grouped together to form nuclei. In the thalamus, the nuclei
that act as the main relays for somatosensory information are the ventral posteromedial (VPM) and the ventral posterolateral
(VPL) nuclei, which receive inputs coming from the head and from the rest of the body, respectively.
In rodents, the somatosensory system is especially important because the whiskers (vibrissae) in the animal’s snout are
actively used to explore the environment. The connections in the pathway linking the whiskers and the cortex are so
specific that a point-to-point correspondence between the vibrissae and individual clusters of cortical cells (‘barrels’)
can be stablished. Because of this, the thalamocortical pathway between VPM and the barrel cortex of the primary
somatosensory area (S1) has been considered a central model for the study of thalamic sensory processing and integration
for the last 50 years. However, fragmentary evidence from population-level anatomical studies suggests that
axonal branching and arborization patterns of cells in the VP complex may be substantially more diverse.

Material & Methods: We systematically performed injections of an anterograde tracer, biotinylated dextran amine
(BDA), to study the axonal projections of small populations of cells in VPM. We also injected the Sindbis pal-eGFP viral
vector to randomly label single neurons within the nucleus, which were manually reconstructed using Neurolucida.
Both micro-populations and single neuron axonal projections were mapped onto a flat map of the somatosensory
areas of the mouse neocortex, and their location analyzed and correlated with the position of the injection.

Results: A detailed somatotopic map of thalamocortical connections linking VPM with the neocortex was drawn.
Whereas anterior VPM targets S1, posterior VPM targets S2 in a manner that is very similar to its anterior counterpart.
However, multi-branched architectures were also present, and we saw that both VPM and VPL can target S1 and S2
with both direct (a neuron targets S1 or S2) and branched projections (a neuron targets both S1 and S2).

Conclusions: a map of the thalamocortical connections from VPM targeting the cortex was drawn, clearly stablishing
a topographic correspondence between the two, based on the regions of the body they represent, and proving that
much of VPM targets not S1, but S2. Axonal projections aimed at S2 could be both specific (barrel-like) or divergent
(reaching S1 and S2 at the same time). This contradicts the stereotypical point-to-point view assumed for all projections
emerging from this nucleus, and also questions how much of a secondary, higher-order area S2 is.

e00060
Distribution of the noradrenaline innervation and Alpha
adrenoceptors in human higher-order thalamic nuclei.

Isabel Pérez-Santos1, Nicola Palomero-Gallagher234, Karl Zilles†235, Carmen Cavada1*. 

Carmen Cavada, Facultad de Medicina, Universidad Autónoma de Madrid. Calle Arzobispo Morcillo 4, Madrid 28029, Spain.
Email: carmen.cavada@uam.es.

Details of affiliation

1Departamento de Anatomía, Histología y Neurociencia; Facultad de Medicina, Universidad Autónoma de Madrid (UAM), 28029
Madrid, Spain.
2Institute of Neuroscience and Medicine (INM-1), Research Centre Jülich, 52425 Jülich, Germany.
3C. & O. Vogt Institute for Brain Research, Heinrich-Heine-University, 40225 Düsseldorf, Germany.
4Department of Psychiatry, Psychotherapy and Psychosomatics, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany.
5JARA-BRAIN, Jülich-Aachen Research Alliance, 52425 Jülich, Germany.

Funding

Chair in Neuroscience UAM-Fundación Tatiana Pérez de Guzmán el Bueno (C.C and I.P.-S.); the Federal Ministry of Education and Research
(01GQ1902 to N.P.-G.); the European Union’s Horizon 2020 Research and Innovation Programme (785907 (Human Bain Project SGA2) and 945539
(Human Brain Project SGA3) to N.P.- G. and K.Z.; Boehringer Ingelheim Fonds (I.P.-S.); Universidad Autónoma de Madrid (I.P.-S.).

Competing Interests:

No competing interest.

Keywords: Noradrenaline, Thalamus, Alpha adrenergic receptors, Primate, Human

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00060

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Isabel Pérez-Santos, Nicola Palomero-Gallagher, Karl Zilles, Carmen Cavada. et al. Distribution of the noradrenaline innervation and Alpha
adrenoceptors in human higher-order thalamic nuclei. IBJ Plus 2021 (S4)e0060:10.24217/2531-0151.21v1s4.000060.

Edited: Madrid, España.

Abstract

Noradrenaline (NA) in the thalamus has relevant neuromodulatory roles. It is also important in pathological conditions
and pharmacological intervention. In fact, thalamic NA has been demonstrated to modulate sensorimotor
gating and prepulse inhibition. Also, brain NA alterations have been shown in neurological and psychiatric disorders.
However, a precise map of the NA innervation in the human higher-order thalamic nuclei is not available yet.
We have used immunohistochemistry against the noradrenaline transporter, a specific marker of the NA phenotype,
to label and describe the distribution of NA in the human mediodorsal (MD) and pulvinar nuclei. To reveal
the Alpha-1 and Alpha-2 adrenergic receptors, we performed autoradiography using the ligands [3H]-Prazosin
(Alpha-1 receptors), [3H]-RX-821002 (whole Alpha-2 adrenoceptor population), and [3H]-UK-14,304 (high-affinity
state Alpha-2 adrenoceptor).
Our results show specific NA innervation in MD and the pulvinar complex. MD NA innervation was moderate,
with the highest densities in the medial and ventral regions of the nucleus. Within the pulvinar complex, the
nucleus with the highest NA innervation was the oral pulvinar (Pul O); the medial pulvinar (Pul M) displayed moderate
densities of NA axons, and the lateral and inferior pulvinar (Pul L, Pul I) presented low NA axon densities.
Receptor distributions were also specific. Alpha-1 receptor concentrations were slightly higher than Alpha-2
concentrations, except for Pul I and Pul L. The highest densities of Alpha-1 receptors were present in the dorsal
and medial regions of MD, and in the medial regions of Pul M. The highest Alpha-2 receptor concentrations were
present in Pul I. Pul O showed different Alpha-2 receptor concentrations depending on whether they were revealed
by [3H]-RX-821002 (high receptor concentrations) or by [3H]-UK-14,304 (rather low receptor concentrations)
pointing to a lower proportion of high-affinity Aplha-2 receptors relative to the total Alpha-2 receptor population
in this nucleus.
The distributions of NA axons and Alpha adrenoceptors in the human MD and pulvinar nuclei suggest critical and
specific roles for NA in modulating limbic, multimodal and sensory association thalamo-cortical circuits.

ABSTRACTS : Other PhD Programmes

e00061
Effect of a workplace program to promote physical activity
on metabolic syndrome risk factors during
the COVID-19 pandemic.

Romero-Caballero, Alejandro1; Crespo-Ruiz, Beatriz2 & Veiga, Oscar L.1. 

*Corresponding author:
Romero Caballero, Alejandro. Department of Physical Education, Sport and Human Movement.
Universidad Autónoma de Madrid (Spain).
E-mail: alexmisterpf@gmail.com

Details of affiliation

1Department of Physical Education, Sport and Human Movement. Universidad Autónoma de Madrid (Spain).
2Universidad de Castilla La Mancha (Spain).

