Abstracts of 5th Symposium on Biomedical Research

e00002
Deficiency in the transcription factor NRF2 worsens inflammatory parameters in a mouse model with combined tauopathy and amyloidopathy.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00002

Copyright:  © 2018 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 I Rojo, Marta Pajares, Angel J García-Yagüe, Izaskun Buendia, Fred Van Leuven, Masayuki Yamamoto, Manuela G Lopez,
Antonio Cuadrado. IBJ Plus 2018 (S1):e0002 doi: 10.24217/2531-0151.18v1s1.00002

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Chronic neuroinflammation is a hallmark of the onset and progression of brain proteinopathies such as Alzheimer disease
(AD) and it is suspected to participate in the neurodegenerative process. Transcription factor NRF2, a master regulator of
redox homeostasis, controls acute inflammation but its relevance in low-grade chronic inflammation of AD is inconclusive
due to lack of good mouse models. We have addressed this question in a transgenic mouse that combines amyloidopathy
and tauopathy with either wild type (AT-NRF2-WT) or NRF2-deficiency (AT-NRF2-KO). AT-NRF2-WT mice died prematurely, at
around 14 months of age, due to motor deficits and a terminal spinal deformity but AT-NRF2-KO mice died roughly 2 months
earlier.

NRF2-deficiency correlated with exacerbated astrogliosis and microgliosis, as determined by an increase in GFAP, IBA1 and
CD11b levels. The immunomodulatory molecule dimethyl fumarate (DMF), a drug already used for the treatment of multiple
sclerosis whose main target is accepted to be NRF2, was tested in this preclinical model.

Daily oral gavage of DMF during six weeks reduced glial and inflammatory markers and improved cognition and motor
complications in the AT-NRF2-WT mice compared with the vehicle-treated animals. This study demonstrates the relevance of
the inflammatory response in experimental AD, tightly regulated by NRF2 activity, and provides a new strategy to fight AD.

e00003
Alterations in the stimulus-secretion coupling related
to aging in the murine model of accelerated senescence
SAMP8.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00003

Copyright: © 2018 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: Andrés M. Baraibar, Carmen Nanclares, Inés Colmena, Isabel Gameiro-Ros, Iris Álvarez-Merz, Alicia Muñoz-Montero, Jesús
M. Hernández-Guijo, Luis Gandía. Alterations in the stimulus-secretion coupling related to aging in the murine model of accelerated
senescence SAMP8. IBJ Plus 2018 (S1):e0003 doi: 10.24217/2531-0151.18v1s1.00003.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The nervous system is especially vulnerable to aging. Its vulnerability is manifested by the existence of
neurodegenerative pathologies like Alzheimer’s disease (AD), Parkinson’s disease (PD) or Amyotrophic Lateral Sclerosis (ALS).
During these diseases, alterations of neurotransmitter systems has been reported. Although, numerous changes can also
be observed in many individuals during non-pathological aging. Our working hypothesis suggests that, with the progression
of age, alterations in the stimulus-secretion coupling can occur, compromising the release of neurotransmitters and causing
cognitive deficits.

Materials and Methods: In this work, mice of the senescence-prone strain 8 (SAMP8) have been used which show agerelated
behavioural deterioration such as deficits in learning and memory, emotional disorders and altered circadian rhythm,
being therefore used as a model of spontaneously occurring Alzheimer’s disease. In parallel, their resistant senescence
(SAMR1) brothers have been used as a control. We used the chromaffin cell as a model of neurosecretion in SAMP8 and
SAMR1 mice at 2, 6 and 12 months of age. By means of the patch-clamp technique, we have studied the ionic currents
involved in the secretory process of catecholamines and in the transmission of the nerve impulse (nicotinic, Na+, Ca2+ and
K+ currents). We have also assessed cell excitability by measuring membrane potential and spontaneous and triggered action
potentials. Moreover, we have studied the release of the neurotransmitters by the amperometric technique using K+ as
stimuli. Finally, we have used the Y-maze to evaluate the cognitive behaviour of the mice.

Results: We have observed that there is an increase in all ionic currents with the age in both, SAMP8 and SAMR1 mice, but
this increase occurs before and more remarkable in SAMP8 mice. Regarding membrane potential and spontaneous and
triggered action potentials we have observed that there is a hyperpolarization of membrane potential in both types of mice
during the aging, moreover, the depolarization that ACh produces is lower throughout the age and again, this phenomenon
occurs before and is more remarkable in SAMP8 mice. Furthermore, the number of action potentials generated at rest and
those produced by depolarizing pulses decreases during the aging, being higher the amplitude and posthyperpolarization
area. Although, the number of action potentials evoked by ACh increases with aging due the less depolarization being more
remarkable in SAMP8 mice.
Regarding the release of the neurotransmitters we have seen that when we stimulate with K+ there is an increase in the
catecholamine secretion with the aging, happening before in SAMP8 mice. Moreover, this increase in the catecholamine
release is accompanied by changes of secretory spikes.
Finally, with Y-maze, we have seen that these alterations in the release of neurotransmitters are associated with a cognitive
deficit, since the SAMP8 mice explore all the arms equally, whereas the R1 always explore more the new arm.

Conclusion: We have found that during the aging the mechanisms of exocytosis of neurotransmitters is altered. These
alterations could be correlated with the changes that occurs in some neurodegenerative diseases and also causing cognitive
deficits.

e00004
Nicotinic receptor subtypes involved in the tobaccomediated
resistance to cisplatin in human non-small cell lung
carcinoma.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00003

Copyright: © 2018 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: Anna Bordas Sánchez, María Extremera Mazuela, José Luis Cedillo Mireles, Carolina Martín-Sánchez, Carmen Montiel López.
Nicotinic receptor subtypes involved in the tobacco-mediated resistance to cisplatin in human non-small cell lung carcinoma. IBJ
Plus 2018 (S1):e0004 doi: 10.24217/2531-0151.18v1s1.00004.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Cigarette smoking not only correlates with the onset and progression of a wide variety of smokingrelated
tumors, but the continued tobacco use after cancer diagnosis is known to decrease the effectiveness of adjuvant
chemotherapy. These tumors express several nicotinic acetylcholine receptor (nAChR) subtypes whose activation by certain
components of tobacco appears to underlie both deleterious effects. One of the tumors most associated with smoking is
non-small cell lung carcinoma (NSCLC), a tumor that accounts for 75-85% of all cases of lung cancer. Nicotine, the addictive
component in tobacco, plays a key role in tobacco-mediated chemoresistance through the activation of a still unknown
nAChR subtype, which triggers signaling pathways that lead to the inhibition of apoptosis. The aim of this study is to
elucidate the nAChR subtype and the apoptotic signaling pathways underlying nicotine-mediated chemoresistance in NSCLC,
as well as to assess whether another component of tobacco, such as the nitrosamine NNK, may also be involved in the above
effect.

Material and Methods: All assays were carried out on A549 cells, a human lung adenocarcinoma cell line. Cell viability and
apoptosis assays were measured using the MTT and Annexin V-FITC kits, respectively. Gene silencing of the nAChR subunits
was performed with small interference RNA (siRNA). Molecules of the pro- and anti-apoptotic signaling pathways triggered
by cisplatin, in the absence or presence of nicotine or NNK, were analyzed by Western Blot.

Results: Nicotine and NNK inhibited cisplatin-induced apoptosis in A549 cells in a dose-dependent manner. The silencing
of the α7 or α5 subunits of the nAChR significantly reduced the above effect of the tobacco components, while silencing of
the dupα7-nAChR subunit increased it. Compared with cells exposed to cisplatin, those also treated with nicotine and NNK
showed an upregulation of anti-apoptotic signaling (pAkt and Akt) and downregulation in the pro-apoptotic protein Bax. The
above effects of nicotine and NNK on the apoptotic signaling pathway were suppressed in cells with the α7 or α5 subunits
silenced and they were increased after the silencing of dupα7 subunits.

Conclusion: These results reveal that some components of tobacco induce resistance to cisplatin by an anti-apoptotic
mechanism and that nAChR subtypes containing α7 or α5 subunits play a key role in the above effect. In addition, our data
also suggest that high dupα7 gene expression levels in NSCLC could reduce tobacco-mediated chemoresistance through
interference with nAChRs containing α7 subunits.

e00005
Effects of olanzapine and aripiprazole on lipolysis in healthy
human subcutaneous adipocytes during short incubations.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00005

Copyright: © 2018 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: Assel Sarsenbayeva, Cátia Marques, Gretha Boersma, Maria João Pereira, Jan Eriksson. Effects of olanzapine and aripiprazole
on lipolysis in healthy human subcutaneous adipocytes during short incubations. IBJ Plus 2018 (S1):e0005 doi: 10.24217/2531-
0151.18v1s1.00005

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Second-generation Antipsychotics (SGAs) have become the treatment of choice over the typical antipsychotics
as they provide excellent efficacy and fewer extrapyramidal symptoms. However, the compliance of the patients to SGAs is
negatively affected by their ability to induce or aggravate metabolic syndrome, namely, weight gain, insulin resistance, and
Type 2 Diabetes. The exact underlying mechanism of metabolic effects of SGAs is not fully elucidated and it is assumed to
be at least partially due to their effect on central nervous system. However, whether SGAs have a direct effect on insulin
action in the tissues is still to be elucidated. The effect of SGAs on body metabolism varies and we have chosen two drugs,
Olanzapine and Aripiprazole, which are associated with high and low risk of metabolic side-effects, respectively.
Our research is focused on studying the effect of both SGAs on insulin resistance in human adipose tissue. Aside from lipid
storage function, adipose tissue has been recognised, as an endocrine organ, producing hormones, such as adiponectin and
leptin, indispensable for energy homeostasis. The set of experiments performed as a part of this study includes measuring
the effect of Olanzapine and Aripiprazole on the lipolysis in human isolated adipocytes.

Methods: Biopsies of subcutaneous adipose tissue (SAT) were collected from 6 patients (3 men, 3 women; age: 20-76 years;
BMI: 20.9-34.5 kg/m2). Subjects were free of antidepressants or antipsychotics treatment. At the moment, only the effect of
Olanzapine has been tested and measured, the experiments with Aripiprazole are in progress.
A 6% adipocyte suspension was incubated with olanzapine (0.004, 0.04, 0.1, 0.2, 2 and 20 μM) or aripiprazole (0.02, 0.2, 0.5,
1, 10 and 100 μM). This was followed by 10 minutes incubation with 4 concentrations of insulin (0.1 μU; 1.0 μU; 10 μU; 100
μU) and then incubated with 0.5 μM ß-adrenergic receptor agonist, Isopretenerol, for 1h 50 min. ß-adrenergic stimulation
activates hormone-sensitive lipase (HSL) enzyme via cAMP-dependent pathway. HSL, in turn, hydrolyses tritriacylglycerol
(TAG), diacylglycerol (DAG) or monoacylglycerol (MAG) molecules producing free fatty acids and glycerol. The supernatant
was then collected and used for glycerol measurement.

