Global DNA Methylation and Hydroxymethylation Levels in PBMCs Are Altered in RRMS Patients Treated with IFN-ß and GA-A Preliminary Study.

Multiple sclerosis (MS) is a chronic disease affecting the central nervous system (CNS) due to an autoimmune attack on axonal myelin sheaths. Epigenetics is an open research topic on MS, which has been investigated in search of biomarkers and treatment targets for this heterogeneous disease. In this study, we quantified global levels of epigenetic marks using an ELISA-like approach in Peripheral Blood Mononuclear Cells (PBMCs) from 52 patients with MS, treated with Interferon beta (IFN-ß) and Glatiramer Acetate (GA) or untreated, and 30 healthy controls. We performed media comparisons and correlation analyses of these epigenetic markers with clinical variables in subgroups of patients and controls. We observed that DNA methylation (5-mC) decreased in treated patients compared with untreated and healthy controls. Moreover, 5-mC and hydroxymethylation (5-hmC) correlated with clinical variables. In contrast, histone H3 and H4 acetylation did not correlate with the disease variables considered. Globally quantified epigenetic DNA marks 5-mC and 5-hmC correlate with disease and were altered with treatment. However, to date, no biomarker has been identified that can predict the potential response to therapy before treatment initiation.

Anti-inflammatory mechanisms and pharmacological actions of phycocyanobilin in a mouse model of experimental autoimmune encephalomyelitis: A therapeutic promise for multiple sclerosis.

Cytokines, demyelination and neuroaxonal degeneration in the central nervous system are pivotal elements implicated in the pathogenesis of multiple sclerosis (MS) and its nonclinical model of experimental autoimmune encephalomyelitis (EAE). Phycocyanobilin (PCB), a chromophore of the biliprotein C-Phycocyanin (C-PC) from Spirulina platensis, has antioxidant, immunoregulatory and anti-inflammatory effects in this disease, and it could complement the effect of other Disease Modifying Treatments (DMT), such as Interferon-ß (IFN-ß). Here, our main goal was to evaluate the potential PCB benefits and its mechanisms of action to counteract the chronic EAE in mice. MOG35-55-induced EAE was implemented in C57BL/6 female mice. Clinical signs, pro-inflammatory cytokines levels by ELISA, qPCR in the brain and immunohistochemistry using precursor/mature oligodendrocytes cells antibodies in the spinal cord, were assessed. PCB enhanced the neurological condition, and waned the brain concentrations of IL-17A and IL-6, pro-inflammatory cytokines, in a dose-dependent manner. A down- or up-regulating activity of PCB at 1 mg/kg was identified in the brain on three (LINGO1, NOTCH1, and TNF-α), and five genes (MAL, CXCL12, MOG, OLIG1, and NKX2-2), respectively. Interestingly, a reduction of demyelination, active microglia/macrophages density, and axonal damage was detected along with an increase in oligodendrocyte precursor cells and mature oligodendrocytes, when assessed the spinal cords of EAE mice that took up PCB. The studies in vitro in rodent encephalitogenic T cells and in vivo in the EAE mouse model with the PCB/IFN-ß combination, showed an enhanced positive effect of this combined therapy. Overall, these results demonstrate the anti-inflammatory activity and the protective properties of PCB on the myelin and support its use with IFN-ß as an improved DMT combination for MS.

Cytotoxic B Cells in Relapsing-Remitting Multiple Sclerosis Patients.

Background: Emerging evidence of antibody-independent functions, as well as the clinical efficacy of anti-CD20 depleting therapies, helped to reassess the contribution of B cells during multiple sclerosis (MS) pathogenesis. Objective: To investigate whether CD19+ B cells may share expression of the serine-protease granzyme-B (GzmB), resembling classical cytotoxic CD8+ T lymphocytes, in the peripheral blood from relapsing-remitting MS (RRMS) patients. Methods: In this study, 104 RRMS patients during different treatments and 58 healthy donors were included. CD8, CD19, Runx3, and GzmB expression was assessed by flow cytometry analyses. Results: RRMS patients during fingolimod (FTY) and natalizumab (NTZ) treatment showed increased percentage of circulating CD8+GzmB+ T lymphocytes when compared to healthy volunteers. An increase in circulating CD19+GzmB+ B cells was observed in RRMS patients during FTY and NTZ therapies when compared to glatiramer (GA), untreated RRMS patients, and healthy donors but not when compared to interferon-ß (IFN). Moreover, regarding Runx3, the transcriptional factor classically associated with cytotoxicity in CD8+ T lymphocytes, the expression of GzmB was significantly higher in CD19+Runx3+-expressing B cells when compared to CD19+Runx3- counterparts in RRMS patients. Conclusions: CD19+ B cells may exhibit cytotoxic behavior resembling CD8+ T lymphocytes in MS patients during different treatments. In the future, monitoring "cytotoxic" subsets might become an accessible marker for investigating MS pathophysiology and even for the development of new therapeutic interventions.

