Design of cyclic peptides as novel inhibitors of ICOS/ICOSL interaction.

Compared to small molecules and antibodies, cyclic peptides exhibit unique biochemical and therapeutic attributes in the realm of pharmaceutical applications. The interaction between the inducible costimulator (ICOS) and its ligand (ICOSL) plays a key role in T-cell differentiation and activation. ICOS/ICOSL inhibition results in a reduction in the promotion of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. Herein, we implement the computational cPEPmatch approach to design the first examples of cyclic peptides that inhibit ICOS/ICOSL interaction. The top cyclic peptide from our approach possessed an IC50 value of 1.87 ± 0.15 µM as an ICOS/ICOSL inhibitor and exhibited excellent in vitro pharmacokinetic properties as a drug candidate. Our work will lay the groundwork for future endeavors in cancer drug discovery, with the goal of developing cyclic peptides that target the ICOS/ICOSL interaction.

Gut Microbial-Derived Metabolites as Immune Modulators of T Helper 17 and Regulatory T Cells.

The gut microbiota and its derived metabolites greatly impact the host immune system, both innate and adaptive responses. Gut dysbiosis and altered levels of microbiota-derived metabolites have been described in several immune-related and immune-mediated diseases such as intestinal bowel disease, multiple sclerosis, or colorectal cancer. Gut microbial-derived metabolites are synthesized from dietary compounds ingested by the host or host-produced metabolites, and additionally, some bacterial products can be synthesized de novo. In this review, we focus on the two first metabolites families including short-chain fatty acids, indole metabolites, polyamines, choline-derived compounds, and secondary bile acids. They all have been described as immunoregulatory molecules that specifically affect the adaptive immune system and T helper 17 and regulatory T cells. We discuss the mechanisms of action and the consequences in health and diseases related to these gut microbial-derived metabolites. Finally, we propose that the exogenous administration of these molecules or other compounds that bind to their immunoregulatory receptors in a homologous manner could be considered therapeutic approaches.

NLRP3 inflammasome as prognostic factor and therapeutic target in primary progressive multiple sclerosis patients.

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Expression of Bone Morphogenetic Proteins in Multiple Sclerosis Lesions.

Bone morphogenetic proteins (BMPs) are secreted proteins that belong to the transforming growth factor-ß superfamily. In the adult brain, they modulate neurogenesis, favor astrogliogenesis, and inhibit oligodendrogenesis. Because BMPs may be involved in the failure of remyelination in multiple sclerosis (MS), we characterized the expression of BMP-2, BMP-4, BMP-5, and BMP-7; BMP type II receptor (BMPRII); and phosphorylated SMAD (pSMAD) 1/5/8 in lesions of MS and other demyelinating diseases. A total of 42 MS lesions, 12 acute ischemic lesions, 8 progressive multifocal leukoencephalopathy lesions, and 10 central nervous system areas from four nonneuropathological patients were included. Lesions were histologically classified according to the inflammatory activity. The expression of BMP-2, BMP-4, BMP-5, BMP-7, BMPRII, and pSMAD1/5/8 was quantified by immunostaining, and colocalization studies were performed. In MS lesions, astrocytes, microglia/macrophages, and neurons expressed BMP-2, BMP-4, BMP-5, and BMP-7; BMPRII; and pSMAD1/5/8. Oligodendrocytes expressed BMP-2 and BMP-7 and pSMAD1/5/8. The percentage of cells that expressed BMPs, BMPRII, and pSMAD1/5/8 correlated with the inflammatory activity of MS lesions, and changes in the percentage of positive cells were more relevant in MS than in other white matter-damaging diseases. These data indicate that BMPs are increased in active MS lesions, suggesting a possible role in MS pathogenesis.

Bone morphogenetic proteins in multiple sclerosis: Role in neuroinflammation.