Results: Short incubations of adipocytes with therapeutic concentrations of Olanzapine show no effect in lipolysis. The
highest concentration of the drug hints at a reduced rate of lipolysis in adipocytes by more than 50% for each insulin
concentration (p<0.0001) and in control conditions (p<0.01).

Conclusions: Therefore, it seems that short-term incubation of adipocytes with 20 μM Olanzapine reduces the rate of
lipolysis, while the therapeutic concentrations do not seem to alter lipolysis in adipocytes.

e00006
Ageing alters the kinetics of exocytosis of both wildtype and
the 3xTg-AD mouse model of Alzheimer´s disease.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00006

Copyright: © 2018 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: Carmen Nanclares, Inés Colmena, Andrés M. Baraibar, Alicia Muñoz-Montero, Iris Álvarez-Merz, Isabel Gameiro-Ros,
Antonio G. García and Luis Gandía. Ageing alters the kinetics of exocytosis of both wildtype and the 3xTg-AD mouse model of
Alzheimer´s disease. IBJ Plus 2018 (S1):e0006 doi: 10.24217/2531-0151.18v1s1.00006.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Alzheimer´s disease (AD) is the most common form of dementia. The alteration of several neurotransmitter
systems has been reported. These alterations in the neurotransmission processes could be correlated with changes in the
synthesis, storage or release of neurotransmitters.

Materials and Methods: In this study we have used a triple transgenic murine model of AD (3xTg-AD). This animal model
contains mutations in the gene encoding the amyloid precursor protein (βAPPSwe), presenilin-1 (PS1M146V) and tauP301L,
which mimics the development of the disease on AD patients. We propose to study here the last steps of exocytosis in
chromaffin cells of 3xTg-AD mice of different ages and their controls (WT) using the amperometric technique. Different ionic
currents involved in the physiological release of catecholamines will be studied using the patch-clamp technique.

Results: We have found significant changes in the exocytosis of catecholamines occurring in 3xTg-AD when compared with
wildtype (WT) mice. These changes show an increase of the number of amperometric spikes during the development of the
disease, as well as an increase in the quantal catecholamine content. Kinetic analysis of secretory spikes shows that as mice
age amperometric spikes are faster in triggering and shorter in duration. However, the release of catecholamines is slower
and longer in duration when comparing 3xTg-AD with WT mice.
Using the patch-clamp technique we found no changes in nicotinic currents, a decrease in sodium currents and an increase in
potassium currents. Some alterations were also found in calcium currents.
Taken together, these findings suggest that throughout the development of 3xTg-AD mice and as Alzheimer’s disease is
established there are changes in chromaffin cell excitability, which causes an increase in neurotransmission.

Conclusion: Although ageing caused some coincident alterations in the kinetics of exocytosis, there were also clear
differences between WT and 3xTg-AD mice. These alterations could have an impact on the response that the organism offers
in a stressful situation.

e00007
Histone modifications associated with nonconventional
systemic drug response in moderate-to-severe psoriasis.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00007

Copyright: © 2018 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: M.C. Ovejero-Benito, A. Reolid, P. Sánchez-Jiménez, M. Saiz-Rodríguez, E. Muñoz-Aceituno, M. Llamas-Velasco, S. Martín-
Vilchez, T. Cabaleiro, M. Román, D. Ochoa, E. Daudén, F. Abad-Santos. Histone modifications associated with nonconventional
systemic drug response in moderate-to-severe psoriasis. IBJ Plus 2018 (S1):e0007 doi: 10.24217/2531-0151.18v1s1.00007.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Epigenetic factors play an important role in psoriasis onset and development. There are different drugs
available to treat moderate-to-severe psoriasis patients resistant to conventional systemic drugs. Although these drugs are
safe and effective for moderate-to-severe psoriasis treatment, some patients do not show an adequate response to them.
Therefore, it is necessary to find biomarkers that could predict patients´ response to these drugs. Objective: To analyse the
association between nonconventional systemic drugs (ustekinumab, secukinumab, adalimumab, ixekizumab and apremilast)
response and histone modifications in moderate-to-severe psoriasis patients.

Materials and methods: Peripheral blood mononuclear cells (PBMCs) were isolated from psoriasis patients treated with
nonconventional systemic drugs before and after the administration of these therapies. PBMCs obtained from healthy
subjects have been used as controls. Afterwards, histones were extracted from PBMCs. Four different histone modifications
(H3 and H4 acetylation, H3K4 and H3K27 methylation) were determined by Enzyme-Linked ImmunoSorbent Assay (ELISA).
Data were analyzed by IBM SPSS v.23.

Results and conclusions: Psoriasis patients presented reduced levels of acetylated H3 and H4 and increased H3K4
methylated levels compared to healthy controls. Global changes in any of the histone modifications analyzed were not
observed in psoriasis patients between before and after treatment administration. Nevertheless, significant changes in
methylated H3K27 were found between responders and non-responders to nonconventional systemic drugs. These results
should be validated in large-scale studies before implementation in clinical practice.

e00008
Effects of olanzapine and aripiprazole on glucose uptake
in healthy human subcutaneous adipocytes during short
incubations.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00008

Copyright: © 2018 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: Cátia Marques, Assel Sarsenbayeva, Gretha Boersma, Angela Valverde, Eugénia Carvalho, Maria João Pereira, Jan Eriksson.
Effects of olanzapine and aripiprazole on glucose uptake in healthy human subcutaneous adipocytes during short incubations. IBJ
Plus 2018 (S1):e0008 doi: 10.24217/2531-0151.18v1s1.00008.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Second-generation Antipsychotics (SGAs) are preferable pharmacological treatment for patients with
Schizophrenia, mainly due to their efficacy and reduced risk of extrapyramidal effects when compared with first generation
antipsychotics. However, there are metabolic side effects associated with the administration of SGAs, namely weight gain,
dyslipidaemia and impaired glucose metabolism. Literature reports that even in the absence of antipsychotic treatment
patients with Schizophrenia have a propensity to develop metabolic changes that can lead to cardiovascular diseases, insulin
resistance, obesity and Type 2 Diabetes. Olanzapine and aripiprazole belong to SGAs and have been reported as drugs with
the highest and the lowest risk of inducing metabolic changes, respectively. However, the pharmacological mechanisms
underlying their metabolic side effects remain unclear and they will be the main focus of our study.
Adipose tissue is not only specialized in storing lipids but it is also an endocrine organ that produces and secretes numerous
biological active compounds that regulate metabolic homeostasis.
Our aim is to evaluate the effect of olanzapine and aripiprazole in the glucose uptake on human isolated adipocytes.

Methods: Biopsies of subcutaneous adipose tissue were collected from 16 healthy volunteers (4 men, 12 women; age: 20-76
years; BMI: 20.9-34.5 kg/m2). Subjects taking antidepressants or antipsychotics were not included. The effect of short-term
incubation (30 min) with different concentrations of olanzapine (0.004, 0.04, 0.1, 0.2, 2 and 20 μM) or aripiprazole (0.02, 0.2,
0.5, 1, 10 and 100 μM) on basal and insulin-stimulated (25 and 1000 μU/ml) D-[U-14C]-glucose uptake of isolated adipocytes
was measured and compared with control.

Results: Short incubation of adipocytes with olanzapine or aripiprazole showed no effect on basal or insulin-stimulated
glucose uptake, with the exception of supra-physiological concentrations of aripiprazole (10 and 100 μM) where we see a
systematic decrease of basal and insulin-stimulated glucose uptake; 10 μM by ͌20-25% (p<0.05) and 100 μM by ͌60-70%
(p<0.01).

Conclusions: Short-term treatment of isolated adipocytes with therapeutic doses of olanzapine or aripiprazole did not
affect glucose uptake, suggesting no acute alteration in insulin activity. These data suggest that the plasma glucose increase
seen in patients taking Olanzapine cannot be justified by acute alteration in insulin-signalling in adipocytes. Incubation with
aripiprazole at 10 and 100 μM decreased the adipocyte glucose uptake but this might be justified by cell death, which will be
explored in future experiments.

e00009
Dasatinib reversibly disrupts endothelial vascular integrity
by increasing non-muscle myosin II contractility in a ROCKdependent
manner.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00009

Copyright: © 2018 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: Anna Kreutzman, Beatriz Colom-Fernández, Ana Marcos Jiménez, Mette Ilander, Carlos Cuesta-Mateos, Yaiza Pérez-García,
Cristina Delgado Arévalo, Oscar Brück, Henna Hakanen, Jani Saarela, Alvaro Ortega-Carrión, Ana de Rosendo, Alba Juanes-García,
Juan Luis Steegmann, Satu Mustjoki, Miguel Vicente-Manzanares, Cecilia Muñoz-Calleja. Dasatinib reversibly disrupts endothelial
vascular integrity by increasing non-muscle myosin II contractility in a ROCK-dependent manner. IBJ Plus 2018 (S1):e0009 doi:
10.24217/2531-0151.18v1s1.00009.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Purpose: Dasatinib is a short-acting dual ABL/SRC family tyrosine kinase inhibitor (TKI), which is frequently used to treat
chronic myeloid leukemia. Although very effective, dasatinib often displays severe adverse effects, including pleural effusions
and increased risk of bleeding primarily in the gastrointestinal tract. The actual causes of these side effects are currently
undetermined. We hypothesize that endothelial cells (ECs) that line the inner walls of blood vessels and control the traffic to
the underlying tissues, might be involved.

Experimental design: The effects of TKIs on ECs were studied by various assays, such as real-time cell impedance
measurements, live-cell microscopy, wound healing, western blot and an in vivo model.

Results: Dasatinib uniquely causes a profound, dose-dependent disorganization of the EC monolayers. Dasatinib promoted
the disassembly of cell-cell contacts, altered cell-matrix contacts and further altered the wound healing. A key observation is
that this effect is fully reversible after drug washout. In line with these in vitro observations, intraperitoneal administration
of dasatinib to mice caused significant vascular leakage in the intestine. The underlying molecular mechanism of dasatinibinduced
reorganization of the actin involves ROCK activation, which increases the amount of the phosphorylation of myosin
light chain and consequently activates the non-muscle myosin II.

Conclusions: Our data are consistent with a scenario in which dasatinib triggers a transient increase in vascular leakage that
probably contributes to adverse effects such as bleeding diathesis and pleural effusions.

e00010
Changes in the Gene Expression Profile of Multiple Myeloma
cell line in response to Immunomodulatory (IMIDs) Drugs:
MYB Gen Target.