Interferon-Beta Injection in Multiple Sclerosis Patients Related to the Induction of Headache and Flu-Like Pain Symptoms: A Systematic Review and Meta-Analysis of Randomised Controlled Trials.

Multiple sclerosis (MS) is a chronic neurodegenerative, inflammatory, and autoimmune disease characterised by the demyelination of the central nervous system. One of the main approaches for treating MS is the use of disease-modifying therapies (DMTs). Among the DMTs are interferons (IFNs), which are cytokines responsible for controlling the activity of the immune system while exerting immunomodulatory, antiviral, and antiproliferative activities. IFN-beta (IFN-ß) is the first-choice drug used to treat relapsing-remitting MS. However, the administration of IFN-ß causes numerous painful adverse effects, resulting in lower adherence to the treatment. Therefore, this study aimed to investigate the headache and flu-like pain symptoms observed after IFNß injection in MS patients using a systematic review and meta-analysis of randomised controlled trials. A total of 2370 articles were identified through research databases. Nine articles were included (three involving IFNß-1b and six involving IFNß-1a). All studies included in the meta-analysis had a low risk of bias. The odds ratio of headache and flu-like pain symptoms increased in MS patients treated with IFN-ß. Thus, the adverse effects of headache and flu-like pain symptoms appear to be linked to IFN-ß treatment in MS. The protocol of the study was registered in the Prospective International Registry of Systematic Reviews (registration number CRD42021227593).

A cluster randomized trial of interferon ß-1a for the reduction of transmission of SARS-Cov-2: protocol for the Containing Coronavirus Disease 19 trial (ConCorD-19).

BACKGROUND: SARS-CoV-2 infection rapidly spreads in populations due to the high rates of community transmission. Interrupting the shedding of SARS-CoV-2 may reduce the incidence of Coronavirus Disease 19 (COVID-19). Herein we provide a protocol for a cluster randomized trial that will examine the effectiveness of treatment with interferon (IFN) ß-1a compared to standard of care in limiting the transmission of SARS-CoV-2. Co-primary objectives are to determine whether IFN therapy reduces (a) the proportion of infected cases shedding SARS-CoV-2 at day 11 post randomization and (b) the incidence of transmission of SARS-CoV-2 infection from index cases to treatment-eligible household post-exposure contacts at day 11 after randomization. Secondary objectives include assessing the impact of IFN treatment on duration of viral clearance, hospitalizations and fatalities, and evaluating the safety of IFN treatment. METHODS: Three hundred and ten households, each including an index case with a recent COVID-19 diagnosis and at least one asymptomatic treatment-eligible household contact, will be randomized to receive 3 doses of 125 µg IFN ß-1a by subcutaneous administration (days 1, 6, and 11), or standard of care. All participants will be followed until day 29. DISCUSSION: The results from this trial will identify whether IFN ß treatment of mild or moderate COVID-19 cases accelerates viral clearance and prevents disease progression and whether IFN ß treatment of post-exposure contacts of COVID-19 cases reduces transmission of infection. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov NCT04552379; date of registration September 17, 2020.