Funding

No external funding sources.

Competing Interests:

Authors declare no conflict of interest.

Keywords: Exercise; Workplace; COVID-19; Metabolic Syndrome.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00061

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Romero-Caballero, Alejandro; Crespo-Ruiz, Beatriz & Veiga, Oscar L, et al. Effect of a workplace program to promote physical activity on
metabolic syndrome risk factors during the COVID-19 pandemic. IBJ Plus 2021 (S4)e0061:10.24217/2531-0151.21v1s4.00061.

Edited: Madrid, España.

Abstract

Introduction: The metabolic syndrome is clinically diagnosed when the accumulation of three or more of the
following risk factors occurs: abdominal obesity, hypertension, dyslipidemia and hyperglycemia. Various epidemiological
studies indicate that these factors increase the predisposition to suffer from different cardiovascular
diseases, type II diabetes, kidney disease, and are related to an increased risk of mortality (1). Physical activity
is considered one of the most promising strategies for both prevention and treatment of metabolic syndrome.
One of the physiological mechanisms that explain it is the release of myokines by skeletal muscle during muscle
contraction, such as BDNF or IL-6, which increase the signaling of the AMPK enzyme complex, thus optimizing the
oxidation of free fatty acids (2). The main studies indicate that during the health crisis caused by COVID-19, especially
during the months in which home confinement was extended, the level of physical activity was reduced,
sedentary time increased and there was a weight increase in the population (3). These changes can negatively
affect the main risk factors for metabolic syndrome. Therefore, the main objective of this study was to analyze
the impact of a workplace program to promote physical activity during the COVID-19 pandemic on metabolic
syndrome risk factors.
Material and methods: 54 office workers (17 women; 47 ± 9.1 years; 26.14 ± 3.95 kg/m2 BMI) participated in an
18-week theory-informed -Behaviour Change Wheel (4)- online program based on education, counseling and individualized
prescription of physical exercise. Both before and after the intervention, blood samples were taken,
waist circumference was evaluated, and blood pressure was measured. Paired samples T-test, Chi Square test and
Cohen’s d were used to evaluate the effect of the intervention program.

Results: After the intervention, significant improvements were observed for waist circumference (p>.001,
d=0.24) and blood pressure (p=.007, d=0.31). Likewise, there was also a statistically significant decrease in the
continuous metabolic risk indicator -MetScore- (p=.021, d=0.16). Finally, the proportion of participants with metabolic
syndrome was reduced (from 7.4% to 1.9%).
Conclusions: This theory informed 18-week online workplace program was effective to improve two of the main
risk factors for metabolic syndrome, such as waist circumference and blood pressure, in addition to reducing total
metabolic risk and the number of subjects with diagnosis of metabolic syndrome during the COVI-19 pandemic.
REFERENCES: (1) Zhang et al., 2017; (2) Kränkel et al., 2019; (3) Deschasaux-Tanguy et al., 2020; (4) Michie et al., 2014.

ABSTRACTS  CIVIS Alliance Universities

e00062
On the improvement of aortic anastomosis.

Danae Manolesou1, Georgia Korompili2, Andreas Lazaris3, Dimitrios Schizas4, Theodore Liakakos5, Τheodore G.
Papaioannou1. 

*Corresponding author:
Danae Manolesou, Biomedical Engineering Unit, First Department of Cardiology, Hippokration Hospital, Medical School, National
and Kapodistrian University of Athens, Athens, Greece. E-mail: d.manolesou@gmail.com

Details of affiliation

1 Biomedical Engineering Unit, First Department of Cardiology, Hippokration Hospital, Medical School, National and Kapodistrian
University of Athens, Athens, Greece.
2 Department of Electrical and Electronic Engineering, University of West Attica, Athens, Greece.
3 Department of Vascular Surgery, Attikon Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.
4 First Department of Surgery, Laiko Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece.

Funding

The research work was supported by the Hellenic Foundation for Research and Innovation (HFRI) under the HFRI PhD Fellowship grant for
DM (Fellowship Number: 101849/2019)..

Competing Interests:

No competing interests.

Keywords: Aorta, Anastomosis, Devices.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00062

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Danae Manolesou, Georgia Korompili, Andreas Lazaris, Dimitrios Schizas, Theodore Liakakos, Τheodore G. Papaioannou, et al. On the
improvement of aortic anastomosis. . IBJ Plus 2021 (S4)e0062:10.24217/2531-0151.21v1s4.00062.

Edited: Madrid, España.

Abstract

Introduction: Despite widespread use of the endovascular repair, open reconstruction of the aorta is still chosen
for a significant number of patients. Hand-sewn anastomosis of the reconstructed aortic segment is technically
demanding and can be time-consuming even for an experienced surgeon. Several alternative aortic anastomotic
devices have been suggested, however, none of them managed to replace the hand-sewn technique in everyday
clinical practice. This study presents a novel surgical needle used to simplify the anastomotic technique for open
aortic reconstruction. The proposed needle has two tips that can be both used for penetration to the tissue. In
that way, the new design aims to eliminate complex maneuvers related to rotation of the tip of the needle using
the needle-holder and the forceps alternately.

Materials and Methods: The needle’s digital 3D model was based on the standard ½ curved, taper point needle.
The geometry of the needle was precisely defined using a standard optical microscope to accurately measure the
needle thickness and a Scanning Electron Microscope (SEM) to investigate the minimum surface of the tip. The
model was designed in SolidWorks with 800 μm maximum body diameter, 12 μm diameter at each one of the
two tips and a 200 μm hole diameter in the middle of the needle body to host the suture. We conducted simulations
of the model in comparison with the conventional needle, in COMSOL environment, to investigate the
effect of the novel design on the needle durability and determine the optimum position, size and orientation of
the hole.

Results: The fatigue factor averaged over the entire needle surface is estimated to be 26% higher for the case of
the horizontally formed hole and 30% higher for the case of the perpendicular hole, compared to the conventional
design, which does not have a hole. In the horizontal orientation, an increase in the hole size from 200 μm to
350 μm increases the fatigue factor less than 3%.

Conclusions: It is expected that the novel design exhibits slightly decreased durability compared to the conventional
design. However, the insertion of the needle by both sides into the tissue will reduce by half the strain
applied on the needle and it will compensate the increased fatigue factor. While manufacturing, characterization
and testing of a working prototype in graft-to-graft anastomosis should be undertaken as a next step to assess
the ergonomics and time requirements of the novel anastomotic technique, the proposed design seems promising
for wide and efficient use in aorta anastomosis.

e00063
Methylglyoxal stress induces a major epigenetic deregulation
leading to a pro-migratory phenotype in breast cancer and
significant clinical relevance.

Gaurav Dube1,#, Assia Tiamiou2,#, Martin Bizet1, Justine Bellier2, Marie-Julie Nokin2, Yasmine Boumahd2, Rachel
Deplus1, Emilie Calonne1, Olivier Peulen2, Vincent Castronovo2, François Fuks1,*, Akeila Bellahcène2,*.