Fernández Lázaro Diego1,2*, Fernández Lázaro César Ignacio2,3, San-Segundo Laura4, Ocio San Miguel Enrique4,5.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00010

Copyright: © 2018 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: Fernández Lázaro Diego, Fernández Lázaro César Ignacio, San-Segundo Laura, Ocio San Miguel Enrique. Changes in the Gene
Expression Profile of Multiple Myeloma cell line in response to Immunomodulatory (IMIDs) Drugs: MYB Gen Target. IBJ Plus 2018
(S1):e0010 doi: 10.24217/2531-0151.18v1s1.00010.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The immunomodulatory drug (IMiD) thalidomide and its structural analogs lenalidomide and pomalidomide
are highly used to treat multiple myeloma (MM), however the molecular mechanism of IMiDs’ action is not well-established.
There before, we performed gene expression profiling analyses using microarray technologies to determine changes in genes
involved in cellular biological pathways of MM induced by IMiDs in monotherapy.

Material and Methods: We used MM1S cell line with concentrations of lenalidomide (1μM) and pomalidomide (100nM)
in six-well plates for 1 and 5 days at 37 °C. After treatment, MM1S cells were evaluated by flow cytometry using Annexin-V
and propidium iodide after selective labelling of plasma cells with CD38 and CD138 antibodies. The samples were analyzed
using Affimetrix Gene-chip Expression Arrays. The absolute expression values for each probe were calculated using the MAS
5.0 software. The comparative analyzes were carried out using the Dchip program, the SAM algorithm, and the Ingenuity
Pathway Analysis software. Expression changes were deemed significant if they were ±2-fold. Ingenuity Pathway Analysis
was used to identify the most relevant biological mechanisms, pathways, and functional categories in the data set of genes
selected by statistical analysis.

Results: After 1-day, in vitro treatment with lenalidomide (1μM), with 5-7% apoptosis, significantly deregulated 10 genes
whereas treatment with pomalidomide (100nM), with 8-10% apoptosis, deregulated 19 genes. Among these genes, 6
genes were exclusively deregulated by lenalidomide, 15 were deregulated by pomalidomide, and 4 genes were regulated
by both lenalidomide and pomalidomide. After 5-days, in vitro treatment with lenalidomide (1μM), with 20-22% apoptosis,
significantly deregulated 39 genes whereas treatment with pomalidomide (100nM), with 25-27% apoptosis, deregulated 359
genes. Among these genes, 8 genes were exclusively deregulated by lenalidomide, 328 were deregulated by pomalidomide,
and 31 genes were regulated by both lenalidomide and pomalidomide. These dysregulated genes are involved in the most
relevant biological pathways for MM.

Conclusion: We observed changes in the gene expression profile of MM cells after treatment with lenalidomide and
pomalidomide, resulting pomalidomide with greater anti-MM effect. The under-expression of the MYB oncogene —
regulator of transcription with an essential role in the control of the proliferation and differentiation of hematopoietic
progenitor cells and their tumorigenesis— was commonly deregulated in both IMiDs lenalidomide and pomalidomide in
monotherapy. Future studies may consider MYB as a new pharmacogenetic therapeutic target in MM.

e00011
Anticholinergics otilonium and pinaverium trigger
mitochondrial-mediated apoptosis in rat cortical neurons.

F. García-Alvarado1, A.G. García2*

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00011

Copyright: © 2018 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: F. García-Alvarado, A.G. García. Anticholinergics otilonium and pinaverium trigger mitochondrial-mediated apoptosis in rat
cortical neurons. IBJ Plus 2018 (S1):e0011 doi: 10.24217/2531-0151.18v1s1.00011.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: In the frame of a Repositioning Drug Programme with cholinergic medicines in current medical use, searching
for neuroprotection, we found that anticholinergics otilonium (OTI) and pinaverium (PIN) had the opposite properties that is,
neurotoxic effects. Here we have explored the mechanism underlying such neurotoxicity.

Material and methods: All experiments were carried out according with European and Spanish Directive for laboratory
animal experimentation. Primary cultures of rat brain embryo cortical neurons were used. Cell viability was estimated with
MTT and apoptosis with flow cytometry. Cytosolic Ca2+ was monitored with Fluo-4 AM.

Results: Fig.1 Shows microphotographs of cortical neurons and neurons incubated with otilonium and pinaverium at
different times; note the cell damage elicited by both compounds.

Fig. 1 Microphotographs at 20X of cultured rat embryonic cortical cells of 8 DIV, in the first column cells under basal
conditions, in the second and third with cells exposed to OTI 10μM and PIN 3μM at different times 0,4,8 and 24 hours.
Cell damage was not mimicked by other cholinergic drugs and was unaffected by catalase, glutathione, N-acetyl-cysteine or
melatonin. Cell death was elicited by apoptosis and necrosis. The apoptotic cell death elicited by OTI and PIN was prevent
by cyclosporine A (CSA) a blocker of the mitochondrial transition pore and by Ac-LEHD-CHO an inhibitor of caspase-3 and
caspase-9.

Conclusion: Data are compatible with the idea that OTI and PIN caused neuronal cell death by activating the intrinsic
apoptotic pathway linked to mitochondria. These compounds may be used as new chemical tools to elicit neuronal
apoptosis.

e00012
Studies on the anti-tumoral activity of 3-substituted indoles
and azaindoles in B cell malignancies

Gema Pérez-Chacón1*, Andrea Unión1, Kaline Arnauts1, Andrea de Andrés1, Cristóbal de los Rios2,3, Juan M. Zapata1*.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00012

Copyright: © 2018 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: Gema Pérez-Chacón, Andrea Unión, Kaline Arnauts, Andrea de Andrés, Cristóbal de los Rios, Juan M. Zapata. Studies on the
anti-tumoral activity of 3-substituted indoles and azaindoles in B cell malignancies. IBJ Plus 2018 (S1):e0012 doi: 10.24217/2531-
0151.18v1s1.00012.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Indole-3-carbinol (3-hydroxymethylindole; I3C) is a natural product found in broadly consumed plants of the Brassica genus,
such as broccoli, cabbage, and cauliflower, which exhibits anti-tumor effects through poorly defined mechanisms. I3C can be
orally administered and clinical trials have demonstrated that I3C is safe in humans. We have described that I3C efficiently
induces apoptosis in cell lines derived from EBV-positive Burkitt’s lymphomas (EBV+BL) (Perez-Chacon et al, 2014 Pharmacol.
Res. 89:46) and in leukemic cells from chronic lymphocytic leukemia (CLL) patients. Interestingly, I3C was able to induce cell
death of CLL cells and to synergize with fludarabine even in cells from patients with refractory CLL (Perez-Chacon et al, 2016.
Clin. Cancer Res 22:134).
We have studied whether other 3-substituted indoles also have a deleterious effect on the viability of the EBV+ BL cell
lines Raji and BL60.2, and of CLL cells. Our results show that 3-substituted indoles with either carboxylic, methylcarboxylic,
aminocarboxylic, cyanomethyl, carboxaldehide, dimethylaminomethyl, methoxymethyl or carboxamide groups failed to have
any effect on the viability of the EBV+BL and CLL cells at concentrations up to 200 μM. Interestingly, not even 3-hydroxyethylindole
and indole-2-methanol showed any cytotoxic effect, thus underlying the key role of the hydroxylmethyl group at
3 position for the anti-tumoral activity of I3C. It is noteworthy that indole-3-ol was cytotoxic in some of the tested cell
lines. However, while I3C cytotoxicity was prevented by the caspases inhibitor zVAD-fmk, thus indicating that I3C induces
apoptosis, zVAD-fmk failed to prevent indole-3-ol-induced cell death, thus suggesting that I3C and indol-3-ol kill by different
mechanisms.
In addition, we have also demonstrated that 7-azaindole and 3-hydroxymethyl-7-azaindole lack of any deleterious effect
on the viability of B cell malignancies under study. This result indicates that the replacement of C for N at 7-position of the
indole ring abolishes its anti-tumor activity.
Finally, we have also observed that C6-methylated-I3C was more potent than I3C in inducing cell death in both EBV+ BL cell
lines and in CLL cells, thus suggesting that substitution(s) at the benzene-fused ring (positions 4, 5, and/or 6) might enhance
the anti-tumor activity of I3C and open the possibility to develop new I3C derivatives with improved anti-tumor activity.

e00013
Mitochondria function and morphology alterations precede
neurosecretion impairment in chromaffin cells of the
SOD1G93A mouse model of amyotrophic lateral sclerosis.

Iago Méndez-lópez1*, Carmen Martínez-Ramírez1, Antonio G. García1, Fernando Padín Nogueira1,2.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00013

Copyright: © 2018 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: Iago Méndez-lópez, Carmen Martínez-Ramírez, Antonio G. García, Fernando Padín Nogueira. Mitochondria function and
morphology alterations precede neurosecretion impairment in chromaffin cells of the SOD1G93A mouse model of amyotrophic lateral
sclerosis. IBJ Plus 2018 (S1):e0013 doi: 10.24217/2531-0151.18v1s1.00013.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Amyotrophic lateral sclerosis (ALS) is characterized with a selective loss of motor neurons that cause paralysis
and respiratory failure. Hyperexcitability and Ca2+-dependent glutamate excitotoxicity has been hypothesized to be involved
in ALS pathogenesis. Exploring the chromaffin cell (CC) of SOD1G93A mouse model of familiar ALS, we found that the fusion
pore kinetics of exocytosis is slowed but with higher catecholamine quantal size when the disease is already established
(Calvo-Gallardo et al., Am J Physiol Cell Physiol 2015;308:C1-C19). To go further in the study of these neurosecretion
alterations, we investigate the exocytosis before the disease onset (30 days postnatal), and we focus in the study of
mitochondrial ultrastructure and function as a crucial organelle involved in the process.

Material and Methods: Mitochondrial ultrastructure was explored by transmission electron microscopy (TEM) and analyzed
with ImageJ software. Luminometer was used to measure ATP levels in CC cultures with the commercial CellTiter-Glo® kit.
Reactive oxygen species (ROS) production was monitored 30 minutes with the fluorescent dye H2DCFDA. Fusion pore kinetic
was studied by amperometry, eliciting exocytosis by 1 minute acetylcholine stimulus.

Results: TEM showed that mitochondria from SOD1G93A CCs have the following alterations with respect to wildtype CCs:
i) more number and small sized; ii) increased mitochondrial intermembrane space; iii) lower number and swollen cristae.
These ultrastructural changes suggesting mitochondrial fission and ultrastructure damage were accompanied by lower ATP
production and a higher rate of ROS generation. However, we fail in observe such significant differences in the fusion pore
kinetics.
Conclusion: The described mitochondrial alterations shown an interesting non-motor neuron degeneration in this ALS
model at presymptomatic stages. However, the kinetic of the exocytotic fusion pore have not been affected, contrary to
the slowed secretion observed once the paralysis is already established. These results evidence that this mitochondrial
alterations precede the functional changes linked to neurotransmitter release. In spite of having lower clinical relevance than
in later stages, it could generate some clues about the initiation and progression of the disease. Our data consolidate the
mitochondria as a potential target and the sympathetic-adrenal system affectation as an interesting new approach for ALS
diagnosis.

e00014
Prediction Models for Voriconazole Pharmacokinetics Based
on Pharmacogenetics: An Exploratory Study Nested to a
Bioequivalence Trial of Two Voriconazole Formulations of
200 mg.