Interferón en infección por COVID-19 / Interferon in COVID-19 infection

CONTEXTO CLÍNICO: La Enfermedad por el Coronavirus 2019 (COVID­19, por su sigla en inglés Coronavirus Disease 2019) es una enfermedad respiratoria de humanos producida por un nuevo coronavirus identificado con la sigla SARS-CoV-2. El 11 de marzo de 2020 la Organización Mundial de la Salud (OMS) declaro la COVID-19 como uma pandemia. Desde ese momento hasta este octubre 2020 su circulación se ha reportado en 205 países reportándose más de 36 millones de casos y más de un millón de muertes. El período de incubación de la infección por 2019­nCoV es de 2 a 14 días. La mayor parte de los contagios se producen persona a persona, siendo altamente transmisible. La clínica varía desde casos asintomáticos a cuadros febriles con tos y dificultad respiratoria, neumonía y distrés respiratorio. También puede acompañarse de alteraciones gastrointestinales.2 En los casos con mal pronóstico, el paciente presenta un importante deterioro respiratorio en 4-8 días. Las imágenes radiológicas muestran generalmente neumonía focal o generalizada semejante al síndrome de distress respiratório agudo. La mayoría de los casos graves requieren ingreso hospitalario, siendo mayoritariamente casos primarios en pacientes de edad avanzada y con comorbilidades (diabetes, enfermedad crónica renal, hipertensión, enfermedad cardiaca y enfermedad pulmonar crónica). La tasa media de letalidad de los pacientes ingresados a UTI es cercana al 49%, siendo los valores más elevados en pacientes masculinos de más de 50 años con comorbilidades múltiples. Actualmente el tratamiento de la COVID­19 es sintomático y de sostén no existiendo hasta el momento tratamiento farmacológico específico curativo. Los tratamientos que se han propuestos son: inhibidores de la ARN polimerasa dependiente de ARN (remdesivir, favipiramir), inhibidores de la neuraminidasa (oseltamivir), inhibidores de la protease (lopinavir/ritonavir, desulfura, inhibidores de la enzima convertidora de angiotensina 2, inhibidores de quinasa (imatinib, baricitinib, ribavirin), inmunomoduladores (plasma de convaleciente, anticuerpos ante receptores IL-6 como tocilizumab y otros, dentro de los cuales se incluye el interferón, los glucorticoides y el umifenovir. En otras pandemias con virus SARS y MERS se ha usado el interferón, dado que múltiples estudios in vitro demuestran acción antiviral. Estudios in vitro específicos en el estudio del virus SARS-CoV-2 definen que la cinética del interferón podría ser variable en los distintos grados de enfermedad y sobre todo en la definición que cuando comenzar su uso, dado el desconocimiento de las curvas virales del SARS-CoV-2 y ventanas terapéuticas apropriadas. El uso asociado de interferón con otros antivirales surge de los experiencia de algún beneficio en estúdios realizados en el tratamiento de los virus MERS-CoV. Dada la falta de vacunas o tratamentos específicos para el nuevo coronavirus SARS-CoV-2, se postula el uso de interferón para el tratamento de pacientes COVID-19 positivos. TECNOLOGÍA: Los distintos tipos de Interferones (IFN) tienen acciones inmunomoduladoras y antivirales uniéndose a receptores celulares, realizando procesos transcripcionales que activan elsistema de citoquinas ante infecciones virales. Los IFN se clasifican en tipo I, II y III. Los IFN tipo I (IFN-α y IFN-ß) son los que tienen mayor actividad antiviral. Los IFN producen expresión de genes que estimulan el estado antiviral celular. Este mecanismo es el que los propuso para los ensayos clinicos en los virus MERS-CoV y SARS-CoV. OBJETIVO: El objetivo del presente informe es evaluar la evidencia disponible acerca de la eficacia, seguridad y aspectos relacionados a las políticas de cobertura del uso de interferón beta en infección por COVID-19. MÉTODOS: Se realizó una búsqueda en las principales bases de datos bibliográficas, en buscadores genéricos de internet, y financiadores de salud. Se priorizó la inclusión de revisiones sistemáticas (RS), ensayos clínicos controlados aleatorizados (ECAs), evaluaciones de tecnologías sanitarias (ETS), evaluaciones económicas, guías de práctica clínica (GPC) y recomendaciones de diferentes organizaciones de salud. CONCLUSIONES: No se ha encontrado evidencia del uso de interferón como mono droga para el tratamiento de pacientes con infección por COVID-19. Evidencia de muy baja calidad proveniente de un ensayo clínico de pocos pacientes y varios sesgos que evaluó el uso de interferón asociado a otros antivirales (lopinavir-ritonavir o atazanavir-ritonavir) en comparación con cualquiera de estas asociaciones, no ha demostrado una reducción de la mortalidad ni mejora clínica a los 28 días en pacientes con neumopatía severa sintomática por COVID-19. Ninguna de las guías o protocolos gubernamentales relevados y/o de sociedades científicas americanas, latinoamericanas y europeas recomiendan el uso interferón como mono droga o uso combinado con otros antivirales. Solo está autorizado su uso en el contexto de investigación clínica. Algunos ensayos clínicos aleatorizados que evalúan la eficacia y seguridad de esta droga em pacientes con cuadros respiratorios por COVID-19 se encuentran en curso, por lo que podrían cambiar la evidencia actualmente disponible.

Effectiveness of first-line treatment for relapsing-remitting multiple sclerosis in Brazil: A 16-year non-concurrent cohort study.