*Corresponding author:
Akeila Bellahcène, Metastasis Research Laboratory, GIGA-Cancer, University of Liège, Liège, Belgium. E-mail: a.bellahcene@ulg.ac.be

Details of affiliation

1Laboratory of Cancer Epigenetics, U-CRC, Université Libre de Bruxelles, Brussels, Belgium.
2Metastasis Research Laboratory, GIGA-Cancer, Université de Liège, Liège, Belgium.
#Equal Contribution

Funding

Fonds de la Recherche Scientifique (FNRS)- GD, AT; Télévie- GD

Competing Interests:

Authors declare no competing interests

Keywords: Methylglyoxal, Breast Cancer, DNA Epigenetics, Methylyglyoxal Signature, Metastasis, Clinical Association

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00063

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Gaurav Dube, Assia Tiamiou, Martin Bizet, Justine Bellier, Marie-Julie Nokin, Yasmine Boumahd, Rachel Deplus1, Emilie Calonne, Olivier
Peulen, Vincent Castronovo, François Fuks, Akeila Bellahcène, et al. Methylglyoxal stress induces a major epigenetic deregulation leading to a promigratory
phenotype in breast cancer and significant clinical relevance. IBJ Plus 2021 (S4)e0063:10.24217/2531-0151.21v1s4.00063

Edited: Madrid, España.

Abstract

Introduction: Cancer cells are largely dependent on glycolysis for their energy supply. One underestimated consequence
of this glycolytic switch is the production of methylglyoxal (MG). MG is a highly reactive dicarbonyl metabolite
that reacts with lipids, nucleic acids and proteins and creates a MG stress. Glyoxalase I enzyme (GLO1) detoxifies MG
and reduces the MG stress. On the other hand, DNA methylation is one of the most studied and important epigenetic
modification in relation with cancer. In particular, the silencing of tumor suppressor genes through DNA methylation is
a well-known pro-oncogenic process. Our work is aimed at understanding the role of MG stress in breast cancer progression
with a specific focus on epigenetic regulation and the characterization of a gene signature of MG stress with
clinical usefulness.

Materials & Methods: We performed transcriptomic (RNAseq) and DNA methylation (Infinium 850K) experiments
using GLO1-depleted breast cancer cells and mouse xenografts samples. Differential expression analysis was performed
using Kallisto-Sleuth pipeline and differential methylation analysis was carried out using in-house pipeline.
Pathway analysis was performed using GSEA tool. Integration of expression, methylation and pathway results was
performed using R scripting.

Results & Conclusion: Analysis of transcriptomic data revealed 1070 differentially expressed genes including key epigenetic
regulators – DNMT3A and DNMT3B. Differential methylation analysis of 1749 CpGs showed a striking DNA hypermethylation
pattern in cells as well as xenografts, indicating a potential link between MG stress and DNA methylation.
DNMT3A and DNMT3B protein levels were increased upon exogenous MG treatment and decreased in presence of MG
potent scavengers such as carnosine. The integration of expression and methylation data resulted in 60 genes composing
MG stress signature. Further refinement of this latter led to a 25-gene-signature that was found to be clinically
relevant in term of predicting drug response and poor survival using publicly available breast cancer patient data.

Conclusion: This study demonstrates a novel link between MG stress and DNA methylation that could be involved in
tumor progression. Our work points towards MG scavengers as promising epigenetic regulatory drugs. Finally, our
results lead to a 25-gene based MG signature with significant clinical relevance.

e00064
Combinations of BH3 mimetics with the TKI nilotinib
synergistically induce apoptosis in blast phase chronic
myeloid leukaemia cells.

Narissa Parry1, Helen Wheadon1, Mhairi Copland1
1.

*Corresponding author:
Narissa Parry, University of Glasgow, Glasgow, United Kingdom. E-mail: n.parry.1@research.gla.ac.uk

Details of affiliation

1 Paul O’Gorman Leukaemia Research
Centre, University of Glasgow, 21 Shelley
Road, Glasgow, United Kingdom, G12 0ZD

Funding

No funding to declare.

Competing Interests:

Mhairi Copland: research funding (Cyclacel Ltd, Novartis, BMS, Incyte), advisory board member (BMS, Novartis, Incyte, Pfizer,
Daiichi Sankyo), and honoraria (Astellas, BMS, Novartis, Incyte, Pfizer, Takeda, Celgene).

Keywords: CML, BH3 mimetics, apoptosis

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00064

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Narissa Parry, et al. Combinations of BH3 mimetics with the TKI nilotinib synergistically induce apoptosis in blast phase chronic myeloid
leukaemia cells. IBJ Plus 2021 (S4)e0064:10.24217/2531-0151.21v1s4.00064.

Edited: Madrid, España.

Abstract

Introduction: Dysregulation of the BCL-2 family is highly implicated in protecting chronic myeloid leukaemia
(CML) cells from intracellular damage and BCR-ABL1-inhibition with tyrosine kinase inhibitors (TKI). There is
growing evidence that this family is a viable therapeutic target in blast phase (BP) CML, for which there are
limited treatment options. BH3 mimetics, a class of small molecule inhibitors with high-specificity against the
prosurvival members of the BCL-2 family, have displayed clinical promise in the treatment of chronic lymphocytic
and acute myeloid leukaemia as single agents and in combination with standard-of-care therapies. Here we categorise
the functional BCL-2 family dependence of BP-CML cell lines and primary patient samples and investigate
the treatment of CML cells with BH3 mimetics alone and in combination with TKIs.

Material and Methods: The effect of BH3 mimetics in four BP-CML cell lines and six BP-CML patient samples
was evaluated by phosphatidylserine (PS) presentation, caspase-3 activation, and the presence of CD34 by flow
cytometry, gene expression analyses by qPCR, and colony-forming unit assays. Three healthy donor samples were
used as controls. Resazurin reduction assays were used to investigate synergy.

Results: Co-treatment of four BP-CML cell lines with the TKI nilotinib and inhibitors of BCL-2 (venetoclax), MCL-1
(S63845), or BCL-xL (A-1331852) resulted in a synergistic reduction in metabolic activity and cell count, and an
increase in PS presentation. The same combinations in six BP-CML patient samples triggered increased induction
of apoptosis over nilotinib alone, and a reduction in colony-forming capacity and primitive CD34+ cell population,
to a greater degree than in healthy samples.
While BH3 mimetics showed little efficacy as single agents, dual-inhibition of BCL-2 prosurvival proteins dramatically
induced apoptosis in all cell lines tested, including two TKI-resistant cell lines, and three patient samples.
For all TKI-sensitive cell lines, the most potent combination was BCL-xL/MCL-1, while TKI-resistant cells showed
greater sensitivity to dual-inhibition of either BCL-2/MCL-1 or BCL-2/BCL-xL.

Conclusions: BH3 mimetics show promise in sensitising cell lines and patient samples to apoptosis induced by TKI
treatment. Moreover, our results suggest that BH3 mimetics have potential to eliminate TKI-resistant cells when
used in combination. Against the backdrop of the impressive clinical success of venetoclax in recent years, these
results represent a promising avenue towards improved treatment options for patients with BP-CML.

e00065
Heterotypic cell-cell communication regulates
glandular stem cell multipotency.