Bone morphogenetic proteins (BMPs) are growth factors that represent the largest subgroup of signalling ligands of the transforming growth factor beta (TGF-ß) superfamily. Their participation in the proliferation, survival and cell fate of several cell types and their involvement in many pathological conditions are now well known. BMP expression is altered in multiple sclerosis (MS) patients, suggesting that BMPs have a role in the pathogenesis of this disease. MS is a demyelinating and neurodegenerative autoimmune disorder of the central nervous system (CNS). MS is a complex pathological condition in which genetic, epigenetic and environmental factors converge, although its aetiology remains elusive. Multifunctional molecules, such as BMPs, are extremely interesting in the field of MS because they are involved in the regulation of several adult tissues, including the CNS and the immune system. In this review, we discuss the extensive data available regarding the role of BMP signalling in neuronal progenitor/stem cell fate and focus on the participation and expression of BMPs in CNS demyelination. Additionally, we provide an overview of the involvement of BMPs as modulators of the immune system, as this subject has not been thoroughly explored even though it is of great interest in autoimmune disorders. Moreover, we describe the data on BMP signalling in autoimmunity and inflammatory diseases, including MS and its experimental models. Thus, we aim to provide an integrated view of the putative role of BMPs in MS pathogenesis and to open the field for the further development of alternative therapeutic strategies for MS patients.

Catalytic Asymmetric Diamination of Styrenes.

An enantioselective catalytic vicinal diamination of styrenes is reported, which proceeds under entirely intermolecular reaction control. It relies on a chirally modified aryliodine(I) catalyst and proceeds within an iodine(I/III) manifold with conventional 3-chloroperbenzoic acid as a terminal oxidant. An environmentally benign solvent combination not only adds to the attractiveness of the process but also slows down the rate of the undesired background reaction. A total of 30 examples are presented, which consistently provide high enantiomeric excesses in the range 91-98%.

Heavy metals fractionation and desorption in pine bark amended mine soils.

The European Community Bureau of Reference method (BCR) was used for evaluating the effects of pine bark amendment (0, 24 and 48 g kg-1) and ageing (1 and 30 days) on Cd, Cu, Ni, Pb and Zn fractionation, on samples from an acid mine soil. In addition, the stirred flow chamber technique was applied for analyzing heavy metals desorption from the unamended and pine bark amended mine soil. When the unamended soil were not subjected to ageing, the added heavy metals were mainly accumulated as soluble fraction (>90% for Cd, Ni and Zn; 71% for Cu; and 45% for Pb). Pine bark amendment and ageing had little effect on Cd, Ni and Zn fractionation, whereas important changes were detected for Cu and Pb in response to both pine bark amendment and ageing (decrease in the soluble fractions, and increase in less mobile fractions). Desorption experiments showed that both pine bark amendment and ageing decreased heavy metals release from the mine soil. The results of this study indicate that pine bark amendment could be used to increase heavy metals retention (especially in the case of Cu and Pb) in acid mine soils, thus reducing the risks of metal transfer to uncontaminated environmental zones.

Valorization of biosorbent obtained from a forestry waste: Competitive adsorption, desorption and transport of Cd, Cu, Ni, Pb and Zn.

Bark from Pinus pinaster is one of the most abundant forestry wastes in Europe, and among the proposed technologies for its reutilization, the removal of heavy metals from wastewater has been gaining increasing attention. In this work, we have studied the performance of pine bark for heavy metal biosorption on competitive systems. Pb, Cu, Ni, Zn and Cd sorption and desorption at equilibrium were studied in batch experiments, whereas transport was studied in column experiments. Batch experiments were performed adding simultaneously different concentrations (0.08-3.15mM) of two or more metals in solution to pine bark samples. Column experiments were performed with 10mM solutions of two metals or a 5mM solution of the five metals. In general, the results under competitive conditions were different to those obtained in monoelemental experiments. The multi-metal batch experiments showed the adsorption sequence Pb≈Cu>Cd>Zn>Ni for lower metal doses, Pb>Cu>Cd>Zn>Ni for intermediate doses, and Pb>Cu>Cd≈Zn≈Ni for high metal doses. Desorption followed the sequence PbCu>Zn>Cd>Ni. The presence of a second metal affected the transport of all the elements studied except Pb, and confirmed the strong influence of Pb and Cu on the retention of the other metals. These results can help to appropriately design decontamination systems using this forestry waste.

Metabolic responses of CHO cells to limitation of key amino acids.