Irene Dapía1,2, Irene García3, Jose Carlos Martinez3, Pedro Arias1,2, Pedro Guerra3, Lucía Díaz3, Alberto García3, Jair
Tenorio1,2, Elena Ramírez3, Gema Gordo1,2, Jesús Frías3, Pablo Lapunzina1,2, Antonio J Carcas3*, Alberto M Borobia3*.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00014

Copyright: © 2018 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: Irene Dapía, Irene García, Jose Carlos Martinez, Pedro Arias, Pedro Guerra, Lucía Díaz, Alberto García, Jair Tenorio, Elena
Ramírez, Gema Gordo, Jesús Frías, Pablo Lapunzina, Antonio J Carcas, Alberto M Borobia. Prediction Models for Voriconazole
Pharmacokinetics Based on Pharmacogenetics: An Exploratory Study Nested to a Bioequivalence Trial of Two Voriconazole
Formulations of 200 mg. IBJ Plus 2018 (S1):e0014 doi: 10.24217/2531-0151.18v1s1.00014.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Individualization of the therapeutic strategy for the oral antifungal voriconazole (VCZ) is highly important
for treatment optimization. This is due to its narrow therapeutic range and its wide interpatient variability in serum
concentrations, which are directly related to both VCZ efficacy and the occurrence of adverse drug reactions (ADRs). Part of
this variability is explained by genetic variation in genes coding enzymes involved in the metabolic pathway of VCZ. To date,
the European Clinical Pharmacogenetics Consortium and the US Food and Drug Administration include CYP2C19 as the only
major PGx biomarker in their dosing guidelines and protocols; however, the effect of other genes might be important for VCZ
dosing prediction. We developed an exploratory pharmacogenetics (PGx) study nested to a bioequivalence trial of two VCZ
formulations of 200 mg to identify new biomarkers related to VCZ pharmacokinetics (PK) that could be included in clinical
practice.

Materials and Methods: Forty-six healthy volunteers were included. Cmax, Tmax and AUC0-∞ were obtained from VCZ
PK analysis and Cmax and AUC0-∞ were adjusted to dose/weight administered. Molecular analysis was performed for the
selected SNPs among the CYP2C19, ABCB1, CYP3A4, CYP3A5, CYP2C9, POR, NR1I2, FMO3 genes using the custom SNParray
genotyping platform PharmArray®. After molecular and PK analysis, we first designed a Clinical Practice VCZ AUC0-
∞ prediction model based on CYP2C19 to be used as a reference model for comparison. We then performed a statistical
analysis in three steps to design new prediction models by the incorporation of additional biomarkers.

Results: Due to the strong influence of CYP2C19 on VCZ AUC0∞ heterogeneity we propose that its major effect might
be masking the influence of other metabolic enzymes and transporters in VCZ metabolism and that this influence could
be unmasked by a stratification of the study population into CYP2C19 phenotypic subgroups. We here propose three
different predictive models that improve the clinical practice model predictive rates by the incorporation of complementary
biomarkers to CYP2C19.

Conclusion: Due to the small sample size, further research is needed for cross-validation of the proposed models. However,
the CYP2C9 gene was proposed as a consistent biomarker for VCZ AUC prediction in addition to the well-established CYP2C19
gene.

e00015
Agranulocytosis after metamizole use: assessment of drug
causality with algorithm versus Lymphocyte Transformation
Test.

García García I1*, Díaz García L1, Rodríguez Mariblanca A1 , Hernández Zabala R1 , Queiruga Parada J1, Ramírez García
E1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00015

Copyright: © 2018 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: García García I, Díaz García L, Rodríguez Mariblanca A, Hernández Zabala R, Queiruga Parada J, Ramírez García E.
Agranulocytosis after metamizole use: assessment of drug causality with algorithm versus Lymphocyte Transformation Test. IBJ Plus
2018 (S1):e0015 doi: 10.24217/2531-0151.18v1s1.00015.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

INTRODUCTION: Metamizole is a non-narcotic analgesic/antipyretic widely used in some countries but prohibited in others
due to suspected risk of agranulocytosis. The mechanism of this side effect has not been completely clarified, but it is
generally accepted that it is immune-mediated. The lymphocyte transformation test (LTT) has been proposed as a diagnostic
method to determine if a drug is the causal agent of an immune-mediated drug reaction. We present a case of drug-induced
agranulocytosis in which Spanish Pharmacovigilance System probability algorithm of drug causality was applied and in vitro
LTT was performed.

CLINICAL CASE: A 30-year-old woman was admitted to our hospital with fever and severe weakness. Eighteen days before
she had received knee surgery and was prescribed metamizole (MTZ) along with dexketoprofeno (DK) alterned every four
hours. She was also on esomeprazole (ESO) and bemiparine treatment since the surgical procedure. Two days after the onset
of treatment she presented fever up to 37.8ºC which was followed by increasing weakness in the next days. On day 6 after
surgery, she was diagnosed tonsillitis and started therapy with amoxicillin-clavulanic. Body temperature values kept rising
and weakness remained after amoxicillin-clavulanic therapy had been completed. Not showing any clinical improvement,
she decided to come to our Hospital. Laboratory tests revealed severe neutropenia of 0.03×10e3/μL. Blood smear showed
no alterations of the red cells, scarce neutrophils and no evidence of dysplasia. Haematological, infectious diseases and
other alternative causes were excluded leading to drug suspicion. Possible causal drugs were discontinued and antibiotic
prophylaxis in addition to filgastrim were started. The algorithm of Spanish pharmacovigilance system was conducted to
MTZ, DK, ESO and bemiparine.

RESULTS: Applying the algorithm, MTZ, DK and ESO obtained 4 points, which implies “possible causality”. Bemiparine
pointed a “conditional causality” (2 points), and was consequently excluded. After drug withdrawal of (MTZ, DK, ESO) and
filgastrim therapy, she showed clinical and analytical improvement and was discharged from Hospital. We performed LTT to
the remaining suspected drugs. The test had a positive result for MTZ, and was negative for DK and ESO. A recommendation
was made to the patient and stated on clinical records not to rechallenge to metamizole or other pyrazolones again.
CONCLUSIONS: This is to our concern the first case of agranulocytosis by metamizole in which LTT has been performed. The
positive result elucidated metamizole as causal agent and supports the hypothesis of immunological mechanism for this
adverse reaction.

e00016
Drug induced cholestasis: assessment of drug causality with
algorithm versus Lymphocyte Transformation Test.

García García I1*, Rodríguez Mariblanca A1, Díaz García L1, Martínez De Soto L1, Hernández Zabala R1, Ramírez García E1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00016

Copyright: © 2018 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: García García I, Rodríguez Mariblanca A, Díaz García L, Martínez De Soto L, Hernández Zabala R, Ramírez García E. Drug
induced cholestasis: assessment of drug causality with algorithm versus Lymphocyte Transformation Test. IBJ Plus 2018 (S1):e0016
doi: 10.24217/2531-0151.18v1s1.00016.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

INTRODUCTION: Cholestatic drug-induced liver injury (DILI) can be a diagnostic challenge due to the varying clinical and
laboratory features, in addition to the extensive differential diagnosis. It can be divided into two categories regarding the
underlying pathogenic mechanism: direct hepatic injury caused by the drug itself or idiosyncratic. The majority of cases
are related to idiosyncratic reactions that develop independently of drug dose, route, or duration of administration. Its
pathogenesis is not yet clear, but among other hypothesis it includes immunological mechanisms. We present a case of drug
induced cholestasis in a patient with rechallenge to suspected drugs in which RUCAM (Roussel Uclaf Causality Assessment
Method) algorithm was applied and in vitro LTT (Lymphocyte Transformation Test) was performed.

CLINICAL CASE: A 88-year-old woman was admitted to our hospital presenting right hypochondrium pain. She was diagnosed
right kidney ischemia which was conservatively treated. Relevant history included cephalosporin allergy, hypertension,
hyperlipidaemia, type 2 diabetes diet controlled, cholelithiasis, arthrosis and sixth nerve ischemic neuropathy fully
recovered. Her medication included hydrochlorothiazide, atenolol and salicylic acid. Six years before she suffered atlas and
wrist fracture and was prescribed metamizole (MTZ), acetaminophen (ACP) and pantroprazole (PAN). Eighteen days later she
was diagnosed dissociated cholestasis, which was related to MTZ, ACP and PAN treatment. A recommendation was made not
to rechallenge to this drugs. The patient claimed not had been re-exposed.
During the current hospitalization she received atenolol, salicylic acyd, MTZ, ACP, omeprazole (OMZ), metoclopramide
(MTC), acenocumarol, amoxicillin clavulanic, enalapril, enoxaparin, furosemide and ranitidine. Thirteen days after the onset
of treatment alkaline phosphatase, alanil transferase and aspartate aminotransferase began to rise.

RESULTS: Alternative causes for dissociated cholestasis were excluded. Taking into account rechallenge, MTZ, ACP and OMZ
were classified as probable (7 points) by RUCAM algorithm. MTC obtained 0 points and was excluded. The rest of medication
was excluded considering dates of administration and rise of liver enzymes. After drug withdrawal of MTZ, ACP and OMZ
liver enzymes levels began to lower. LTT was performed to MTZ, ACP and OMZ with a positive result for MTZ, and with lower
values to OMZ. A recommendation was made to the patient and stated on clinical records not to rechallenge to pyrazolones
or proton pump inhibitors.

CONCLUSIONS: LTT is of great help to elucidate the culprit agent when several drugs are suspected, allowing us to make
more precise recommendations.

e00017
Drug induced hypersensitivity by simvastatin: peripheral
polyneuropathy, eosinophilia, fever and skin eruption.

García García I1*, Herrero Gil C2, Rodríguez Mariblanca A1, Monserrat Villatoro J1 , Rodríguez Dávila MA2, Ramírez
García E1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00017

Copyright: © 2018 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: García García I, Herrero Gil C, Rodríguez Mariblanca A, Monserrat Villatoro J, Rodríguez Dávila MA, Ramírez García E. Drug
induced hypersensitivity by simvastatin: peripheral polyneuropathy, eosinophilia, fever and skin eruption. IBJ Plus 2018 (S1):e0017
doi: 10.24217/2531-0151.18v1s1.00017.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

INTRODUCTION: Statins adverse effects are infrequent, and although myopathy is well known by clinicians, few attention
is paid to its other adverse reactions, which makes it difficult to establish drug causality. Through our Prospective
Pharmacovigilance Program from Laboratory Signals at Hospital, we detect abnormal laboratory values which indicate severe
reactions that are frequently related to the use of drugs. We are reporting a case of induced hypersensitivity caused by
simvastatin (SMV).

