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1.
Lipids Health Dis ; 22(1): 97, 2023 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37420217

RESUMO

BACKGROUND: The common inflammatory disease multiple sclerosis (MS) is a disease of the central nervous system. For more than 25 years autologous hematopoietic stem cell transplantation (AHSCT) has been used to treat MS. It has been shown to be highly effective in suppressing inflammatory activity in relapsing-remitting MS (RRMS) patients. This treatment is thought to lead to an immune system reset, inducing a new, more tolerant system; however, the precise mechanism behind the treatment effect in MS patients is unknown. In this study, the effect of AHSCT on the metabolome and lipidome in peripheral blood from RRMS patients was investigated. METHODS: Peripheral blood samples were collected from 16 patients with RRMS at ten-time points over the five months course of AHSCT and 16 MS patients not treated with AHSCT. Metabolomics and lipidomics analysis were performed using liquid-chromatography high-resolution mass spectrometry. Mixed linear models, differential expression analysis, and cluster analysis were used to identify differentially expressed features and groups of features that could be of interest. Finally, in-house and in-silico libraries were used for feature identification, and enrichment analysis was performed. RESULTS: Differential expression analysis found 657 features in the lipidomics dataset and 34 in the metabolomics dataset to be differentially expressed throughout AHSCT. The administration of cyclophosphamide during mobilization and conditioning was associated with decreased concentrations in glycerophosphoinositol species. Thymoglobuline administration was associated with an increase in ceramide and glycerophosphoethanolamine species. After the conditioning regimen, a decrease in glycerosphingoidlipids concentration was observed, and following hematopoietic stem cell reinfusion glycerophosphocholine concentrations decreased for a short period of time. Ceramide concentrations were strongly associated with leukocyte levels during the procedure. The ceramides Cer(d19:1/14:0) and Cer(d20:1/12:0) were found to be increased (P < .05) in concentration at the three-month follow-up compared to baseline. C16 ceramide, Cer(D18:2/16:0), and CerPE(d16:2(4E,6E)/22:0) were found to be significantly increased in concentration after AHSCT compared to prior to treatment as well as compared to newly diagnosed RRMS patients. CONCLUSION: AHSCT had a larger impact on the lipids in peripheral blood compared to metabolites. The variation in lipid concentration reflects the transient changes in the peripheral blood milieu during the treatment, rather than the changes in the immune system that are assumed to be the cause of clinical improvement within RRMS patients treated with AHSCT. Ceramide concentrations were affected by AHSCT and associated with leukocyte counts and were altered three months after treatment, suggesting a long-lasting effect.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Humanos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/etiologia , Resultado do Tratamento , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante Autólogo/métodos
2.
Acta Neurol Scand ; 143(6): 602-607, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33626181

RESUMO

OBJECTIVE: To identify serum proteins associated with MS and affected by interferon beta treatment. METHODS: Plasma samples from 29 untreated relapsing-remitting MS patients and 15 healthy controls were investigated with a multiplexed panel containing 92 proteins related to inflammation. Follow-up samples were available from 13 patients at 1 and 3 months after initiation of treatment with interferon beta-1a. RESULTS: Ten proteins were differentially expressed in MS patients. Five of these were altered by treatment with IFN-ß 1a: uPA, CX3CL1, CCL2, TRAIL and IL18. CONCLUSION: CCL2 and TRAIL were confirmed to be modulated with interferon beta treatment in MS. As novel findings, we now report that uPA and CX3CL1 were differentially expressed in MS and increased after IFN-beta-1a treatment. Conflicting results have been reported on how interferon beta affects IL-18.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Citocinas/sangue , Citocinas/efeitos dos fármacos , Interferon beta-1a/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Quimiocina CCL2/sangue , Quimiocina CCL2/efeitos dos fármacos , Quimiocina CX3CL1/sangue , Quimiocina CX3CL1/efeitos dos fármacos , Feminino , Humanos , Interleucina-18/sangue , Masculino , Proteínas de Membrana/sangue , Proteínas de Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Ligante Indutor de Apoptose Relacionado a TNF/sangue , Ligante Indutor de Apoptose Relacionado a TNF/efeitos dos fármacos
3.
Biol Blood Marrow Transplant ; 26(1): 50-57, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31525494

RESUMO

Little is known about the inflammatory milieu in the blood during autologous hematopoietic stem cell transplantation (AHSCT) and how it is affected by the stem cell mobilization, collection, and reinfusion and conditioning regimen. In this study, we analyzed 92 proteins connected to inflammation at 10 time points during and after AHSCT in 16 patients with multiple sclerosis (MS). Serum from 29 patients with newly diagnosed MS and 15 healthy controls were included for comparative analysis. There were no significant differences in inflammatory serum protein levels between patients with newly diagnosed MS and healthy controls, but 29 out of 73 detectable proteins were significantly altered between at least 2 adjacent sampling time points during AHSCT. The predominant changes occurred after the conditioning regimen had been administered, whereas stem cell mobilization, collection, and reinfusion appeared to have less impact. Two distinct response patterns could be discerned, likely representing loss of basal cytokine production and homeostasis. The analyzed serum proteins gradually returned to baseline levels after treatment, with no remaining differences at 3 months after AHSCT. We conclude that treatment with AHSCT has a major but transient impact on the inflammatory milieu of peripheral blood.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla/sangue , Esclerose Múltipla/terapia , Condicionamento Pré-Transplante , Adulto , Autoenxertos , Biomarcadores/sangue , Feminino , Humanos , Inflamação/sangue , Inflamação/etiologia , Masculino
4.
Proc Natl Acad Sci U S A ; 111(28): 10305-10, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24982147