Alessia Centonze1, #, Shuheng Lin1, #, Elisavet Tika1, Alejandro Sifrim2, 3, Marco Fioramonti1, Milan Malfait1, Yura
Song1, Anne Dannau1, Gaelle Bouvencourt1, Christine Dubois1, Nina Dedoncker2, Viviane de Maertelaer4, Alexandra
Van Keymeulen1, Thierry Voet2, 3, Cédric Blanpain1, 5, *.

# These authors contributed equally to this work.
*Corresponding author: Cedric Blanpain cedric.blanpain@ulb.ac.be

Details of affiliation

1Laboratory of Stem Cells and Cancer, Université Libre de Bruxelles (ULB), Brussels, Belgium.
2Department of Human Genetics, University of Leuven, KU Leuven, Leuven, Belgium.
3Sanger Institute-EBI Single-Cell Genomics Centre, Wellcome Trust Sanger Institute, Hinxton, UK.
4 IRIBHM, Université Libre de Bruxelles (ULB), Brussels, Belgium
5WELBIO, Université Libre de Bruxelles (ULB), Brussels B-1070, Belgium.

Funding

This work was supported by the ERC and the FNRS. A.C. is supported by the FNRS/FRIA. S.L. is a long term EMBO fellow. C.B is supported by
WELBIO, FNRS, TELEVIE, Fond Erasme, Fondation Contre le Cancer, ULB Foundation, European Research Council, the Foundation Baillet Latour.

Competing Interests:

The authors declare no competing interests

Keywords: stem cell, mammary gland, multipotency.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00065

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Centonze A., et al. Heterotypic cell-cell communication regulates glandular stem cell multipotency. IBJ Plus 2021 (S4)e0065:10.24217/2531-
0151.21v1s4.00065

Edited: Madrid, España.

Abstract

Glandular epithelia including the mammary gland (MG) and the prostate glands are composed of basal cells (BCs)
and luminal cells (LCs). Many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced
in adult life by distinct pools of unipotent stem cells. However, adult unipotent BSCs can reactivate multipotency in
regenerative conditions and upon oncogene expression. This suggests that an active mechanism restricts BSC multipotency
during physiological conditions, although the nature of this mechanism is unknown. Here we show that the
ablation of LCs reactivated the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids.
Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal
cell differentiation program before giving rise to LCs, reminiscent of the genetic program that regulates multipotency
during embryonic development. By predicting ligand-receptor pairs from single-cell data, we find that TNF, which is
secreted by LCs, restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and
EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating
TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication
between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.

e00066
High-Sensitivity Detection and Genotyping
of Yellow Fever Virus.

Mario Di Donato* 1, Riccardo De Santis 1, Giovanni Faggioni 1, Florigio Lista1.

*Corresponding author:
Army Medical Center, Scientific Department, Roma, Italy. E-mail: mario.didonato@uniroma1.it

Details of affiliation

1Army Medical Center, Scientific Department, Roma, Italy

Funding

The authors declare that they have no competing interests

Competing Interests:

This work was funded by Italian Ministry of Defence

Keywords: Flavivirus; YFV; hemorrhagic illness.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00066

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mario Di Donato, Riccardo De Santis, Giovanni Faggioni, Florigio Lista, et al. High-Sensitivity Detection and Genotyping of Yellow Fever Virus.
IBJ Plus 2021 (S4)e0066:10.24217/2531-0151.21v1s4.00066.

Edited: Madrid, España.

Abstract

Yellow fever is a severe hemorrhagic illness with a high mortality rate. The causative agent, a mosquito-borne
RNA virus, is endemic in tropical regions of Africa and South America. Despite the use of a safe and effective
vaccine for over a half century, an alarming resurgence of yellow fever outbreaks has been recorded in endemic
areas in the last decades. Active surveillance, immunization programs and early detection of yellow fever outbreaks
are essential tools to fight and minimize the spread of infections. The aim of this study was to develop a
high sensitivity specificity reverse-transcription PCR for the detection of all the yellow fever virus strains.
Several pairs of primer were designed and tested on a conserved region of yellow fever genome. A Real-Time
PCR has shown a very high sensitivity and specificity. The employed primers were also evaluated in a classical
endpoint PCR. The method did not show a significant loss of performance. Sequencing analysis of the resulting
amplicon allowed to identify the yellow fever virus genotype.
This novel methods might represent a further improvement in YFV molecular analysis. The opportunity to
choose between a Real-Time PCR or a classic endpoint PCR for the detection of yellow fever virus could better
meet the needs of laboratories. Moreover, sequencing analysis of the amplicon allows to identify the yellow
fever virus genotype.

e00067
Chemerin regulates normal angiogenesis and
hypoxia-driven neovascularization.

Cyrine Ben Dhaou1,2, Kamel Mandi1, Michaël Frye1, Angela Acheampong3, Ayoub Radi1, Benjamin De Becker3,
Mathieu Antoine1, Nicolas Baeyens4, Valérie Wittamer1, Marc Parmentier1.

Details of affiliation

1WELBIO and I.R.I.B.H.M., Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Brussels, Belgium.
2University of Tours, INRA Centre Val-de-Loire UMR-85, CNRS UMR-1247,
Physiologie de la Reproduction et Comportements, FRANCE.
3 Erasme Hospital, Université Libre de Bruxelles, Route de Lennik 808, 1070 Anderlecht
4 Laboratoire de Physiologie et Pharmacologie, Université Libre de Bruxelles,
Campus Erasme, 808 route de Lennik, B-1070 Brussels, Belgium

Funding

Competing Interests:

Keywords:

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00067

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Cyrine Ben Dhaou, Kamel Mandi, Michaël Frye, Angela Acheampong, Ayoub Radi, Benjamin De Becker, Mathieu Antoine, Nicolas Baeyens,
Valérie Wittamer, Marc Parmentier, et al. Chemerin regulates normal angiogenesis and hypoxia-driven neovascularization. I IBJ Plus 2021 (S4)
e0067:10.24217/2531-0151.21v1s4.00067.

Edited: Madrid, España.

Abstract

Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte
populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral
factor inhibiting the vascularization of tumors. We show here that overexpression of bioactive chemerin in mice results
in a reduction of the density of the retinal vascular network. Chemerin did not affect vascular sprouting during
the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This
phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating that the destabilizing role of chemerin
on the retinal network is mediated by this receptor. We also demonstrate that the neoangiogenesis process in
a model of pathological proliferative retinopathy, and in response to hind limb ischemia, is significantly reduced in
mice overexpressing chemerin. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the
density of the retinal vasculature. These results suggest that inhibition of the PI3-kinase/AKT pathway is involved in
the chemerin-induced vessel regression process.

e00068
Identification of an immune profile able to improve IMDC
stratification in mRCC patients.

Alessandra Di Filippo1, Ilaria Grazia Zizzari1, Marianna Nuti1.

*Corresponding author:
Alessandra Di Filippo. E-mail: alessandra.difilippo@uniroma1.it

Details of affiliation

1Laboratory of Tumor Immunology and Cell Therapy, Department of Experimental Medicine, Policlinico Umberto I, “Sapienza”
University of Rome, 00161 Rome, Italy.