CLINICAL CASE: Laboratory Signals are reviewed each week; among others, peripheral eosinophilia >700x10e3/μL is detected
and prospectively evaluated. We identified eosinophilia in a 58 year old male patient. His medical history was remarkable for
hypertension, hyperlipidaemia under SMV 10 mg once a day treatment and type 2 diabetes mellitus diet controlled. In 2015
he received surgery and radiotherapy for a non-functional pituitary adenoma. Symptoms started in 2017 with two episodes
of urticaria followed by dissociated cholestasis, fever up to 39ºC, weakness, loss of weight and numbness in his right hand
with subsequent pain, cramps, paresthesia and hypoesthesia of his feet. Infectious or inflammatory diseases, malignant
diseases and other alternative diagnosis were excluded. Electromyography showed predominantly axonal sensory-motor
peripheral neuropathy. When a definitive diagnosis had not yet been established he started treatment with corticosteroids,
showing 24 hours later, improvement in constitutional symptoms and normalization of body temperature. Taking all this into
account SMV was established as suspected cause. We made contact with Internal Medicine Service and SMV therapy was
discontinued maintaining corticosteroids treatment.

RESULTS: On review three weeks after the withdrawal of SMV, he presented resolution of weakness and fever, along with
weight gain. Two months later he showed improvement of peripheral neuropathy symptoms, muscle cramps had reduced
and he had no pain, although numbness and sensation of pins and needles in his feet persisted.
Eosinophilia, urticaria, fever and neuropathy are described as side effects of SMV in the drug label and while a literature
review revealed several cases reported, no patient has been described as having all of them at the same time. The clinical
and laboratory features suggest a Drug reaction with eosinophilia and systemic symptoms (DRESS), with an atypical
presentation regarding that nervous system damage is an unusual manifestation of DRESS. A lymphocyte transformation test
will be performed to confirm SMV as causal agent.

CONCLUSIONS: Drug adverse reactions comprise a wide range of entities and we should always take account of medication
once alternative diagnosis are excluded.

e00018
A Cell-Permeable Peptide Corresponding to the Calmodulin-
Binding Domain of Grb7 Inhibits Proliferation and Migration
of A431 Cells.

Juan Alcalde1, María González-Muñoz1, Iván Madroñal1, Antonio Villalobo1*

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00018

Copyright: © 2018 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: Juan Alcalde, María González-Muñoz, Iván Madroñal, Antonio Villalobo. A Cell-Permeable Peptide Corresponding to the
Calmodulin-Binding Domain of Grb7 Inhibits Proliferation and Migration of A431 Cells. IBJ Plus 2018 (S1):e0018 doi: 10.24217/2531-
0151.18v1s1.00018.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Grb7 (growth factor receptor bound 7) is a mammalian adaptor protein that transduces signals from tyrosine
kinase receptors. This protein is overexpressed, together with ErbB2, in different human carcinomas. Grb7 is implicated
in cell migration, contributing to the invasiveness and metastatic capacity of tumour cells. Also, Grb7 controls cell
proliferation and tumour-associated angiogenesis. Our group demonstrated that calmodulin (CaM) binds to the proximal
region of the pleckstrin homology (PH) domain of human Grb7 in a Ca2+-dependent manner, comprising the sequence
243RKLWKRFFCFLRRS256 [1, 2]. A deletion mutant of Grb7, lacking its CaM-binding domain (CaM-BD), impairs the adhesion
and migratory capacity of transfected cells [3]. Moreover, the expression of this deletion mutant in rat C6 glioblastoma
cells strongly inhibited its proliferation, the growth of brain tumours derived from stereotaxically implanted tumour cells,
and tumour-associated angiogenesis [4]. Therefore, the CaM-BD of Grb7 could be a potential target to inhibit tumour cells
invasiveness and metastasis development [5].

Materials and Methods: Human carcinoma epidermoid A431 cells were used in all the experiments. Custommade synthesis
of peptides (>99% purity) with the amino acid sequence RKLWKRFFCFLRRS, and a derivative with a myristoyl group at
its N-terminus (Myr-RKLWKRFFCFLRRS) to facilitate cell internalization, were manufactured by Wuxi Nordisk Biotech Ltd.
(China). Cell migration was measured by artificial wound healingassays and time-lapse microscopy, and cell proliferation was
measured by Crystal Violet staining. The assays were done in the absence and presence of 10 nM EGF, and 20-50 μg/ml of
the different peptides, or 10-50 μM W-7 and W-12 (CaM antagonists).

Results: The addition of EGF first induced a 12-18 h lag phase in which the migration of A431 cells was arrested, and a
significant retraction of the border of the artificial wound was observed. Thereafter, the migration rate of the cells increased
several folds as compared to the rate of migration of cells in the absence of EGF. The RKLWKRFFCFLRRS peptide inhibited the
migration of A431 cells both in the absence and presence of EGF.
This inhibitory effect was more evident using the Myr-RKLWKRFFCFLRRS peptide. The high affinity CaM antagonist W-7, and
in lesser extent its low affinity analogue W-12, incremented the lag phase in which the migration of the cells was arrested
upon addition of EGF and decreased its subsequent migration rate. As previously demonstrated [6], EGF induced a significant
inhibition of cell proliferation in A431 cells, and both the RKLWKRFFCFLRRS and Myr-RKLWKRFFCFLRRS peptides further
inhibited the proliferative response.

Conclusion: The mechanism of action of the peptides under study could be due to the capacity to sequester CaM and/or to
the prevention of Grb7 dimerization, possibilities that are under study. These peptides, upon selected delivery to tumour
cells, could have potential therapeutic effects against cancer and metastasis development.

e00019
Pharmacogenetic testing in a suspected drug-induced
parkinsonism related with antipsychotic treatment: a case
report.

Díaz García L1, Dapia I2, Garcia Garcia I1, Arias P2, Borobia Alberto M1*, Carcas Antonio J1*

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00019

Copyright: © 2018 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 García L, Dapia I, Garcia Garcia I, Arias P, Borobia Alberto M, Carcas Antonio J. Pharmacogenetic testing in a suspected
drug-induced parkinsonism related with antipsychotic treatment: a case report. IBJ Plus 2018 (S1):e0019 doi: 10.24217/2531-
0151.18v1s1.00019.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The establishment of a pharmacogenetics unit with the intention of facilitating the integration of
pharmacogenetic testing into clinical practice is important to personalise treatments and clinical recommendations and to
carry out pre-emptive genotyping in risk populations. In our Clinical Pharmacogenetics Unit at La Paz University Hospital
we classified pharmacogenetics tests into three groups. One of these groups are drugs with well-known evidence but
without a well-defined protocol. In these cases, we evaluate a specific therapeutic problem and determine whether the
pharmacogenetics test is recommended. We presented the case of a patient with suspected drug-induced parkinsonism
(DIP) related with antipsychotic treatment.

Material and Methods: DIP is the most common movement disorder induced by antipsychotics and reduces quality of
life favouring non-compliance of treatments in patients with psychotic disorders with risk of morbidity and mortality.
We evaluated a patient being treated with several drugs (sertraline, paroxetine, risperidone, quetiapine, zuclopenthixol,
haloperidol and aripiprazole) who had developed parkinsonism one year earlier. Information about the pharmacogenetic
testing variants and their impact on drug response was gathered mostly from the variant and clinical annotations in
PharmGKB. We analysed single nucleotide polymorphism (SNP) associated with the metabolism and response of these drugs.
We selected CYP2D6 and CYP2C19 as the most important biomarkers involved (all drugs have metabolism by CYP2D6, except
sertraline which has metabolism mainly by CYP2C19). Pharmacogenetic test was performed and 21 SNP were analysed with
OpenArray technology (PharmArray®).

Results: Our patient presented the genotype CYP2C19*1/*17 (Rapid Metabolizer) and CYP2D6*4/*41 (Intermediate
Metabolizer). So, sertraline metabolism is increased and the metabolism of the other antipsychotic drugs are reduced and
the higher plasma concentrations may increase the probability of side effects, among them DIP.

Conclusions: These results could explain the higher risk of DIP observed in our patient due to the pharmacokinetic
interaction at the level of CYP450. We are aware of the limitations of the CYP2D6 technique analysis but in the case of our
patient it is important to consider these results in the evaluation of clinical symptoms due to the severity of the side effects
of this type in therapeutic efficacy and the optimal treatment of antipsychotic disorders. In addition, it is important to point
out the implementation of clinical pharmacogenetics programs in the clinical practice to carry out individualization of clinical
recommendations and pre-emptive genotyping in risk populations.

e00020
KV1.3 channel inhibition by indolic compounds in chronic
lymphocytic leukemia cells.

Baena-Nuevo M1,2*, Vera-Zambrano A1,2, Martinez-Laperche C3, Muñoz-Calleja C4, Buño I3, Zapata JM2,5, Perez-Chacon
G2,5 and Gonzalez T1,2,5

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00020

Copyright: © 2018 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, Vera-Zambrano A, Martinez-Laperche C, Muñoz-Calleja C, Buño I, Zapata JM, Perez-Chacon G, Gonzalez T.
KV1.3 channel inhibition by indolic compounds in chronic lymphocytic leukemia cells. IBJ Plus 2018 (S1):e0020 doi: 10.24217/2531-
0151.18v1s1.00020.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Chronic lymphocytic leukemia (CLL) is the most common leukemia in western countries and it is based on B
cell clonal expansion. This disease has no cure and the appearance of pharmacological resistance is very common. We have
previously identified indole-3-carbinol (I3C) and its main metabolite, 3,3’-diindolylmethane (DIM), as active compounds with
pharmacological activity against CLL. KV1.3 potassium channels are involved in B and T cells function controlling plasmatic
membrane potential, Ca2+ entry and cellular proliferation. Therefore, these channels could be a new therapeutic target
against CLL. Here, we have analysed if these indolic compounds can act, in part, by modulating the KV1.3 function.

Material and Methods: we assessed the activity and effect of the indolic compounds on KV1.3 channels in CLL cells that are
either sensitive or resistant to I3C cytotoxicity. Currents were registered by whole-cell patch-clamp. Statistical significance
was determined by t-Student test or by nonparametric Mann-Whitney test.

Results: The KV1.3 current magnitude on CLL cells correlated with their sensitivity to the cytotoxic effect of I3C; I3C resistant
CLL (IR-CLL) cells exhibiting ≈2.5-fold higher KV1.3 current amplitude than sensitive (IS-CLL) cells. Both I3C and DIM inhibited
KV1.3 current in CLL cells, DIM being ≈4-fold more potent than the parent compound. However, a non-cytotoxic compound,
indole-3-carboxylic acid (I3CA), did not inhibit the KV1.3 current.