BACKGROUND: Relapsing-remitting multiple sclerosis (RRMM) is a chronic, progressive, inflammatory and immune-mediated disease that affects the central nervous system and is characterized by episodes of neurological dysfunction followed by a period of remission. The pharmacological strategy aims to delay the progression of the disease and prevent relapse. Interferon beta and glatiramer are commonly used in the Brazilian public health system and are available to patients who meet the guideline criteria. The scenario of multiple treatments available and in development brings the need for discussion and evaluation of the technologies already available before the incorporation of new drugs. This study analyses the effectiveness of first-line treatment of RRMS measured by real-world evidence data, from the Brazilian National Health System (SUS). METHODS AND FINDINGS: We conducted a non-concurrent national cohort between 2000 and 2015. The study population consisted of 22,722 patients with RRMS using one of the following first-line drugs of interest: glatiramer or one of three presentations of interferon beta. Kaplan-Meier analysis was used to estimate the time to treatment failure. A univariate and multivariate Cox proportional hazard model was used to evaluate factors associated with treatment failure. In addition, patients were propensity score-matched (1:1) in six groups of comparative first-line treatments to evaluate the effectiveness among them. The analysis indicated a higher risk of treatment failure in female patients (HR = 1.08; P = 0,01), those with comorbidities at baseline (HR = 1.20; P<0,0001), in patients who developed comorbidities after starting treatment (i.e., rheumatoid arthritis-HR = 1.65; P<0,0001), those exclusive SUS patients (HR = 1.31; P<0,0001) and among patients using intramuscular interferon beta (IM ßINF-1a) (28% to 60% compared to the other three treatments; P<0,0001). Lower risk of treatment failure was found among patients treated with glatiramer. CONCLUSIONS: This retrospective cohort suggests that glatiramer is associated with greater effectiveness compared to the three presentations of interferon beta. When evaluating beta interferons, the results suggest that the intramuscular presentation is not effective in the treatment of multiple sclerosis.

Informe diário de evidências COVID-19: busca referente aos dias 4, 5 e 6 de julho de 2020 / COVID-19 daily evidence report: search for July 4, 5 and 6, 2020

O Informe Diário de Evidências é uma produção do Ministério da Saúde que tem como objetivo acompanhar diariamente as publicações científicas sobre tratamento farmacológico e vacinas para a COVID-19. Dessa forma, são realizadas buscas estruturadas em bases de dados biomédicas, referente ao dia anterior desse informe. Não são incluídos estudos pré-clínicos (in vitro, in vivo, in silico). A frequência dos estudos é demonstrada de acordo com a sua classificação metodológica (revisões sistemáticas, ensaios clínicos randomizados, coortes, entre outros). Para cada estudo é apresentado um resumo com avaliação da qualidade metodológica. Essa avaliação tem por finalidade identificar o grau de certeza/confiança ou o risco de viés de cada estudo. Para tal, são utilizadas ferramentas já validadas e consagradas na literatura científica, na área de saúde baseada em evidências. Cabe ressaltar que o documento tem caráter informativo e não representa uma recomendação oficial do Ministério da Saúde sobre a temática. Foram encontrados 17 artigos.

Polymorphisms in the CIITA -168A/G (rs3087456) and CIITA +1614G/C (rs4774) may influence severity in multiple sclerosis patients.

It is currently unknown how genetic factors may influence the clinical course of multiple sclerosis (MS). OBJECTIVE: We examined the impact of CIITA polymorphisms -168A/G (rs3087456) and +1614G/C (rs4774) on the risk of disability progression, severity and on responses to first-line immunomodulator treatments. METHODS: Genomic DNA was extracted from blood samples. We used ABI3730xl and GeneMapper v.4.0 software to identify genotype variations. All patients were followed up and clinically reassessed at three-month intervals. Disability progression was measured by the Expanded Disability Status Scale and disease severity by the Multiple Sclerosis Spasticity Scale (MSSS). RESULTS: We included 37 men and 80 women. We found no evidence regarding the influence of the single nucleotide polymorphisms studied in the Expanded Disability Status Scale or therapeutic response of the evaluated drugs. We performed a logistic regression analysis with the MSSS and found that a less severe MS course was associated with wild type CIITA -168AA and CIITA +1614GG, as the chance of the patient progressing to MSSS2 and MSSS3 decreased in 61% and 75% with CIITA -168AA and 66% and 75% with CIITA +1614GG, respectively (p < 0.0001). Although less significant, the CIITA +1614 GC also pointed to a less severe MS course and the chance of the patient progressing to MSSS3 decreased 79% (p = 0.015). We also observed that the CIITA -168GG genotype was more frequent in MSSS2 and MSSS3 and had 40% lower odds ratio to becoming more severe MS. CONCLUSION: These data suggest that CIITA -168AA, CIITA +1614GG and CIITA +1614 GC polymorphisms may be associated with a better MS clinical course. This knowledge may be useful for a better understanding of MS and its therapeutic management.