RESUMO

Over 40 susceptibility loci have been identified for type 1 diabetes (T1D). Little is known about how these variants modify disease risk and progression. Here, we combined in vitro and in vivo experiments with clinical studies to determine how genetic variation of the candidate gene cathepsin H (CTSH) affects disease mechanisms and progression in T1D. The T allele of rs3825932 was associated with lower CTSH expression in human lymphoblastoid cell lines and pancreatic tissue. Proinflammatory cytokines decreased the expression of CTSH in human islets and primary rat ß-cells, and overexpression of CTSH protected insulin-secreting cells against cytokine-induced apoptosis. Mechanistic studies indicated that CTSH exerts its antiapoptotic effects through decreased JNK and p38 signaling and reduced expression of the proapoptotic factors Bim, DP5, and c-Myc. CTSH overexpression also up-regulated Ins2 expression and increased insulin secretion. Additionally, islets from Ctsh(-/-) mice contained less insulin than islets from WT mice. Importantly, the TT genotype was associated with higher daily insulin dose and faster disease progression in newly diagnosed T1D patients, indicating agreement between the experimental and clinical data. In line with these observations, healthy human subjects carrying the T allele have lower ß-cell function, which was evaluated by glucose tolerance testing. The data provide strong evidence that CTSH is an important regulator of ß-cell function during progression of T1D and reinforce the concept that candidate genes for T1D may affect disease progression by modulating survival and function of pancreatic ß-cells, the target cells of the autoimmune assault.


Assuntos
Catepsina H/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Secretoras de Insulina/metabolismo , Adolescente , Alelos , Animais , Apoptose/genética , Catepsina H/genética , Linhagem Celular , Criança , Pré-Escolar , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Knockout , Ratos
5.
Diabetologia ; 59(3): 492-501, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26602422

RESUMO

AIMS/HYPOTHESIS: It is thought that T cells play a major role in the immune-mediated destruction of beta cells in type 1 diabetes, causing inflammation of the islets of Langerhans (insulitis). The significance of insulitis at the onset of type 1 diabetes is debated, and the role of the T cells poorly understood. METHODS: In the Diabetes Virus Detection (DiViD) study, pancreatic tissue from six living patients with recent-onset type 1 diabetes was collected. The insulitis was characterised quantitatively by counting CD3(+) T cells, and qualitatively by transcriptome analysis targeting 84 T and B lymphocyte genes of laser-captured microdissected islets. The findings were compared with gene expression in T cells collected from kidney biopsies from allografts with ongoing cellular rejection. Cytokine and chemokine release from isolated islets was characterised and compared with that from islets from non-diabetic organ donors. RESULTS: All six patients fulfilled the criteria for insulitis (5-58% of the insulin-containing islets in the six patients had ≥ 15 T cells/islet). Of all the islets, 36% contained insulin, with several resembling completely normal islets. The majority (61-83%) of T cells were found as peri-insulitis rather than within the islet parenchyma. The expression pattern of T cell genes was found to be markedly different in islets compared with the rejected kidneys. The islet-infiltrating T cells showed only background levels of cytokine/chemokine release in vitro. CONCLUSIONS/INTERPRETATION: Insulitis and a significant reserve reservoir for insulin production were present in all six cases of recent-onset type 1 diabetes. Furthermore, the expression patterns and levels of cytokines argue for a different role of the T cells in type 1 diabetes when compared with allograft rejection.


Assuntos
Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/imunologia , Insulina/sangue , Pâncreas/cirurgia , Linfócitos T/fisiologia , Adulto , Subpopulações de Linfócitos B/metabolismo , Diabetes Mellitus Tipo 1/cirurgia , Feminino , Humanos , Masculino , Adulto Jovem
6.
Am J Pathol ; 185(1): 129-38, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25524212