Funding

Competing Interests:

Keywords:

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00068

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Alessandra Di Filippo, Ilaria Grazia Zizzari, Marianna Nuti, et al. Identification of an immune profile able to improve IMDC stratification in
mRCC patients. IBJ Plus 2021 (S4)e0068:10.24217/2531-0151.21v1s4.00068.

Edited: Madrid, España.

Abstract

Chemerin is a multifunctional protein initially characterized in our laboratory as a chemoattractant factor for leukocyte
populations. Its main functional receptor is CMKLR1. We identified previously chemerin as an anti-tumoral
factor inhibiting the vascularization of tumors. We show here that overexpression of bioactive chemerin in mice results
in a reduction of the density of the retinal vascular network. Chemerin did not affect vascular sprouting during
the post-natal development of the network, but rather promoted endothelial cell apoptosis and vessel pruning. This
phenotype was reversed to normal in CMKLR1-deficient mice, demonstrating that the destabilizing role of chemerin
on the retinal network is mediated by this receptor. We also demonstrate that the neoangiogenesis process in
a model of pathological proliferative retinopathy, and in response to hind limb ischemia, is significantly reduced in
mice overexpressing chemerin. Mechanistically, PTEN and FOXO1 antagonists could almost completely restore the
density of the retinal vasculature. These results suggest that inhibition of the PI3-kinase/AKT pathway is involved in
the chemerin-induced vessel regression process.

ABSTRACTS University of Malaya

e00069
Investigation of capillary leak syndrome induced by the
eastern (Daboia siamensis) and western (Daboia russelii)
Russell’s viper venoms.

Thava Malar Changra Lingam1, Kae Yi Tan1, Choo Hock Tan2*.

*Corresponding author:
Choo Hock Tan, Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
E-mail: tanch@um.edu.my

Details of affiliation

1Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.
2Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia.

Funding

BKS003-2020.

Competing Interests:

Keywords: Russell’s viper; Daboia siamensis; Daboia russelii; Capillary leak syndrome; Capillary permeability; Hematocrit.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00069

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Lingam,TMC., et al. (2021). Investigation of capillary leak syndrome induced by the eastern (Daboia siamensis) and western (Daboia russelii)
Russell’s viper venom. IBJ Plus 2021 (S4)e0069:10.24217/2531-0151.21v1s4.00069.

Edited: Madrid, España.

Abstract

Introduction: Russell’s viper consists of two allopatric species; i.e. Daboia siamensis and Daboia russelii. Both
species are venomous snakes of medical importance in Asia as categorized by the WHO. Envenomation by Russell’s
viper commonly causes hemotoxicity and, in some cases, capillary leak syndrome (CLS) which has not been
well characterized. This study aims to investigate the CLS activity of D. siamensis and D. russelii venoms from
different geographical locales and its neutralization by regional antivenoms.

Methods: Russell’s viper venoms were intradermally inoculated in ICR mice which had been pre-administered
with Evan’s blue solution. The diameter and intensity of Evan’s blue extravasation surrounding the venom-inoculation
site and the hematocrit level of each mouse were examined. In the neutralization study, various doses of
antivenom were used to neutralize the CLS effect applying a pre-incubation and a challenge-rescue approach.

Results: Both D. siamensis and D. russelii caused significant vascular permeability, reflected by extensive Evan’s
blue extravasation. The Evan’s blue extravasation caused by D. russelii was generally less variable (76,000-86,000
CLS unit) than caused by D. siamensis venom (33,000 – 88,000 CLS unit). The hematocrit levels were, however
higher in mice inoculated with D. siamensis venom (60-67%) compared with D. russelii venom (53-58%). In the
preincubation neutralization study, regional antivenoms (DsMAV-Thai, DsMAV-Taiwan and VPAV-India) were able
to reduce the CLS-inducing activity of Russell’s viper venoms by 2-14 folds. In the challenge-rescue study, antivenoms
administered intravenously post-envenomation were less effective in neutralizing the CLS-inducing activity
of the venoms, implying that CLS has a rapid onset that preceded the neutralizing activity of the antivenom, and/
or, the antivenom had a limited biodistribution to the venom inoculation site.

Conclusion: D. siamensis and D. russelii venoms induced potent CLS in the mouse model. Antivenoms generally
showed limited efficacy in neutralizing the CLS effect. Innovative treatment for CLS is needed

e00070
Gut microbiota dysbiosis in survivors of childhood acute
lymphoblastic leukemia and the association with immune
dysregulation and metabolic derangement.

Ling Ling Chua1, Reena Rajasuriar2, Yvonne Ai Lian Lim3, Yin Ling Woo4, P’ng Loke5,6, Hany Ariffin1,7*.

*Corresponding author:
Hany Ariffin, University of Malaya, Kuala Lumpur, Malaysia. E-mail: hany@ummc.edu.my

Details of affiliation

1 Department of Paediatrics, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
2 Department of Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
3 Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
4 Department of Obstetrics and Gynaecology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
5 Department of Microbiology, New York University School of Medicine, New York, New York, USA.
6 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
7 Department of Paediatrics, University of Malaya Medical Centre, Kuala Lumpur, Malaysia.

Funding

UMRG grant RP049A-17HTM and UM.C/HIR/MOHE/MED/12 (HA); UM.C/625/HIR/MOE/MED/23 (YLAL and PL); PPP grant PG346-2016A
(WYL and LLC); UMRG RP029-14HTM (RR and WYL) and Merdeka Award International Attachment grant (LLC).

Competing Interests:

The authors declare that they have no competing interests.

Keywords: Childhood acute lymphoblastic leukemia, childhood cancer survivors, microbiota dysbiosis, immune dysregulation, metabolic disorders,
Faecalibacterium.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00070

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Ling Ling Chua, Reena Rajasuriar, Yvonne Ai Lian Lim, Yin Ling Woo, P’ng Loke, Hany Ariffin, et al. Gut microbiota dysbiosis in survivors
of childhood acute lymphoblastic leukemia and the association with immune dysregulation and metabolic derangement. IBJ Plus 2021 (S4)
e0070:10.24217/2531-0151.21v1s4.00070.

Edited: Madrid, España.

Abstract

Introduction Survivors of childhood acute lymphoblastic leukaemia (ALL) are at risk of developing therapy-related
late effects including inflammation-related comorbidities (e.g. metabolic disorders). Gut microbiota plays important
roles in maintaining health including regulating immune responses and energy metabolism. Gut microbial community
can be perturbed by various external factors including chemotherapy and antibiotic, two key components in ALL
treatment. This study aims to investigate whether gut microbiota is restored or remains perturbed after the cessation
of cancer treatment and its implication on immune and metabolic derangements in long-term survivors of ALL.

Method We performed (1) a longitudinal study to examine changes in the gut microbiota of children with ALL
assessed through anal swabs whilst undergoing and after cessation of treatment and (2) a cross-sectional study to
examine microbiota differences between long-term survivors of childhood ALL (≥5 years post treatment) with adult
controls. Changes of microbiota profile during chemotherapy and its association with immune and metabolic derangement
among the survivors was investigated. Microbiota profiling was performed with 16s RNA gene sequencing.