Combination of cytokine-enhanced vaccine and chemo-gene therapy as surgery adjuvant treatments for spontaneous canine melanoma.

After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-ß gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.

Interferon-Beta Treatment Differentially Alters TLR2 and TLR4-Dependent Cytokine Production in Multiple Sclerosis Patients.

OBJECTIVE: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-ß treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients. METHODS: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). RESULTS: In patients treated with IFN-ß, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam3Cys) was lower than in healthy controls and untreated MS patients. However, IFN-ß treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-ß treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls. CONCLUSIONS: Our data suggest that IFN-ß treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-ß treatment.

Distinct Roles of Direct Transduction Versus Exposure to the Tumor Secretome on Murine Endothelial Cells After Melanoma Gene Therapy with Interferon-ß and p19Arf.

Tumor vasculature plays a central role in tumor progression, making it an attractive therapeutic target. In this study, we explore the antiangiogenic potential of our melanoma gene therapy approach combining interferon ß (IFNß) and p19Arf gene transfer. Since these proteins are modulators of tumor vasculature, we explore the impact of IFNß and p19Arf gene transfer on murine endothelial cells (tEnd). Adenovirus-mediated gene transfer of p19Arf to tEnd cells inhibited proliferation, tube formation, migration, and led to increased expression of genes related to the p53 cell death pathway, yet IFNß gene transfer had no significant impact on tEnd viability. Alternatively, tEnd cells were exposed to the factors generated by transduced B16 (mouse melanoma) cells using either coculture or conditioned medium. In either case, transduction of B16 cells with the IFNß vector, whether alone or in combination with p19Arf, resulted in endothelial cell death. Strikingly, treatment of tEnd cells with recombinant IFNß did not induce death, demonstrating that additional factors produced by B16 cells contributed to the demise of tEnd cells. In this work, we have shown that our melanoma gene therapy strategy produces desirable negative effects on endothelial cells, possibly correlating with antiangiogenic activity.

Polymorphisms in the CIITA -168A/G (rs3087456) and CIITA +1614G/C (rs4774) may influence severity in multiple sclerosis patients / Os polimorfismos no gene CIITA - 168A/G (rs3087456) e CIITA +1614G/C (rs4774) podem influenciar a gravidade em pacientes com esclerose múltipla

ABSTRACT It is currently unknown how genetic factors may influence the clinical course of multiple sclerosis (MS). Objective: We examined the impact of CIITA polymorphisms −168A/G (rs3087456) and +1614G/C (rs4774) on the risk of disability progression, severity and on responses to first-line immunomodulator treatments. Methods: Genomic DNA was extracted from blood samples. We used ABI3730xl and GeneMapper v.4.0 software to identify genotype variations. All patients were followed up and clinically reassessed at three-month intervals. Disability progression was measured by the Expanded Disability Status Scale and disease severity by the Multiple Sclerosis Spasticity Scale (MSSS). Results: We included 37 men and 80 women. We found no evidence regarding the influence of the single nucleotide polymorphisms studied in the Expanded Disability Status Scale or therapeutic response of the evaluated drugs. We performed a logistic regression analysis with the MSSS and found that a less severe MS course was associated with wild type CIITA −168AA and CIITA +1614GG, as the chance of the patient progressing to MSSS2 and MSSS3 decreased in 61% and 75% with CIITA −168AA and 66% and 75% with CIITA +1614GG, respectively (p < 0.0001). Although less significant, the CIITA +1614 GC also pointed to a less severe MS course and the chance of the patient progressing to MSSS3 decreased 79% (p = 0.015). We also observed that the CIITA −168GG genotype was more frequent in MSSS2 and MSSS3 and had 40% lower odds ratio to becoming more severe MS. Conclusion: These data suggest that CIITA −168AA, CIITA +1614GG and CIITA +1614 GC polymorphisms may be associated with a better MS clinical course. This knowledge may be useful for a better understanding of MS and its therapeutic management.