Chinese hamster ovary (CHO) cells are the predominant host for production of therapeutic glycoproteins. In particular, the glutamine-synthetase (GS) expression system has been widely used in the biopharmaceutical industry for efficient selection of high-yielding clones. However, much remains unclear on how metabolic wiring affects culture performance. For instance, asparagine and serine have been observed to be the largest nitrogen sources taken up by GS-CHO cells, but their roles in biosynthesis and energy generation are poorly understood. In this work, a comprehensive profiling of extracellular metabolites coupled with an analysis of intracellular label distributions after 1-(13) C-pyruvate supplementation were used to trace metabolic rearrangements in different scenarios of asparagine and serine availability. The absence of asparagine in the medium caused growth arrest, and was associated with a dramatic increase in pyruvate uptake, a higher ratio of pyruvate carboxylation to dehydrogenation and an inability for de novo asparagine synthesis. The release of ammonia and amino acids such as aspartate, glutamate, and alanine were deeply impacted. This confirms asparagine to be essential for these GS-CHO cells as the main source of intracellular nitrogen as well as having an important anaplerotic role in TCA cycle activity. In turn, serine unavailability also negatively affected culture growth while triggering its de novo synthesis, confirmed by label incorporation coming from pyruvate, and reduced glycine and formate secretion congruent with its role as a precursor in the metabolism of one-carbon units. Overall, these results unfold important insights into GS-CHO cells metabolism that lay a clearer basis for fine-tuning bioprocess optimization.

Inhibitors of Immune Checkpoints: Small Molecule- and Peptide-Based Approaches.

The revolutionary progress in cancer immunotherapy, particularly the advent of immune checkpoint inhibitors, marks a significant milestone in the fight against malignancies. However, the majority of clinically employed immune checkpoint inhibitors are monoclonal antibodies (mAbs) with several limitations, such as poor oral bioavailability and immune-related adverse effects (irAEs). Another major limitation is the restriction of the efficacy of mAbs to a subset of cancer patients, which triggered extensive research efforts to identify alternative approaches in targeting immune checkpoints aiming to overcome the restricted efficacy of mAbs. This comprehensive review aims to explore the cutting-edge developments in targeting immune checkpoints, focusing on both small molecule- and peptide-based approaches. By delving into drug discovery platforms, we provide insights into the diverse strategies employed to identify and optimize small molecules and peptides as inhibitors of immune checkpoints. In addition, we discuss recent advances in nanomaterials as drug carriers, providing a basis for the development of small molecule- and peptide-based platforms for cancer immunotherapy. Ongoing research focused on the discovery of small molecules and peptide-inspired agents targeting immune checkpoints paves the way for developing orally bioavailable agents as the next-generation cancer immunotherapies.

[Effects of the COVID-19 pandemic on the mental health of patients with pre-existing psychopathology]. / Efectos de la pandemia de la COVID-19 en la salud mental de pacientes con psicopatología previa.

OBJECTIVE: Several studies stated greater impact on mental health among psychiatric population during the first wave of the COVID-19 pandemic. The aim of this paper was to study the pandemic effects more than a year after its outbreak on the mental state of adult patients with pre-existing psychopathology attending a Mental Health Center in Madrid (Spain). METHODS: A cross-sectional observational study was carried out with a sample of fifty-eight patients using a questionnaire that collected different descriptive variables. Results of the PHQ-9 Depression Scale, the GAD-7 Anxiety Scale, the IES-R Impact of Event Scale-Revised, the Clinical Global Impression scale CGI-GI, and the assessment of the professionals responsible for the patients about their clinical state were also collected. A descriptive analysis and binary logistic regression models were performed. RESULTS: There was a prevalence of 70% anxiety, 76% depression and 57% stress due to a stressful event more than one year after the start of the pandemic. Likewise, associations were found between the symptoms and certain clinical, mediating variables and gender. CONCLUSIONS: The psychological state of patients with pre-existing psychopathology attended at the Mental Health Center is negatively affected by the COVID-19 pandemic up to a year and a half after its onset.