Conclusions: IR-CLL cells exhibit greater KV1.3 current magnitude than IS-CLL cells, which can be due to the existence of
different canalosomes in these cells. The KV1.3 inhibitory effect of the indolic compounds tested, correlates with their
cytotoxic effect on CLL cells. Our results suggest that KV1.3 channel could be involved in CLL cells pharmacological resistance
mechanisms and its inhibition could be part of the cytotoxic effect of I3C and DIM, which open new venues to the treatment
of this disease.

e00021
Sirolimus monitoring in airway compromise vascular tumor:
a case report.

Rodríguez Mariblanca A1*, Díaz García L1, García García I1, Borobia A1, Carcas Sansuán AJ1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00021

Copyright: © 2018 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: Rodríguez Mariblanca A, Díaz García L, García García I, Borobia A, Carcas Sansuán AJ. Sirolimus monitoring in airway
compromise vascular tumor: a case report. IBJ Plus 2018 (S1):e0021 doi: 10.24217/2531-0151.18v1s1.00021.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Neonates with vascular anomalies causing airway compromise require early initiation of therapy. Treatment
options are limited. In the last years, sirolimus has shown to be an effective treatment. Correct dosing in these patients, who
have a reduced hepatic metabolism of sirolimus, is difficult. Standard dosing can cause supratherapeutic levels in patients
under 7 months of age.

Methods: Female preterm, born at 34 weeks in a gemelar delivery, with a policystic multicompartimental lymphatic
malformation with cervical airway compromise, required intubation. Sirolimus dosing in neonates is not clear, but
therapy with 0.8mg/m2 every 12 hours was started the day after the born. After five days the through concentration
was supratherapeutic (>90ng/ml), being the goal trough 5-15ng/ml. The dose was decreased to 0.1mg/kg once daily,
but supratherapeutic levels persisted for three more days. Sirolimus was discontinued to avoid toxicity. Moreover, the
pharmacogenetics study was carried out in order to pick up some mutation which could explain the supratherapeutic levels.

Results: Sirolimus levels started to go down the fourth day after the suspension. Ten days after the last dose, the through had
decreased until 9.21ng/ml. Estimated sirolimus half-life, calculated with the last concentration points after discontinuation,
was 55 hours, which is similar to the half-life observed previously. After that, sirolimus 0,05mg/kg/day was restarted,
remaining in the within the therapeutic range. The genotype determination of CYP3A4 (rs55785340, rs4646438, rs2740574,
rs35599367, rs28371759, rs138105638, rs67666821), CYP3A5 (rs776746, rs55965422, rs10264272, rs41303343, rs41279854),
CYP3A7 (rs45465393, rs11568824, rs45494802, rs45575938, rs45467892, rs11568825, rs11568826, rs45446698, rs55798860,
rs28451617, rs2257401, rs779179631) and CYP2C8 (rs11572080, rs10509681, rs1058930, rs11572103) was performed. It did
not show any mutation. Sirolimus is a substrate of CYP3A4, whose expression is very low in neonates, instead they express
CYP3A7. Maturation of hepatic enzyme composition occurs over the first 6 months of life, with rapid changes in the first
3-6 weeks. Some studies have modelled the pharmacokinetic of sirolimus in infants and children, demonstrating significant
variability attributed to body size and organ maturation processes, including bioavailability and hepatic metabolism.

Conclusion: The cause of the supratherapeutic sirolimus levels was probably the immaturity, as the genetic test showed a
normal metabolizer genotype. Important uncertainty exists about the appropriate dosage and schedule in the first postnatal
days/weeks. More studies are needed to clarify these questions and others like the efficacy and safety.

e00022
Cyclosporine-induced lymphoproliferative disorder in toxic epidermal necrolysis (net): a case report.

Rodríguez Mariblanca A1*, García García I1, Martínez de Soto L1, Díaz García L1, Ramírez García E1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00022

Copyright: © 2018 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:Rodríguez Mariblanca A, García García I, Martínez de Soto L, Díaz García L, Ramírez García E. Cyclosporine-induced
lymphoproliferative disorder in toxic epidermal necrolysis (net): a case report. IBJ Plus 2018 (S1):e0022 doi: 10.24217/2531-
0151.18v1s1.00022.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are a delayed-type hypersensitivity
reaction to drugs. They are medical emergencies and an early recognition and appropriate management is decisive. The
mortality rates have decreased, but may be as high as 20-30%. It is very important the immediate withdrawal of causative
drugs and prompt referral to a burn unit for specific supportive treatment. Several immunomodulatory agents are used in
the treatment of TEN, but evidence of their efficacy is limited, only retrospective observational open-label single-centre
studies are available. Glucocorticoids and intravenous immunoglobulin were not found to be superior to supportive care.
Cyclosporine is associated with a mortality reduction in a systematic review with a meta-analysis of TEN.

Description of the case: Female 58-year-old patient was moved to La Paz Burn Care Unit from Caceres, suspecting a NET.
Complementary studies didn’t show an infectious causative of TEN, a palmeatte hair lotion was considered the culprit drug.
Cyclosporine therapy 175mg every 12 hours was started the same day she was admitted to our centre. It was keep out for
9 days. Supratherapeutic cyclosporine levels were obtained after nine days (869,8ng/ml), being withdrawn. It was restarted
50mg every 12 hours 3 days later, remaining in goal trough until re-epithelialization. The genotype determination of CYP3A4
and CYP3A5 was performed in order to detect a possible mutation related with the metabolism of cyclosporine. It did not
show any mutation. After nine months since the patient was discharged, she was diagnosed with lymphoma. In the tests
done during the hospitalization, we found a positive PCR for Epstein-Barr virus (EBV) and several adenopathies in the body
computed tomography.

Results: Cyclosporine seems to be a promising therapy in TEN, but it has been well documented in the literature an
increased incidence of neoplastic disorders, specifically lymphoproliferative disorders. We have not found any other case
of cyclosporine induced-lymphoma in TEN because of the low TEN incidence, but cyclosporine has been widely used as
immunosuppressive agent for transplant recipients. Most lymphomas associated with cyclosporine have occurred within 1
year of initiating the drug, being lymph nodes one of the predominant sites. It is well known an association between EBV and
polyclonal lymphoproliferation.

Conclusion: The CT and EBV results suggest the lymphoma is related to cyclosporine therapy. The increased incidence
of lymphoproliferative disorders is related to high blood levels not to a specific cyclosporine dose. Acyclovir use and/or
decrease the dose of cyclosporine may decrease the proliferation of lymphomas.

e00023
CYP2C19 defines clopidogrel response in patients undergoing
percutaneous neurointervention procedure.

Miriam Saiz-Rodríguez1*, Daniel Romero-Palacián1, Carlos Villalobos-Vilda1, José Luis Caniego2, Carmen Belmonte1, 3,
Dora Koller1, Eduardo Bárcena2, María Talegón1, Francisco Abad-Santos1, 3, 4.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00023

Copyright: © 2018 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:Miriam Saiz-Rodríguez, Daniel Romero-Palacián, Carlos Villalobos-Vilda, José Luis Caniego, Carmen Belmonte, Dora
Koller, Eduardo Bárcena, María Talegón, Francisco Abad-Santos. CYP2C19 defines clopidogrel response in patients undergoing
percutaneous neurointervention procedure. IBJ Plus 2018 (S1):e0023 doi: 10.24217/2531-0151.18v1s1.00023

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Clopidogrel is a widely prescribed thienopyridine prodrug which inhibits platelet aggregation. It is prescribed
to prevent atherothrombotic and thromboembolic events in patients who are given a stent implant in carotid, vertebral or
cranial arteries. CYP2C19 is the most studied enzyme involved in clopidogrel metabolism. The most common CYP2C19 no
function polymorphisms (*2 and *3) have been associated with hyporesponse to clopidogrel, showing lower levels of the
active metabolite. On the contrary, the presence of the increased function allele (*17) has demonstrated enhanced platelet
inhibition and clopidogrel hyperresponse.

Methods: This observational retrospective study assessed antiplatelet response and clinical events after clopidogrel
treatment in patients who underwent percutaneous neurointervention, related to CYP2C19 metabolizer status (normal (NM),
intermediate/poor (IM-PM) and ultra-rapid (UM); inferred from *2, *3 and *17 allele determination by real-time PCR).

Results: One hundred twenty-three patients were analysed, of which 83% had cardiovascular risk factors. The most
common type of intervention was angioplasty with stent. According to the aggregation value, 58.7% of the patients were
responders to clopidogrel; moreover, 4.1% required dose reduction and 12.2% change of treatment. CYP2C19 IM-PM had
higher aggregation value (201.1 vs 137.6 NM, 149.4 UM, p<0.05) and lower response rate (37.5% vs. 69.8% NM, 61.1% UM),
along with higher treatment change rate (25% vs. 5.7% NM, 10.5% UM). Moreover, 20% of the patients suffered from a
subsequent clinical event. The highest ischemic events incidence occurred in NM (11.3% vs. 6.3% IM, 10.5% UM; p=0.358)
and haemorrhagic events in UM (13.2% vs. 0% IM and 3.8% NM; p=0.041). No differences found regarding ischemic events’
onset time, while haemorrhagic events’ frequency in UM was higher with shorter onset time (p=0.047). Additionally, 53% of
the patients were receiving concomitant treatment with proton-pump inhibitors (PPIs), which showed significantly higher
aggregation value when compared to those not receiving PPI concomitant treatment (178.1 vs. 134.4; p=0.009).

Conclusion: CYP2C19 no function and increased function alleles defined clopidogrel response. CYP2C19 genotyping and
platelet reactivity quantification help to determine whether a patient could be at risk of ischemic or haemorrhagic event.
CYP2C19 UM patients have increased bleeding risk after percutaneous neurointervention. Therapeutic recommendations
should include an alternative therapeutic option in IM-PM or UM patients.

e00024
Modulation of SIRT1 by IL-1β/NFκB signaling during
Acetaminophen-induced hepatotoxicity.

Patricia Rada1,2, Virginia Pardo1,2 , Maysa A. Mobasher1,2,3, Irma García-Martínez1,2, Laura Ruiz1,2, Águeda González-
Rodríguez4, Cristina Sanchez-Ramos1, Jordi Muntané5,6, Susana Alemany1, María Monsalve1, M. Pilar Valdecantos 1,2*,
Ángela M. Valverde 1,2*

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00024

Copyright: © 2018 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:Patricia Rada, Virginia Pardo, Maysa A. Mobasher, Irma García-Martínez, Laura Ruiz, Águeda González-Rodríguez, Cristina
Sanchez-Ramos, Jordi Muntané, Susana Alemany, María Monsalve, M. Pilar Valdecantos, Ángela M. Valverde. Modulation of
SIRT1 by IL-1β/NFκB signaling during Acetaminophen-induced hepatotoxicity. IBJ Plus 2018 (S1):e0024 doi: 10.24217/2531-
0151.18v1s1.00024.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The liver is the main organ in charge of drug catabolism and also the major site susceptible to drug injury.
Sirtuin 1 (SIRT1), a NAD+-dependent histone deacetylase, is a key player in liver physiology and a therapeutic target against
hepatic inflammation. In this study, we evaluated the role of SIRT1 in the pro-inflammatory context and oxidative stress
during acetaminophen (APAP)-mediated hepatotoxicity.