RESUMO Atualmente não se sabe como os fatores genéticos podem influenciar o curso clínico da esclerose múltipla (EM). Objetivo: Examinamos o impacto dos polimorfismos CIITA −168A/G (rs3087456) e CIITA +1614G/C (rs4774) no risco de progressão da incapacidade, gravidade e resposta aos tratamentos imunomoduladores de primeira linha. Métodos: O DNA genômico foi extraído de amostras de sangue. Utilizamos o software ABI3730xl e GeneMapper v.4.0 (Applied Biosystems) para identificar variações genotípicas. Todos os pacientes foram acompanhados e reavaliados clinicamente em intervalos de três meses. A progressão da incapacidade foi medida pela EDSS e a gravidade da doença pelo MSSS. Resultados: Incluímos 37 homens e 80 mulheres. Não encontramos evidências sobre a influência dos SNPs estudados no EDSS e na resposta terapêutica aos fármacos avaliados. Realizamos uma análise de regressão logística com o MSSS e observamos uma evolução menos grave da EM associada aos tipos selvagens CIITA −168AA e CIITA +1614GG, pois a chance do paciente atingir MSSS2 e MSSS3 diminuiu em 61%/75%, e 66/75% respectivamente (p < 0,0001). Embora menos significativo, o CIITA +1614GC também foi relacionado com evolução menos grave da EM e a chance do paciente atingir o MSSS3 diminuiu 79% (p = 0,015). Nós também observamos que o genótipo CIITA −168GG foi mais frequente no MSSS2 e MSSS3 e teve uma razão de chance 40% menor para atingir forma mais grave da EM. Conclusão: Estes dados sugerem que os polimorfismos CIITA −168AA, CIITA +1614GG e CIITA +1614GC podem estar associados a um melhor curso clínico da EM. Este conhecimento pode ser útil para uma melhor compreensão da EM e o seu manejo terapêutico.

Differential Transcription of SOCS5 and SOCS7 in Multiple Sclerosis Patients Treated with Interferon Beta or Glatiramer Acetate.

The participation of proinflammatory cytokines in the progression of Multiple Sclerosis (MS) has been well documented. Cytokines activate the JAK-STAT pathway, in which the suppressors of cytokine signaling (SOCS) exert a negative feedback. This paper analyzes the levels of SOCS5 and SOCS7 transcripts, quantified by RT-qPCR, in MS patients, and the concentrations of proinflammatory cytokines, IFN-γ, IL17, and IL6, determined by ELISA. Samples of peripheral blood were obtained from MS patients in the relapsing-remitting phase, treated with IFN-ß or glatiramer acetate (GA), and from healthy individuals. SOCS7 mRNA was significantly higher in patients treated with GA (1.36 ± 0.23) than in those treated with IFN-ß (0.65 ± 0.1). Regarding gender, the level of SOCS5 and SOCS7 transcripts were similar between MS and healthy females; in MS males, the level of SOCS7 transcripts were significantly lower (0.59 ± 0.03) than in healthy males (1.008 ± 0.05). Plasmatic levels of IFN-γ were significantly higher in MS patients (60 pg/mL, range 0-160) than in healthy subjects (0 range, 0-106). The same pattern was observed in MS patients treated with IFN-ß (68 pg/mL, range 0-160) compared to patients treated with GA (51 pg/mL, range 0-114), and in MS females (64 pg/mL, range 0-161) compared to healthy females (0, range 0-99). We hypothesize that the increase in SOCS7 transcription in patients treated with GA could partially explain the action mechanism of this drug, while the increase in the concentration of IFN-γ in MS patients could help elucidate the immunopathology of the disease.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer.

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-ß cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Pediatric-onset multiple sclerosis in Brazilian patients: Clinical features, treatment response and comparison to pediatric neuromyelitis optica spectrum disorders.