OBJETIVO: Numerosos estudios han confirmado que las personas con un diagnóstico psiquiátrico se vieron más afectadas en su salud mental que la población general en una primera fase de la pandemia de la COVID-19. El objetivo de este trabajo fue estudiar los efectos de la pandemia, más de un año después de su inicio, en el estado psíquico de pacientes adultos con psicopatología previa de un Centro de Salud Mental (CSM) en Madrid. METODOS: Se realizó un estudio observacional transversal en una muestra de cincuenta y ocho pacientes mediante un cuestionario que recogía diferentes variables descriptivas. Se administraron la Escala de Depresión PHQ-9, la Escala de Ansiedad GAD-7, la Escala Revisada de Impacto del Estresor EIE-R, la Escala de Impresión Clínica Global CGI-GI y la valoración de los facultativos responsables de los pacientes sobre su estado clínico. Se realizó un análisis descriptivo y se aplicaron modelos de regresión logística binaria. RESULTADOS: Hubo una prevalencia de 70% de ansiedad, 76% de depresión y 57% de estrés por evento estresante más de un año después de iniciarse la pandemia. Asimismo, se encontró asociación de los síntomas clínicos con determinadas variables clínicas, con variables mediadoras y con el género. CONCLUSIONES: El estado psíquico de los pacientes con psicopatología previa atendidos en el CSM se ve negativamente afectado por la pandemia de la COVID-19 hasta año y medio después de su inicio.

Impact of universal use of a hyperangulated videolaryngoscope as the first option for all intubations in the ICU: A prospective before-after study.

BACKGROUND: Tracheal intubation in ICU is associated with high incidence of difficult intubations. The study aimed to investigate whether the "universal" use of a hyperangulated videolaryngoscope would increase the frequency of "easy intubation" in ICU patients compared to direct laryngoscopy. METHODS: A prospective before-after study was conducted. The pre-interventional period (36 months) involved tracheal intubations using direct laryngoscopy as the first intubation option. In the interventional period (18 months) a hyperangulated videolaryngoscope was the first intubation option. The primary outcome was the percentage of patients with "easy intubation" defined as intubation on the first attempt and easy laryngoscopy (modified Cormack-Lehane glottic view of I-IIa). Secondary outcomes included difficult laryngoscopy, operator technical difficulty, and complications. RESULTS: We enrolled 407 patients, 273 in non-interventional period, and 134 in interventional period. Tracheal intubation in the interventional period was associated with higher incidence of "easy intubation" (92.5%) compared with the non-interventional period (75.8%); P < 0.001)). Glottic visualization improved in the interventional period, with a reduced incidence of difficult laryngoscopy (1.5% vs. 22.5%; P < 0.001). The proportion of first-success rate intubation was 92.5% in the interventional period, and 87.8% in the non-interventional period (P = 0.147). Moderate and severe technical difficulty of intubation reported decreased in the interventional period (6% vs. 17.6%; P < 0.001). There was no significant difference between both periods in the incidence of complications. CONCLUSION: "Universal" use of hyperangulated videolaryngoscopy for tracheal intubation in patients admitted in ICU improves the percentage of easy intubation compared to direct laryngoscopy.

Effect of early vs. delayed extubation on functional outcome among patients with acute ischemic stroke treated with endovascular thrombectomy under general anesthesia: the prospective, randomized controlled EDESTROKE trial study protocol.

BACKGROUND: Recent meta-analyses and randomized studies have shown that among patients with acute ischemic stroke undergoing endovascular thrombectomy, general anesthesia with mechanical ventilation is associated with better functional status compared to local anesthesia and sedation, and they recommend its use. But once the procedure is completed, when is the optimal moment for extubation? Currently, there are no guidelines recommending the optimal moment for extubation. Prolonged mechanical ventilation time could potentially be linked to increased complications such as pneumonia or disturbances in cerebral blood flow due to the vasodilatation produced by most anesthetic drugs. However, premature extubation in a patient who has suffered a stroke could led to complications such as agitation, disorientation, abolished reflexes, sudden fluctuations in blood pressure, alterations in cerebral blood flow, respiratory distress, bronchial aspiration, and the need for reintubation. We therefore designed a randomized study hypothesizing that early compared with delayed extubation is associated with a better functional outcome 3 months after endovascular thrombectomy treatment under general anesthesia for acute ischemic stroke. METHODS: This investigator-initiated, single-center, prospective, parallel, evaluated blinded, superiority, randomized controlled trial will include 178 patients with a proximal occlusion of the anterior circulation treated with successful endovascular thrombectomy (TICI 2b-3) under general anesthesia. Patients will be randomly allocated to receive early (< 6 h) or delayed (6-12 h) extubation after the procedure. The primary outcome measure is functional independence (mRS of 0-2) at 90 days, measured with the modified Rankin Score (mRS), ranging from 0 (no symptoms) to 6 (death). DISCUSSION: This will be the first trial to compare the effect of mechanical ventilation duration (early vs delayed extubation) after satisfactory endovascular thrombectomy for acute ischemic stroke under general anesthesia. TRIAL REGISTRATION: The study protocol was approved April 11, 2023, by the by the Santiago-Lugo Research Ethics Committee (CEI-SL), number 2023/127, and was registered into the clinicaltrials.gov clinical trials registry with No. NCT05847309. Informed consent is required. Participant recruitment begins on April 18, 2023. The results will be submitted for publication in a peer-reviewed journal and presented at one or more scientific conferences.