Result In the longitudinal cohort (n=7, age 2-6 years), microbiota dysbiosis occurred even prior to start of chemotherapy,
characterized by enrichment of Bacteroidetes (p=0.001). After completion of chemotherapy (median 6
months), microbial composition remained distinct from that of healthy controls, suggesting incomplete microbiota
restoration. Microbiota dysbiosis was also observed among the long-term survivors (n=73, median age=26 years)
characterized by lower bacterial diversity (p<0.01) and alteration in bacterial composition with depletion of Faecalibacterium
and Ruminococcaceae, and enriched with Peptoniphilus, Finegoldia and Anaerococcus [all q<0.05] compared
to controls (n=61, median age=23 years). Among long-term survivors, Finegoldia and Anaerococcus were positively
correlated with T-cell activation (HLA-DR+ cells, p<0.05) while Faecalibacterium and Ruminococcaceae were
negatively correlated with systemic inflammation (IL-6, p<0.05; CRP, p<0.001). Notably, survivors with obesity had
lower abundance of Faecalibacterium (<0.1%) and higher plasma levels of IL6 and C-reactive protein (all p<0.05).

Conclusion This study is the first to provide a broad insight into the dynamics of gut microbiota in children undergoing
chemotherapy for ALL and the relationship between microbiota dysbiosis with immune and metabolic late
effects in long-term survivors. We speculate that interventions to restore the microbial community may reduce the
risk of late effects in survivors of childhood leukemia.

e00071
Real-time Impedance Monitoring of Biofilm Formation from
Dual Flagellar Systems in Aeromonas dhakensis.

Tien-Tien Vicky Lau1, Suat-Moi Puah1, Jin-Ai Mary Anne Tan2, Savithri Devi Ampalam Puthucheary3, Kek-Heng Chua1*

*Corresponding author:
Kek-Heng Chua, Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
E-mail: khchua@um.edu.my

Details of affiliation

1Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. 2Academy of
Sciences Malaysia, 50480 Kuala Lumpur, Malaysia.
3Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

Funding

This study was funded by the University of Malaya Research grant (UMRG RP039B-15HTM).

Competing Interests:

The authors declare that no competing interests exist.

Keywords: Aeromonas dhakensis, biofilm formation, cell index, polar and lateral flagella, real-time impedance

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00071

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Tien-Tien Vicky Lau, Suat-Moi Puah, Jin-Ai Mary Anne Tan, Savithri Devi Ampalam Puthucheary, Kek-Heng Chua et al. Real-time
Impedance Monitoring of Biofilm Formation from Dual Flagellar Systems in Aeromonas dhakensis. . IBJ Plus 2021 (S4)e0071:10.24217/2531-
0151.21v1s4.00071.

Edited: Madrid, España.

Abstract

Introduction: Aeromonas dhakensis is an emerging human pathogen that causes severe wound and skin infections,
and septicemia. It has dual flagellar systems for motility under different circumstances. Flagellum is one of the
virulence factors involved in biofilm formation of many pathogens. Biofilms are communities of microorganisms attached
to a surface encased in self-produced polymeric substances which protect bacteria from antimicrobial agents.
Thus far, the role of polar and lateral flagella in biofilm formation of A. dhakensis remains unknown. This study aims
to elucidate the role of 5 flagellar genes in A. dhakensis biofilm formation.

Material and Methods: Five unmarked
deletion mutants (polar flagellar: ΔflaH, Δmaf-1; lateral flagellar: ΔlafB, ΔlafK and ΔlafS) and complemented strains
(cflaH, cmaf-1, clafB, clafK and clafS) were constructed using a clinical A. dhakensis strain isolated from burn
wound infection. Bacterial culture of wild type (WT), mutants, and complemented strains were diluted in LB broth to
OD600= 0.05 and inoculated into E-plate (ACEA, USA). E-plate was incubated at 30°C and monitored on xCELLigence
Real-Time Cell Analyzer at 10-min time intervals for 48 hr. Biofilm formation was quantitated based on adherence of
bacteria and produced polymeric substances to gold microelectrodes as the impedance signal which is then converted
into cell index (CI) value.

Results: Biofilm formation in WT began at 6 hr and progressed until 21 hr, comprising of
3 stages (early, middle, and late) of CI increments. At 22-23 hr, WT biofilms achieved maturation with the highest CI
value (0.0746). At 24 hr onward, WT biofilms began to disperse with decreasing CI values (lowest CI value= 0.0554).
All mutants displayed different CI curves of biofilm formation when compared to the WT. Of the 5 studied genes,
ΔflaH, Δmaf-1, ΔlafK and ΔlafS formed biofilms earlier (t=3-4 hr) than the WT (t=6 hr). At 6-24 hr, Δmaf-1, ΔlafB,
ΔlafK and ΔlafS showed reduced CI values as compared to the WT, indicating less biofilm formation. On the contrary,
ΔflaH demonstrated CI values that were comparable to the WT, suggesting it did not affect biofilm formation. Full
restoration of biofilm formation in maf-1 (polar) and lafB, lafK and lafS (lateral) were observed at 11-18 hr as their CI
values were comparable to the WT, suggesting that these genes are involved in the middle and late stages of biofilm
formation, possibly by providing mechanical support to biofilm development and structure. Conclusion: Polar (maf-
1) and lateral flagella (lafB, lafK and lafS) play role in the middle and late stages of A. dhakensis biofilm formation.

e00072
The Burden of Multidrug-Resistant Tuberculosis
in Malaysian TB Patients.

Mahindran Rajendran¹, Nasrin Aghamohammadi¹*, Rafdzah Ahmad Zaki2, Zamzurina Abu Bakar3.

*Corresponding author:
Nasrin Aghamohammadi, Centre for Epidemiology and Evidence-Based Practice, Department of Social and Preventive Medicine,
Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
E-mail: nasrin@ummc.edu.my; nasrianm01@gmail.com

Details of affiliation

1 Centre for Epidemiology and Evidence-Based Practice, Department of Social and Preventive Medicine, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia
2 Institute for Respiratory Medicine, Jalan Pahang, Pekeliling, 53000 Kuala Lumpur, Malaysia.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit organizations..

Competing Interests:

Keywords: Multidrug-resistant TB, prevalence, risk factors, tuberculosis, TB surveillance.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00072

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Mahindran Rajendran, Nasrin Aghamohammadi, Rafdzah Ahmad Zaki, Zamzurina Abu Bakar, et al. Manuscript´s full Title. The Burden of
Multidrug-Resistant Tuberculosis in Malaysian TB Patients. IBJ Plus 2021 (S4)e0072:10.24217/2531-0151.21v1s4.00072.

Edited: Madrid, España.

Abstract

Background. Multidrug-resistant tuberculosis (MDR-TB) is indeed a global epidemic across the world.
Objective. The aim of this study is to determine the prevalence of MDR-TB among tuberculosis (TB) patients in Malaysia.