BACKGROUND: Multiple Sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are acquired demyelinating syndromes of the central nervous system more frequently in young adults and their beginning before 18 years of age is rare. They are autoimmune diseases with distinct pathophysiology, clinical presentation, treatment and prognoses. During childhood these conditions often present similar clinical features and differential diagnosis among pNMOSD, pMS and acute disseminated encephalomyelitis (ADEM) is still difficult at disease onset. The aim of this article is to describe the epidemiologic and clinical features, to evaluate the response to treatment and to compare the mains characteristics between the patients with MS and NMOSD who had the first event prior to 18 years of age followed at the Universidade Federal de São Paulo (UNIFESP). METHODS: Retrospective analysis of patients with MS and NMOSD who started the disease before 18 years of age followed for at UNIFESP. All patients fulfilled the McDonald 2010 criteria for MS and the IPND 2015 criteria or 2006 diagnostic criteria for NMOSD. For treatment analysis, we select patients with a follow-up of more than 6 months. RESULTS: Sixty-eight patients fulfilled the inclusion criteria for MS and were selected for analysis. Mean age of onset was 15 years, 73.5% were female and the mean follow-up was 6.7 years. Mean annualized relapse rate (aRR) observed was 0,82 relapse/year and mean progression index (PI) was 0.31 EDSS points/year. The multivariate analysis showed a significant association between the EDSS on first appointment and total number of relapses with neurological disabilities in long term in patients with MS. The treatment with interferon-beta (IFN-ß) and glatiramer acetate (GA) was safe and patients treated with high dose IFN-ß and GA had a statistically significant reduction in disability progression. Eleven patients fulfilled the inclusion criteria for NMOSD: mean age of onset was 14 years, 72.7% were female and the mean follow-up was 6.3 years. Mean aRR observed was 1.5 relapse/year and mean PI was 2.2 EDSS points/year. The treatment with azathioprine was safe and significant halts disability progression. Patients with NMOSD reached EDSS 6 prior than those with MS. CONCLUSIONS: Pediatric demyelinating diseases in Brazil are similar to the diseases described abroad. In patients with pMS, the EDSS score at the first appointment and the total number of relapses were associated with poor prognosis. NMOSD is more severe than MS in pediatric patients. Treatment with DMD and azathioprine was well tolerated and effective in reducing relapse rate and disability.

Decreased serum levels of sCD40L and IL-31 correlate in treated patients with Relapsing-Remitting Multiple Sclerosis.

The CD40/CD40L system is a binding key for co-stimulation of immune cells. Soluble form of CD40L has been widely studied as marker of inflammatory and autoimmune diseases. Here we analyze serum concentrations of sCD40L, as well as 14 cytokines, in patients with Multiple Sclerosis (MS) treated with Glatiramer acetate or Interferon beta. In the healthy control group, we found in serum a highly positive correlation between sCD40L and Interleukin (IL)-31, an anti-inflammatory Th2 cytokine. Additionally, an important reduction in IL-31 and sCD40L serum levels, as well as a significant reduction in CD40 mRNA expression and complete depletion of CD40L mRNA, detected from peripheral blood cells, was found in treated patients with MS. Therefore, sCD40L and IL-31 must be taken into account as possible prognostic markers when analyzing the disease progress of MS in order to provide more personalized treatment.

Eficacia y seguridad de alemtuzumab en pacientes con esclerosis múltiple remitente-recurrente, enfermedad altamente activa y falla al uso de interferón beta-ia y natalizumab / Efficacy and safety of alemtuzumab in patients with relapsing-remitting multiple sclerosis, highly active disease and failure to use interferon beta-ia and natalizumab