Material and Methods: SIRT1 expression was analyzed in APAP-induced liver failure in humans and mice. Hepatotoxicity was
assessed in wild-type and transgenic mice overexpressing SIRT1 (SIRT1 Tg) poisoned with APAP (300 mg/kg). Raw 264.7 and
peritoneal macrophages were treated with APAP and conditioned medium (CM) was added to mouse hepatocytes. siRNA
was used to reduce inflammatory mediators in hepatocytes.

Results: SIRT1 protein levels decreased in human and mouse livers following APAP overdose. SIRT1-Tg mice maintained
higher levels of SIRT1 upon APAP injection than wild-type mice and were protected against hepatotoxicity by modulation
of antioxidant systems and restrained inflammatory responses, with decreased oxidative stress, pro-inflammatory cytokine
mRNA levels, nuclear factor kappa B (NFκB) signaling, and cell death. Mouse hepatocytes stimulated with conditioned
medium of APAP-treated macrophages (APAP-CM) showed decreased SIRT1 levels; an effect mimicked by interleukin 1β
(IL1β), an activator of NFκB. This negative modulation was abolished by neutralizing IL1β in APAP-CM or silencing p65-NFκB
in hepatocytes. APAP-CM of macrophages from SIRT1-Tg mice failed to downregulate SIRT1 protein levels in hepatocytes.
In vivo administration of the NFκB inhibitor BAY 11-7082 preserved SIRT1 levels and protected from APAP-mediated
hepatotoxicity.

Conclusion: SIRT1 protein levels are downregulated by IL1β/NFκB signaling in APAP hepatotoxicity, resulting in inflammation
and oxidative stress. Thus, maintenance of SIRT1 during APAP overdose by inhibiting NFκB might be clinically relevant. Our
work evidenced the unique role of SIRT1 in APAP hepatoprotection by targeting oxidative stress and inflammation.

e00025
Neuroprotective effects of new compounds directed to PP2A,
a promising therapeutic target for Alzheimer’s disease.

Raquel L. Arribas1*, Rocío Lajarín Cuesta1, Cristóbal de los Ríos Salgado1,2

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00025

Copyright: © 2018 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 L. Arribas, Rocío Lajarín Cuesta, Cristóbal de los Ríos Salgado. Neuroprotective effects of new compounds directed to
PP2A, a promising therapeutic target for Alzheimer’s disease. IBJ Plus 2018 (S1):e0025 doi: 10.24217/2531-0151.18v1s1.00025.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Alzheimer’s disease (AD) is a progressive neurological disease that causes a progressive memory loss.
Main histopathological hallmarks of AD are senile plaques, and neurofibrillary tangles, generated by aggregation of the
microtubule associated protein tau. In addition to these pathological characteristics, there are other alterations, such as
oxidative stress or loss of cholinergic transmission, among many others. One of the most promising approaches in AD
treatment is to inhibit neurofibrillary degeneration produced by an abnormal tau hyperphosphorylation. In this sense,
serine/threonine phosphoprotein phosphatase 2A (PP2A) is the major phosphatase in brain that accounts for over 70% of
tau dephosphorylation. It has been shown that PP2A activity is significantly decreased in postmortem AD brains, partly due
to the increase of endogenous inhibitors that bind the PP2A catalytic subunit C.

Hypothesis: Okadaic acid (OA) is a natural toxin capable of inhibiting PP2A, leading to tau hyperphosphorylation. Our
working hypothesis is based on antagonizing the inhibitory effects of OA on PP2A by designing analogues of OA, capable to
bind to PP2A but without exerting inhibition, and thus preventing the attachment of endogenous inhibitors.

Material y Methods: The compounds synthesized, analogues to C19-C27 OA fragment, have been pharmacologically
studied, evaluating them in several in vitro models of AD, such as: tau hyperphosphorylation induced by OA, oxidative stress
caused by the toxic cocktail rotenone and oligomycin A or the glutamate induced excitotoxicity, all of them by the MTT
method. Furthermore, we have measured the cellular phosphatase activity by the pNPP method. In order to carry out these
objectives, we have used SH-SY5Y neuroblastoma cells and rat embryonic cortical neurons. Finally, we confirm our theory by
docking studies.

Results and conclusions: Our molecules are not toxic in SH-SY5Y cells or in cortical neurons, and they are capable of reducing
the neurotoxicity induced by OA. Some of them also showed good profile in the cell stress model induced by R/O A in SHSY5Y
cells, and in the glutamate-induced excitotoxicity in cortical neurons. The new compounds maintained the serine/
threonine phosphatase activity, depressed by the action of two PP2A inhibitors: OA and citostatin. Molecular docking studies
indicated that compound ITH12680 is capable of binding to PP2A similarly to OA, but it does not interact with the catalytic
site, thus confirming our starting hypothesis. Taking into account these results, we conclude that our compounds have
potential indication for the treatment of neurodegenerative diseases based on the maintenance of PP2A activity, which
prevents tau hyperphosphorylation.

e00026
Identification of genetic and molecular determinants
associated with cardiotoxicity by anthracyclines and taxanes
according to age.

Gómez Vecino A1,2,R, Corchado-Cobos R1,2,R, Blanco-Gómez A1,2,R, Prieto C3, Patino-Alonso C2,4, Galindo-Villardón P2,4,
Fraile-Martín S5, Rodrigues-Teixeira T5, García-Macías C5, Galvis-Jiménez M1,2,6, García-Sánchez A2,6, Isidoro-García
M2,6, García-Cenador MB2,8, García-Criado FJ2,8, García-Sancha N1,2, Holgado-Madruga M2,9, Sánchez Fernández PL 2,10,C,
Pérez Losada J1,2,C*

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00026

Copyright: © 2018 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: Gómez Vecino A, Corchado-Cobos R, Blanco-Gómez A, Prieto C, Patino-Alonso C, Galindo-Villardón P, Fraile-Martín S,
Rodrigues-Teixeira T, García-Macías C, Galvis-Jiménez M, García-Sánchez A, Isidoro-García M, García-Cenador MB, García-Criado FJ,
García-Sancha N, Holgado-Madruga M, Sánchez Fernández PL, Pérez Losada J. Identification of genetic and molecular determinants
associated with cardiotoxicity by anthracyclines and taxanes according to age. IBJ Plus 2018 (S1):e0026 doi: 10.24217/2531-
0151.18v1s1.00026.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Cardiotoxicity due to anthracyclines (CDA) is a very common problem in cancer patients, with great repercussion on
their quality of life, which limits chemotherapy treatment and has consequences in the final prognosis of the oncologic disease
itself. The susceptibility and degree of cardiotoxicity by anthracyclines is very heterogeneous among patients, and who will suffer
this complication is unknown. CDA is a complex trait, thus follows a model of quantitative genetics, whose polygenic component
is mostly unidentified. In addition, as a complex trait, CDA heterogeneity is explained by the variability among subphenotypes that
would participate in its pathogenesis. Thus, anthracyclines exert their toxicity through DNA damage, so that among these subphenotypes
would be the molecular pathways involved in the response to it, and a series of signaling pathways that promote and
others that protect from that heart damage anthracyclines have a pro-genotoxicity effect. Differences in these pathways with the
genetic variants linked with them could contribute to different susceptibility to CDA among individuals.

Material and Methods: we identified the genetic and molecular determinants of cardiotoxicity in a simplified model of controlled
genetic and phenotypic heterogeneity, generated by a backcross of two mouse strains of divergent phenotypic behavior, FVB and
C57BL/6. We evaluated cardiac damage at histopathological level and also quantified different subphenotypes such as signaling
pathways associated with cardiac damage and protection, genotoxicity pathways, TGFβ levels, telomere length, and expression of
miRNAs in the myocardium.

Results: We quantified anthracycline cardiotoxicity in a heterogeneous cohort of mice with breast cancer generated by a backcross.
Cardiotoxicity was higher in old mice, was higher in the combined treatment with taxanes, was higher in the subendocardial zone
and was influenced by the genetic background. We have identified multiple QTL associated with CDA in these different conditions
studied. Differences in the grade of anthracycline cardiotoxicity at the histopathological level were accompanied by differences
in the molecular levels in the myocardium of different molecular components of the pathways of response to damage at DNA,
signaling pathways, miRNAs and telomere length. QTLs associated with these subphenotypes help to define CDA variability. Lastly,
we have also defined CDA by multivariate models.

Conclusion: The identification of genetic and molecular factors responsible for the increased risk of CDA will contribute to a better
understanding of their pathophysiology, which could lead to new approaches to predict, prevent and treat this serious complication
of chemotherapy.

e00027
Using omics approaches to develop a biomarker signature
for anti-psychotic drug toxicity.

Sangeetha Bupalan1, Afshin Samali2, Adrienne M. Gorman2

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00027

Copyright: © 2018 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:Sangeetha Bupalan, Afshin Samali, Adrienne M. Gorman. Using omics approaches to develop a biomarker signature for antipsychotic
drug toxicity. IBJ Plus 2018 (S1):e0027 doi: 10.24217/2531-0151.18v1s1.00027.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Schizophrenia (SCZ) is a chronic brain disorder with symptoms of hallucinations, delusions, disturbed behavior
and emotional and cognitive impairment. These symptoms are managed by administration of antipsychotic drugs (APDs),
either as a single APD or a combination of different APDs depending on the individual. It is always recommended to take a
maintenance dose of APD even after the reduction of SCZ symptoms which makes it a lifelong treatment. APD treatment
is currently known to be the only effective treatment against SCZ. However, they are observed to trigger metabolic
dysfunctions such as insulin resistance, obesity, coronary heart diseases and dyslipedemia in SCZ patients at the later stage
during APD treatment. The main aim of the project is to identify a biomarker signature that could be detected in blood to
diagnose the metabolic dysfunction due to APD administration at an early stage. The talk will provide an insight into omic
approaches for biomarker discovery and a worked example of these approaches using relevant existing data sets from public
repositories.

Materials and Methods: The public repository Array Express (AE) (https://www.ebi.ac.uk/arrayexpress/) was used to shortlist
the datasets from transcriptomic studies of primary human hepatocytes (PHH) that has been treated with any antipsychotic
drug. R studio was used to process the data and shortlist genes that had a minimum fold change of 2. Bioinfominer online
tool (https://bioinfominer.com/) was used for Gene ontology enrichment analysis.