Method. Between 2009 and 2019, data from Malaysia’s National Tuberculosis Information System was retrieved for
a retrospective report. The data was analyzed using SPSS version 20 statistical tools (SPSS Inc, Chicago, Ill, USA).

Results. The overall estimated prevalence of MDR-TB infection among TB patients in Malaysia was 0.34%, according
to data from the National TB Surveillance in Malaysia (TBIS). There was a significant variation in the prevalence of
MDR-TB between male and female patients, according to the findings. The occurrence of MDR-TB is more common
in single patients compared to married patients. The prevalence of MDR-TB in ethnic’s groups shows Malay and
Indian (p<0.001). In comparison to non-alcoholic patients, the alcoholic patients have a higher risk of contracting
MDR-TB, followed by smoking patients.

Conclusion. As a result of the analysis of MDR-TB prevalence and risk factors, health experts in Malaysia will be able
to implement more effective public health policies.
Acknowledgements. The authors would like to acknowledge the University of Malaya (UM), Secretariat of National
Institutes of Health (NIHSEC) of Ministry of Health, Malaysia (MOH) and Institute for Respiratory Medicine (IPR)
Kuala Lumpur, Malaysia for this study.
Ethical approval. National Institutes of Health and Medical Research Ethics Committee. NMRR-19-1460-46943

e00073
The effects of attitude, self-efficacy and perceived usefulness
on the actual use of personal radiation dosimeter among
multiethnic Malaysian radiology workers.

Siti Farizwana Mohd Ridzwan1,2*, Marzuki Isahak1.

*Corresponding author:
Siti Farizwana Mohd Ridzwan, Faculty of Medicine, University of Malaya and Universiti Kebangsaan
Malaysia, Kuala Lumpur, Malaysia.
E-mail: farizwana@yahoo.com

Details of affiliation

1Department of Social and Preventive Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
2Department of Radiology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia

Funding

The authors declare no funding.

Competing Interests:

None declared.

Keywords: medical radiation worker, radiation monitoring, health behaviour, partial least square, TPB, TAM

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00073

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Siti Farizwana Mohd Ridzwan, Marzuki Isahak. The effects of attitude, self-efficacy and perceived usefulness on the actual use of personal
radiation dosimeter among multiethnic Malaysian radiology workers. IBJ Plus 2021 (S4)e0073:10.24217/2531-0151.21v1s4.00073.

Edited: Madrid, España.

Abstract

Introduction: To ensure medical radiation workers are protected from excessive ionising radiation exposure, they were provided with personal dosimeters to monitor the accumulated radiation dose. However, previous assessments revealed that the use of a personal dosimeter was not satisfactory in many countries. The reasons for non-adherence were previously recorded, including forgetfulness, unavailability, late supply, no supervision, and the device’s physical factor. A few demographic and occupation factors influenced the consistent device use, such as age, gender, education, years of experience, type of hospital and working hour. However, a fixed conclusion cannot be made from these factors because human behaviour is unpredictable and complex. Therefore, we have successfully validated a research model by integrating the leading behaviour theories, Theory of Planned Behaviour (TPB) and Technology Acceptance Model (TAM). This model attempts to explain personal dosimeter use among the medical radiation workers using latent behavioural constructs. We believe this is the first effort to truly understand the workers’ actual practice towards personal radiation monitoring in Malaysia.

Method: A validated survey link was distributed to the institution coordinators (n=73) priorly assigned by the head of departments who
agreed to participate in the study. The coordinators were requested to disseminate the survey link to other eligible
workers in the department on two separate occasions over eight weeks between April and June 2019. The respondents
were assured that all answers were anonymous, and the investigators were blinded to respondents’ identities.
Participants’ responses were accrued through the SurveyMonkey web site, which was only accessible to the investigators.
The final data were exported to MS Excel and Smart-PLS 3 to be analysed further.

Results: The survey link was clicked-through by 411 respondents, but only 379 respondents (92.2%) completed their responses. The proposed
model shows the strongest explanatory factor for actual dosimeter use; attitude (β=0.646, p<0.001). Meanwhile,
self-efficacy (β=0.221, p=0.014) and perceived usefulness (β=0.214, p<0.018) can explain the behaviour minimally.
The overall developed model explained 42.3% of the variance in the tested constructs (R2=0.423).

Conclusion: The findings suggest strategies to the authority regarding the successful radiation monitoring practice. Continuous education is vital to improve workers’ attitude and self-efficacy. Moreover, the authority should also upgrade the device to help workers adhere to the requirement.

e00074
Profiling of the Malayan blue coral snake (Calliophis bivirgata
flaviceps) venom through an integrated -omics approach.

Praneetha Palasuberniam1,3, Kae Yi Tan2, Choo Hock Tan1,*ar3..

*Corresponding author:
Choo Hock, Tan, Department of Pharmacology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia,
Email: tanch@um..edu.my

Details of affiliation

1Department of Pharmacology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia
2Department of Molecular Medicine, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia
3Department of Biomedical Sciences & Therapeutics, Faculty of Medicine & Health Sciences, Universiti Malaysia Sabah, Kota
Kinabalu, Sabah, Malaysia

Funding

The work was supported by ST011-2020

Competing Interests:

Authors declare no competing of interest

Keywords: proteomics; three-finger toxins, delta-neurotoxins

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00074

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Palasuberniam, P., et al (2021) Profiling of the Malayan blue coral snake (Calliophis bivirgata flaviceps) venom through an integrated -omics
approach. IBJ Plus 2021 (S4)e0074:10.24217/2531-0151.21v1s4.00074

Edited: Madrid, España.

Abstract

Introduction: The Malayan blue coral snake, Calliophis bivirgata flaviceps (C. bivirgata flaviceps), is a medically
important venomous snake widely distributes in the subcontinent region of Southeast Asia. It is an elapid species
of the Old World coral snakes, and snakebite envenomation caused by this species can result in clinical toxicity.
However, there has not been a comprehensive profiling of the protein composition of its venom to provide further
insights into the pathophysiology of envenomation. To bridge this knowledge gap, this study aims to investigate the
venom composition of C. bivirgata flaviceps through an integrated -omics approach.
Materials and Methods: The venom proteins of C. bivirgata flaviceps were decomplexed using C-18 reverse-phase
high performance liquid chromatography (rpHPLC) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
The protein fractions obtained from rpHPLC were then trypsin-digested and subjected to nano-electrospray ionization-
liquid chromatography and tandem mass spectrometry. The analysis of peptide spectra and protein identification
were conducted with the use of an integrated database enriched with the de novo venom-gland transcriptome
of the C. bivirgata flaciveps.
Results: A total of 15 toxin families were identified in the venom proteome. Three finger toxins (3-FTx), comprising
various neurotoxins and cytotoxins, dominated the venom proteome by 54.8% of total venom proteins. The high
abundance of 3-FTx and the unique presence of delta-neurotoxin in C. bivirgata flaviceps venom, correlate with toxic
activities of the venom. Kunitz-type serine protease inhibitors contributed the second most abundant protein family
(25.4%), followed by phospholipase A2 (9.0%) and zinc-metalloproteinase-disintegrin (6.6%). Other minor proteins
which constituted less than 5% of venom proteins, includes vespryn, phosphodiesterase, cystatin, cysteine-rich
venom protein, hyaluronidase, snake venom serine protease and venom growth factor.
Conclusion: The integrated -omics approach elucidated the venom proteome of C. bivirgata flaviceps, unravelling
the complexity of toxins in the venom. The findings provide deeper insights into the pathophysiology of envenomation
caused by this unique species.

e00075
Medication safety: Evaluating and enhancing implementation
of risk minimisation measures in Malaysia.