Metabolic signatures of GS-CHO cell clones associated with butyrate treatment and culture phase transition.

Chinese hamster ovary (CHO) cells are preferred hosts for the production of recombinant biopharmaceuticals. Efforts to optimize these bioprocesses have largely relied on empirical experience and our knowledge of cellular behavior in culture is incomplete. More recently, comprehensive investigations of metabolic network operation have started to be used to uncover traits associated with optimal growth and recombinant protein production. In this work, we used (1) H-nuclear magnetic resonance ((1) H-NMR) to analyze the supernatants of glutamine-synthetase (GS)-CHO cell clones expressing variable amounts of an IgG4 under control and butyrate-treated conditions. Exometabolomic data revealed accumulation of several metabolic by-products, indicating inefficiencies at different metabolic nodes. These data were contextualized in a detailed network and the cellular fluxomes estimated through metabolic flux analysis. This approach allowed comparing metabolic activity across different clones, growth phases and culture conditions, in particular the efficiency pertaining to carbon lost to glycerol and lactate accumulation and the characteristic nitrogen metabolism involving high asparagine and serine uptake rates. Importantly, this study shows that early butyrate treatment has a marked effect on sustaining high nutrient consumption along culture time, being more pronounced during the stationary phase when extra energy generation and biosynthetic activity is fueled to increase IgG formation. Collectively, the information generated contributes to deepening our understanding of CHO cells metabolism in culture, facilitating future design of improved bioprocesses.

Discovery of ICOS-Targeted Small Molecules Using Pharmacophore-Based Screening.

There are currently no small molecules clinically approved as immune checkpoint modulators. Besides possessing oral bioavailability, cell-penetrating capabilities and enhanced tumor penetration compared to monoclonal antibodies (mAbs), small molecules are amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may avoid immune-related adverse events associated with mAbs. The interaction of inducible co-stimulator (ICOS) with its ligand (ICOS-L) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. This study represents the development and validation of a virtual screening strategy to identify small molecules that bind a novel druggable binding pocket in human ICOS. We used a lipophilic canyon in the apo-structure of ICOS and the ICOS/ICOS-L interface individually as templates for molecular dynamics simulation to generate 3D pharmacophores subsequently used for virtual screening campaigns. Our strategy was successful finding a first-in-class small molecule ICOS binder (5P, KD value=108.08±26.76 µM) and validating biophysical screening platforms for ICOS-targeted small molecules. We anticipate that future structural optimization of 5P will result in the discovery of high affinity chemical ligands for ICOS.

Molecular signature associated with cladribine treatment in patients with multiple sclerosis.