Results and conclusion: We found a microarray dataset of PHH that has been treated with Chlorpromazine (APD) from
AE (AE id: E-MTAB-1747). The deregulated gene list from PHH that were treated with CPZ was significantly grouped under
inflammatory and immune response from the gene ontology term enrichment analysis. Since the gene products of the
inflammatory/immune response are mostly secreted, they are likely to be detected in blood which makes them potential
biomarker candidates. However, these candidates were identified from PHH isolated from a single donor, so more work
would be required to confirm them as valid biomarkers. Despite that caveat, the gene list can be considered as a useful
reference list for future experiments.

e00028
Impaired signaling of Transcription factors NF-ƘB and
NRF2 in CX3CR1-deficient microglia leads to altered
neuroinflammation and phagocytosis: implications in
tauopathies.

Sara Castro-Sánchez1*, Ángel J. García-Yagüe1, Marcos Galán-Ganga1, Tresa López-Royo1, Sebastian Kügler2, Isabel
Lastres-Becker1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00028

Copyright: © 2018 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: Sara Castro-Sánchez, Ángel J. García-Yagüe, Marcos Galán-Ganga, Tresa López-Royo, Sebastian Kügler, Isabel Lastres-Becker.
Impaired signaling of Transcription factors NF-ƘB and NRF2 in CX3CR1-deficient microglia leads to altered neuroinflammation and
phagocytosis: implications in tauopathies. IBJ Plus 2018 (S1):e0028 doi: 10.24217/2531-0151.18v1s1.00028.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: TAU protein aggregation is the main characteristic of a group of age-related neurodegenerative diseases called
tauopathies. One of the key hallmarks associated with neurodegeneration is the presence of low-grade chronic inflammation,
indicating a crosstalk between damaged neuron and glial cells. Previously we have shown that TAUP301L overexpressing neurons
released CX3CL1 that activates anti-inflammatory NRF2 signalling in microglial cells in vitro and in vivo. However, the potential role
of CX3CR1 in the context of TAU pathology and its implication neuroinflammation are poorly described.

Material and Methods: Pro-inflammatory markers in immortalized microglia cells (IMG) treated with CX3CL1 were analysed.
We also studied mRNA expression levels of NF-ƘB, anti-inflammatory Nrf2 signalling and TAM receptors (TYRO3, AXL and
MER) in CX3CR1-deficient primary microglia cells. Finally, the effect of sulforaphane (SFN), a NRF2 inducer, was examined on
neuroinflammation in Cx3cr1+/+ and Cx3cr1-/- mice stereotaxically injected in the right hippocampus with an adeno-associated
vector expressing human TAUP301L and treated daily with SFN (50mg/kg, i.p) during three weeks.

Results: In this study we show that CX3CL1 treatment induced NF-ƘB-p65 and pro-inflammatory cytokines expression. On the other
hand, CX3CR1-deficient primary microglia cells present impaired NF-ƘB mRNA expression levels and decreased anti-inflammatory
NRF2 signalling, suggesting a dual role of CX3CL1/CX3CR1 axis in neuroinflammation. Lack of CX3CR1 microglia exhibit decreased
mRNA expression levels of TAM receptors (TYRO3, AXL and MER) that functionally results in a deficiency in phagocytosis. SFN
treatment reverses astrogliosis in Cx3cr1+/+ and Cx3cr1-/-, whereas at microglial level we did not see any improvement in the
Cx3cr1-/- mice.

Conclusions: These findings suggest that CX3CR1-NRF2 axis activation is essential in the modulation of microglial activation
associated with tauopathy, and that the associated polymorphisms of CX3CR1 must be taken into account in the design of
pharmacological strategies for the treatment of taupathies.

e00029
Antitumoral effects of Hispanolone derivatives in
glioblastoma cells.

Vanesa Sánchez-Martin1,2*, Beatriz de las Heras2, Sonsoles Hortelano1

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00029

Copyright: © 2018 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: Vanesa Sanchez-Martin, Beatriz de las Heras, Sonsoles Hortelano. Antitumoral effects of Hispanolone derivatives in
glioblastoma cells. IBJ Plus 2018 (S1):e0029 doi: 10.24217/2531-0151.18v1s1.00029.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: Glioblastoma multiforme (GBM) constitutes the most frequent and aggressive primary brain tumor in adults.
Even vast efforts have been made to develop effective treatments, including the combination of surgery, chemotherapy,
and radiotherapy; the prognosis for the patients is extremely poor, with mean survival of about 14 months. Therefore,
there is still an urgent need for novel and effective therapies for treating these tumors. On this issue, natural product-based
molecules represent interesting therapeutic alternatives. Hispanolone derivatives have been shown to induce apoptosis in
several human cancer cells. Nevertheless, the activity of these compounds against glioblastoma cells remains unclear. In the
present study, we aimed to investigate the effects of a hispanolone derivative, α-hispanolol, on proliferation as well as on the
migration and invasion of human glioblastoma cells.

Material y Methods: Cytotoxicity of α-hispanolol was determined against glioblastoma cancer cells, using the MTT assay.
Flow cytometry was performed to analyze the changes in cell cycle and apoptotic effect, if any. Cells were also studied for
their wound healing and invasive potential as well as for Western blotting of apoptotic genes.

Results: Our results show that α-hispanolol induced cell morphological changes and decreased the cell viability of U87 and
U373 cells in a dose- and time-dependent manner. This inhibitory effect was found to be linked to arrest of cell cycle at the
G0/G1 phase, along with induction of apoptosis and accumulation in the sub-G1 phase. Moreover, α-hispanolol also induced
caspases activities and increased the pro-apoptotic protein (Bax and Bid), and inhibited the anti-apoptotic proteins (Bcl-2 and
Bcl-xl) in GBM cells. In addition, α-hispanolol displayed an inhibitory effect on the migration and invasion of glioma cells by
inhibiting the expression and activity of MMPs.

Conclusion: Our findings suggest that α-hispanolol may have therapeutic potential for the treatment of glioblastomas.

e00030
Treatment with Brusatol inhibits oncogenic transcription
factors NRF2 and TAZ, reducing glioma stem cells survival.

Diego Lastra1, Marta Pajares1, Natalia Robledinos-Antón1, Ricardo Gargini2, Antonio Cuadrado1, Maribel Escoll1.

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00030

Copyright: © 2018 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, Marta Pajares, Natalia Robledinos-Antón, Ricardo Gargini, Antonio Cuadrado, Maribel Escoll. ITreatment with
Brusatol inhibits oncogenic transcription factors NRF2 and TAZ, reducing glioma stem cells survival. IBJ Plus 2018 (S1):e0030 doi:
10.24217/2531-0151.18v1s1.00030.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Glioblastomas are nervous system solid tumours with poor prognosis and hard treatment, since they present glioma stem cells
(GSCs), a subpopulation of cells responsible for resistance to chemotherapy and radiotherapy, which lead to tumour recurrence.
NRF2 and TAZ transcription factors are involved in tumour development, but their role in regulation of cancer stem cells (CSCs)
and their possible crosstalk have not been explored. Our studies demonstrate a correlation between NRF2 and TAZ expression and
the prognosis of patients with glioma. Knock-down of NRF2 in human glioblastoma explants and glioblastoma cell lines, decreased
messenger RNA and protein levels of TAZ and its transcriptional signature. In addition, we identified functional NRF2- binding
sites (Antioxidant Response Element, ARE) in the promoter region of the TAZ encoding gene (WWTR1). Besides, NRF2 knock-down
reduces cell growth both in vitro and in vivo, being rescued with TAZ overexpression.
Consequently, we conclude that at least part of the tumorigenic capacity of NRF2 is TAZ-dependent. Following this evidence, we
have used NRF2 inhibitor Brusatol. This treatment reduced TAZ levels and GSCs growth in the same way as NRF2 knock-down.
Because of this, we propose NRF2 and TAZ pharmacological inhibition as a novel glioblastoma therapy.

e00031
Measurement of polymorphic P-glycoprotein activity in cell
cultures: a review.

Pablo Zubiaur1*, Miriam Saiz-Rodríguez1, Dora Koller1, María C Ovejero-Benito1 , Francisco Abad-Santos1

*Corresponding author:
Pablo Zubiaur, Clinical Pharmacology Department, Hospital Universitario de la Princesa, Instituto Teófilo Hernando, Universidad
Autónoma de Madrid (UAM), Instituto de Investigación Sanitaria la Princesa (IP), Madrid, Spain. E-mail: pablo.zubiaur@salud.madrid.org

Published April 10, 2018.

DOI: 10.24217/2531-0151.18v1s1.00031

Copyright: © 2018 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: Pablo Zubiaur, Miriam Saiz-Rodríguez, Dora Koller, María C Ovejero-Benito, Francisco Abad-Santos. Measurement of
polymorphic P-glycoprotein activity in cell cultures: a review. IBJ Plus 2018 (S1):e0031 doi: 10.24217/2531-0151.18v1s1.00031.

Edited: Madrid, España.

Editor: Alberto M. Borobia Pérez.

Abstract

Introduction: The P-glycoprotein (P-gp) is an efflux pump widely expressed in the organism that exports xenobiotic compounds
out of the tissue where it is expressed. It plays a central role in the Blood-Brain Barrier permeability, being responsible of Central
Nervous System side-effects or ineffectiveness of many drugs. Several single-nucleotide polymorphisms (SNPs) in ABCB1 (the gene
encoding for P-gp) have been identified. The most relevant ones, C3435T, C2677T/A and C1236T have been associated with variable
pharmacokinetic parameters in healthy volunteers that received single oral doses of antidepressants and antipsychotics. There is no
consensus regarding the in vivo effect they have. Here, we compile relevant information in order to simplify the understanding of
materials, methods and cell lines classically used to assess polymorphic P-gp activity in in vitro cell culture models.

Methods: A comprehensive research of the studies performed in this regard has been accomplished. More than 389 articles have
been reviewed, corresponding to the topics “ABCB1 polymorphisms”, “assessment of P-gp function” and “P-gp expression in cell
cultures” published in PubMed Search Engine.

Results: Twenty-four articles have been summarised and classified. Site-directed mutagenesis has been acknowledged as a
convenient approach to obtain cell lines expressing mutant P-gp. Ten different techniques have been identified as key in the
assessment of P-gp function: Transfection; Western Blot; Flow Cytometry; Transcriptional Analysis; TEER measurements; Calcein-
AM, Rhodamine-123 or Radioactivity based accumulation and transport assays; Transwell® inserts; MTT viability assays.

Conclusion: Site-directed mutagenesis performed in plasmids that contain wild-type ABCB1, followed by transfection of the plasmid
into cells (HeLa, Caco-2 cells) may lead to cell lines expressing P-gp with the SNPs of interest (C3435T, C2677T/A and C1236T).
Assessment of P-gp function may be accomplished by Calcein-AM or Rhodamine-123 accumulation assays. The actual effect of
these SNPs in P-gp on antidepressants or antipsychotics efflux through membranes could be assessed by Transwell® insert transport
assays.