Rema Panickar1,2, Adeeba Kamarulzaman1, Zoriah Aziz1,3..

*Corresponding author:
Zoriah Aziz, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Email: zoriah@um.edu.my

Details of affiliation

1Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
2National Pharmaceutical Regulatory Agency, Ministry of Health, Petaling Jaya, Malaysia.
3Faculty of Pharmacy, MAHSA University, Selangor, Malaysia.

Funding

No external funding

Competing Interests:

The authors declare no conflicts of interest.

Keywords: risk communication, pharmacovigilance, allopurinol, SCARs, risk management.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00075

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Rema Panickar, Adeeba Kamarulzaman, Zoriah Aziz. Medication safety: Evaluating and enhancing implementation of risk minimisation
measures in Malaysia. IBJ Plus 2021 (S4)e0075:10.24217/2531-0151.21v1s4.00075.

Edited: Madrid, España.

Abstract

Introduction: Risk minimisation measures (RMMs), such as product information updates and risk communication,
are the steps taken to prevent or reduce the occurrence of adverse drug reactions (ADRs). RMM implementation
places a burden on the healthcare system in terms of cost, personnel, and time. Though it is important to evaluate
the effectiveness of RMMs, this is not routinely done in Malaysia. We aimed to (i) examine the impact of various
RMMs implemented in Malaysia using allopurinol as a model, and (ii) suggest specific methods to enhance the effectiveness
of RMMs, specifically risk communication, in Malaysia.

Methods: Our study comprises two phases: in the first phase, we obtained data for ADR reports associated with
allopurinol, its utilisation and related RMMs from the Ministry of Health, Malaysia. We evaluated the impact of
RMM implementation on allopurinol reporting rate and utilisation. To examine the relation between RMMs and
the ADR reporting rate we used the Pearson χ2 test of independence. In Phase 2, we will conduct a cross-sectional
online study (March-June 2021) involving doctors and pharmacists across Malaysia using an adapted English-language
questionnaire. We will evaluate the usefulness of four medication risk communication measures used in Malaysia
[bulletin, safety alerts, Direct Healthcare Professional Communications (DHPCs), and educational material]. To identify
factors associated with the usefulness of medication risk communication, we will use logistic regression analysis.

Results: For the first phase of the study, 16 RMMs were implemented in Malaysia for allopurinol-related severe
cutaneous adverse drug reactions (SCARs) from 2000 to 2018. Following the implementation of the RMMs, for the
period 2004 to 2018, we noted an overall reduction (21.5%) in allopurinol utilisation. Meanwhile, ADR reporting
rates for all drugs and allopurinol increased. RMMs implemented in August 2014 [χ2(1, N = 258) = 5.32, P = .021] and
October 2016 [χ²(1, N = 349) = 3.85, P = .0499] showed a statistically significant reduction in ADR reports related to
off-label allopurinol use. Preliminary data from the second phase of the study showed a lack of awareness of the
current medication risk communication in Malaysia. We noted that doctors and pharmacists serving in the Malaysian
public healthcare sector are more aware of the bulletin and safety alerts, while the private sector receives more
DHPCs and educational material.

Conclusions: Our Phase 1 study showed that targeted and interactive RMMs have a greater impact on improving
medication safety. Preliminary results of the second phase indicate that public-private collaboration may be required
for the success of risk communication in Malaysia.

e00076
Biosimilars in Malaysia: Regulation and factors associated
with confidence to promote their use among
hospital pharmacists.

Noraisyah Mohd Sani1,2, Adeeba Kamarulzaman1, Zoriah Aziz1,3.

*Corresponding author:
Zoriah Aziz, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Email: zoriah@um.edu.my

Details of affiliation

1Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
2Ministry of Health, Putrajaya, Malaysia.
3Faculty of Pharmacy, MAHSA University, Selangor, Malaysia.

Funding

No external funding

Competing Interests:

The authors declare no conflicts of interest.

Keywords: biosimilars, regulation, pharmacists, confidence, use.

Published April 10, 2018.

DOI: 10.24217/2531-0151.21v1s4.00076

Copyright: © 2021 Author. This is an open-access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite as: Noraisyah Mohd Sani, Adeeba Kamarulzaman, Zoriah Aziz. Biosimilars in Malaysia: Regulation and factors associated with confidence to
promote their use among hospital pharmacists. IBJ Plus 2021 (S4)e0077:10.24217/2531-0151.21v1s4.00076.

Edited: Madrid, España.

Abstract

Introduction: Biosimilars are medicines made from living cells that are used for diseases such as cancer, diabetes
and other inflammatory diseases. Using biosimilars, a similar version of biologic originators in terms of quality, efficacy
and safety, may reduce healthcare expenses. Our study seeks to address the following questions: (1) what is the
current regulatory overview of biosimilars in Malaysia in terms of regulation, approved products and their corresponding
adverse effects (AEs), and completed and ongoing clinical trials and (2) what are the factors associated with
the confidence of Malaysian hospital pharmacists to promote the use of biosimilars.

Methods: Our study consists of two phases. For Phase 1 of the study, we reviewed data based on the data search
from the official websites of the National Pharmaceutical Regulatory Agency (NPRA) Malaysia and three other well-
established agencies. The agencies were the World Health Organisation (WHO), the European Medicines Agency
(EMA), and the United States Food and Drug Administration (US FDA). We searched AEs data from the NPRA AE
database and the World Health Organisation VigiLyze database. For clinical trials data, we extracted them from
ClinicalTrials.gov website by the U.S. National Library of Medicines. For Phase 2 of the study, we will conduct a nationwide
cross-sectional study using anonymous electronic survey distributed to all registered pharmacists working
in hospitals. We refined the questionnaire from previous studies to address the study objectives. Multiple logistic
regression analysis will assess the significance of variables, which predict the likelihood of confidence to promote
the use of biosimilars among the hospital pharmacists.

Results: Findings from Phase 1 showed that Malaysia follows a stringent regulatory pathway for the approval of
biosimilars to maintain the quality, efficacy, and safety of biosimilars in line with the principles of EMA. Since issuing
a biosimilar guideline in 2008 until February 2020, NPRA has approved 24 biosimilar products and received 499 AE
reports, including 43 (8.6%) serious cases. The NPRA has approved ten Phase III clinical trials in Malaysia, with four
still ongoing.

Conclusions: We expect the introduction of biosimilars in Malaysia to have a positive effect on patient care, as their
use may lower the cost of biologic therapies. Our findings may be useful to plan for strategies to improve pharmacists’
confidence to promote the use of biosimilars in clinical practice towards improving their uptake in Malaysia.