Introduction: Little is known about the molecular profiling associated with the effect of cladribine in patients with multiple sclerosis (MS). Here, we aimed first to characterize the transcriptomic and proteomic profiles induced by cladribine in blood cells, and second to identify potential treatment response biomarkers to cladribine in patients with MS. Methods: Gene, protein and microRNA (miRNA) expression profiles were determined by microarrays (genes, miRNAs) and mass spectrometry (proteins) in peripheral blood mononuclear cells (PBMCs) from MS patients after in vitro treatment with cladribine in its active and inactive forms. Two bioinformatics approaches to integrate the three obtained datasets were applied: (i) a multiomics discriminant analysis (DIABLO - Data Integration Analysis for Biomarker discovery using Latent variable approaches for Omics studies); and (ii) a multi-stage integration of features selected in differential expression analysis on each dataset and then merged. Selected molecules from the in vitro study were quantified by qPCR ex vivo in PBMCs from MS patients receiving cladribine. Results: PBMCs treated in vitro with cladribine were characterized by a major downregulation of gene, protein, and miRNA expression compared with the untreated cells. An intermediate pattern between the cladribine-treated and untreated conditions was observed in PBMCs treated with cladribine in its inactive form. The differential expression analysis of each dataset led to the identification of four genes and their encoded proteins, and twenty-two miRNAs regulating their expression, that were associated with cladribine treatment. Two of these genes (PPIF and NHLRC2), and three miRNAs (miR-21-5p, miR-30b-5p, and miR-30e-5p) were validated ex vivo in MS patients treated with cladribine. Discussion: By using a combination of omics data and bioinformatics approaches we were able to identify a multiomics molecular profile induced by cladribine in vitro in PBMCs. We also identified a number of biomarkers that were validated ex vivo in PBMCs from patients with MS treated with cladribine that have the potential to become treatment response biomarkers to this drug.

Therapeutic Effect of IL-21 Blockage by Gene Therapy in Experimental Autoimmune Encephalomyelitis.

The pathogenic role of the interleukin 21 (IL-21) in different autoimmune diseases, such as multiple sclerosis (MS), has been extensively studied. However, its pleiotropic nature makes it a cytokine that may exhibit different activity depending on the immunological stage of the disease. In this study, we developed a gene therapy strategy to block the interaction between IL-21 and its receptor (IL-21R) by using adeno-associated vectors (AAV) encoding a new soluble cytokine receptor (sIL21R) protein. We tested this strategy in a murine model of experimental autoimmune encephalomyelitis (EAE), obtaining different clinical effects depending on the time at which the treatment was applied. Although the administration of the treatment during the development of the immune response was counterproductive, the preventive administration of the therapeutic vectors showed a protective effect by reducing the number of animals that developed the disease, as well as an improvement at the histopathological level and a modification of the immunological profile of the animals treated with the AAV8.sIL21R. The beneficial effect of the treatment was also observed when inducing the expression of the therapeutic molecule once the first neurological signs were established in a therapeutic approach with a doxycyline (Dox)-inducible expression system. All these clinical results highlight the pleiotropicity of this cytokine in the different clinical stages and its key role in the EAE immunopathogenesis.

Immunomodulatory Effects Associated with Cladribine Treatment.

Cladribine is a synthetic deoxyadenosine analogue with demonstrated efficacy in patients with relapsing-remitting multiple sclerosis (MS). The main mechanism of action described for cladribine is the induction of a cytotoxic effect on lymphocytes, leading to a long-term depletion of peripheral T and B cells. Besides lymphocyte toxicity, the mode of action may include immunomodulatory mechanisms affecting other cells of the immune system. In order to induce its beneficial effects, cladribine is phosphorylated inside the cell by deoxycytidine kinase (DCK) to its active form. However, the mechanism of action of cladribine may also include immunomodulatory pathways independent of DCK activation. This in vitro study was designed to explore the impact of cladribine on peripheral blood mononuclear cells (PBMC) subsets, and to assess whether the immunomodulatory mechanisms induced by cladribine depend on the activation of the molecule. To this end, we obtained PBMCs from healthy donors and MS patients and performed proliferation, apoptosis and activation assays with clinically relevant concentrations of cladribine in DCK-dependent and -independent conditions. We also evaluated the effect of cladribine on myeloid lineage-derived cells, monocytes and dendritic cells (DCs). Cladribine decreased proliferation and increased apoptosis of lymphocyte subsets after prodrug activation via DCK. In contrast, cladribine induced a decrease in immune cell activation through both DCK-dependent and -independent pathways (not requiring prodrug activation). Regarding monocytes and DCs, cladribine induced cytotoxicity and impaired the activation of classical monocytes, but had no effect on DC maturation. Taken together, these data indicate that cladribine, in addition to its cytotoxic function, can mediate immunomodulation in different immune cell populations, by regulating their proliferation, maturation and activation.