INTRODUCCIÓN: La presente evaluación de tecnología expone la evidencia científica encontrada acerca de alemtuzumab en pacientes con esclerosis múltiple remitente-recurrente con enfermedad altamente activa que hayan fallado al tratamiento con interferón beta-1a y natalizumab. La esclerosis múltiple (EM) o mielopatia desmielinizante es una enfermedad neurológica inflamatoria crónica autoinmune del sistema nervioso central. Se estima en el Perú, una prevalencia de menos de 5 personas por cada 100,000 habitantes. El patrón más común de esta enfermedad (aproximadamente el 85 % de los pacientes) es el tipo de EM recurrente-remitente (EMRR). TECNOLOGÍA SANITARIA DE INTERÉS: Alemtuzumab (Lemtrada®, Boehringer Ingelheim) es un anticuerpo monoclonal humano, el cual disminuye la expresión del antígeno CD52, al unirse a este en la superficie celular de los linfocitos T, linfocitos B, linfocitos citolíticos naturales. monocitos y macrófagos. El mecanismo de acción exacto del medicamento aún no ha sido descifrado por los investigadores, sin embargo, se estipula que los cambios cuantitativos y cualitativos (depleción y repoblación de los linfocitos) de las células del sistema inmune. reprograman la tolerancia del mismo, reduciendo así la frecuencia de nuevos episodios y recaídas, y por lo tanto retrasan la evolución de la enfermedad. METODOLOGÍA: Se llevó a cabo una búsqueda sistemática de la literatura con respecto a la eficacia y seguridad de alemtuzumab para el tratamiento de esclerosis múltiple remitente recurrente con enfermedad altamente activa que hayan recibido tratamiento con interferón (en falla) y natalizumab. Se realizó tanto una búsqueda sistemática como una búsqueda manual en las páginas web de grupos dedicados a la investigación y educación en salud que elaboran guías de práctica clínica. RESULTADOS: Tras la búsqueda sistemática de evidencia científica, no se encontraron guías de práctica clínica que cumplan con los criterios de inclusión para la evaluación y descripción de la evidencia. Las guías más actuales no contienen recomendaciones acerca del uso de alemtuzumab, ya que su fecha de publicación es previa a la autorización comercial de alemtuzumab en el mercado. Se seleccionaron cinco evaluaciones de tecnologías sanitarias (ETS). las cuales emitieron sus recomendaciones favorables hacia el uso de alemtuzumab como terapia de segunda línea basados principalmente en la evidencia presentada de eficacia por dichos ensayos clínicos. Es de notar que estas recomendaciones fueron basadas todas en dos ensayos clínicos fase III CARE-MS I y CARE-MS II y uno de fase II CAMMS223. Adicionalmente se evaluaron tres revisiones sistemáticas (RS). las cuales también basaron sus cálculos en los mismos ensayos clínicos de las ETS. De los ensayos clínicos mencionados. el único ensayo que responde directamente a nuestra pregunta PICO de interés es el estudio fase III de etiqueta abierta CARE-MS II, en el cual se evaluaron a pacientes con tratamiento previo. La eficacia reportada en CARE-MS II corresponde a una reducción en la tasa anual de recaída en un 49.4 % a favor de alemtuzumab 12 mg frente a interferón beta-1 a con un rafe ratio 0.51, IC 95 % [0.39 - 0.65]; p <0 - 0001. Para el desenlace tiempo hasta la discapacidad a los seis meses el resultado fue favorable para el grupo de alemtuzumab 12 mg en comparación con interferón beta-1 a siendo la diferencia entre ambos grupos del 42%, con un hazard ratio (HR) 0.58; IC 95 % [0.38 ­ 0.87] y con significancia estadística (p = 0.008). Sin embargo, se observó un alto riesgo de sesgo en el CARE-MS II. especialmente por su diseño de etiqueta abierta y la naturaleza subjetiva de sus desenlaces primarios (tasa de recaída y discapacidad acumulada) y las pérdidas en el seguimiento desiguales entre los grupos. Estas características incrementan la incertidumbre con respecto al beneficio clínico neto que ofrecería alemtuzumab en términos de eficacia respecto al interferón. Respecto a la seguridad, los eventos adversos reportados en los ensayos clínicos son de mayor frecuencia (2 o 3 veces más frecuentes) para el grupo de alemtuzumab en comparación con interferón beta-1 a, tales como trastornos tiroideos, trastornos del sistema linfático y sanguíneo e infecciones graves. los cuales califican como eventos adversos serios que ponen en riesgo la vida del paciente e involucran hospitalización e intervenciones médicas adicionales después del tratamiento con alemtuzumab. Como ejemplo, se estima que durante toda la duración de los ensayos clínicos desde el CAMMS223 hasta el término del CARE-MS II (48 meses), la incidencia estimada de trastornos tiroideos para el grupo de alemtuzumab sea del 36%. Alemtuzumab es un medicamento de muy alto costo (el costo por los dos ciclos de tratamiento por paciente en 12 meses es de aproximadamente 300 mil soles), lo que se traduce en un perfil de costo-oportunidad poco ventajoso para nuestro sistema de salud, dada especialmente la incertidumbre en su eficacia y el alto riesgo de eventos adversos serios, con lo que no es posible determinar claramente su balance neto de riesgo-beneficio (seguridad versus eficacia). CONCLUSIONES: Alemtuzumab es un medicamento de muy alto costo (el costo por los dos ciclos de tratamiento por paciente en 12 meses es de aproximadamente 300 mil soles), lo que se traduce en un perfil de costo-oportunidad poco ventajoso para nuestro sistema de salud, dada especialmente la incertidumbre en su eficacia y el alto riesgo de eventos adversos serios, con lo que no es posible determinar claramente su balance neto de riesgo-beneficio (seguridad versus eficacia). Por lo expuesto, el Instituto de Evaluación de Tecnologías en Salud e Investigación (IETSI) no aprueba el uso de alemtuzumab en pacientes con esclerosis múltiple remitente recurrente con enfermedad altamente activa que hayan recibido tratamiento previo con interferón betal a (en falla) y natalizumab.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.