RESUMO

The cause of type 1 diabetes remains unknown. To dissect the link between hyperexpression of human leukocyte antigen (HLA) class I on the islet cells, we examined its expression in subjects with recent-onset type 1 diabetes. IHC showed seemingly pronounced hyperexpression in subjects with recent-onset type 1 diabetes, as well as in some nondiabetic subjects. In all subjects, HLA class I expression on exocrine tissue was low. However, no difference in the level of HLA class I expression was found between islet and exocrine tissue using Western blot, flow cytometry, real-time quantitative PCR, or RNA sequencing analyses. Also, the level of HLA class I expression on the messenger level was not increased in islets from subjects with recent-onset type 1 diabetes compared with that in nondiabetic subjects. Consistently, the HLA class I specific enhanceosome (NLRC5) and related transcription factors, as well as interferons, were not enhanced in islets from recent-onset type 1 diabetic subjects. In conclusion, a discrepancy in HLA class I expression in islets assessed by IHC was observed compared with that using quantitative techniques showing similar expression of HLA class I in islets and exocrine tissue in subjects with recent-onset type 1 diabetes, nor could any differences be found between type 1 diabetic and nondiabetic subjects. Results presented provide important clues for a better understanding on how this complex disease develops.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Genes MHC Classe I , Ilhotas Pancreáticas/metabolismo , Pâncreas Exócrino/metabolismo , Adolescente , Adulto , Idoso , Criança , Diabetes Mellitus Tipo 1/diagnóstico , Humanos , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pessoa de Meia-Idade , Fatores de Transcrição/metabolismo , Transcriptoma , Adulto Jovem
7.
Mult Scler Relat Disord ; 84: 105482, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38341978

RESUMO

BACKGROUND: Mitochondrial DNA (mtDNA) is a pro-inflammatory damage-associated molecular pattern molecule and could be an early indicator for inflammation and disease activity in MS. Autologous hematopoietic stem cell transplantation (aHSCT) is a potent treatment for MS, but its impact on mtDNA levels in cerebrospinal fluid (CSF) remains unexplored. OBJECTIVES: To verify elevated CSF mtDNA concentrations in MS patients and assess the impact of aHSCT on mtDNA concentrations. METHODS: Multiplex droplet digital PCR (ddPCR) was used to quantify mtDNA and nuclear DNA in 182 CSF samples. These samples were collected from 48 MS patients, both pre- and post-aHSCT, over annual follow-ups, and from 32 healthy controls. RESULTS: CSF ccf-mtDNA levels were higher in patients with MS, correlated to multiple clinical and analytical factors and were normalized after intervention with aHSCT. Differences before aHSCT were observed with regard to MRI-lesions, prior treatment and number of relapses in the last year prior to aHSCT. CONCLUSION: Our findings demonstrate elevated CSF mtDNA levels in MS patients, which correlate with disease activity and normalize following aHSCT. These results position mtDNA as a potential biomarker for monitoring inflammatory activity and response to treatment in MS.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Esclerose Múltipla , Humanos , Esclerose Múltipla/tratamento farmacológico , DNA Mitocondrial/líquido cefalorraquidiano , DNA Mitocondrial/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante Autólogo/métodos , Mitocôndrias
8.
Brain Commun ; 5(1): fcad011, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36756308

RESUMO

Multiple sclerosis has been established as an inflammatory disease of the central nervous system. Many aspects of the pathophysiology are still unknown and it is presently unclear how different treatments affect the immunopathology of multiple sclerosis. In this study, we explored cytokines discriminating between individuals with multiple sclerosis and healthy controls and then how these cytokines were affected by treatment intervention with autologous haematopoietic stem cell transplantation or intrathecal rituximab. CSF from individuals with multiple sclerosis and healthy controls were analysed with a proximity extension assay to simultaneously determine the level of 92 cytokines and other inflammation-related proteins. In total, CSF from 158 multiple sclerosis patients and 53 healthy controls were analysed. Sixty-four patients with relapsing-remitting multiple sclerosis and 27 with progressive multiple sclerosis took part in a cross-sectional study and underwent lumbar puncture on a single occasion. Forty-five patients with relapsing-remitting multiple sclerosis were treated with autologous haematopoietic stem cell transplantation and underwent lumbar puncture at baseline and then at follow-up visits made at 1-, 2- and 5 years. Twenty-two patients with progressive multiple sclerosis were treated with intrathecal rituximab and followed with lumbar punctures at baseline and then at follow-up visits made at 3-, 6- and 12 months. Of the 92 studied cytokines, 16 were found to be altered in multiple sclerosis and 11 were decreased after treatment with autologous haematopoietic stem cell transplantation. None of the studied cytokines was affected by treatment with intrathecal rituximab for progressive multiple sclerosis. Some proteins were highly associated with each other. Therefore, a cluster analysis was made and then the highest-ranked protein from the four highest-ranked clusters was used for the subsequent analyses. CCL3, IL-12B, CXCL10 and IL-8 discriminated between multiple sclerosis patients and controls, but only IL-12B differed between patients with relapsing-remitting and progressive multiple sclerosis. The CSF concentrations of CCL3, IL-12B and CXCL10 were decreased after autologous haematopoietic stem cell transplantation, whereas IL-8 appeared to be unaffected by this intervention. High concentrations of IL-8 were associated with worse outcome in both treatment groups. Overall, the results suggest a profound effect of autologous haematopoietic stem cell transplantation on the inflammatory milieu of the CSF in multiple sclerosis.

9.
Brain Commun ; 5(3): fcad164, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37274830

RESUMO

Multiple sclerosis is a highly complex and heterogeneous disease. At the onset it often presents as a clinically isolated syndrome. Thereafter relapses are followed by periods of remissions, but eventually, most patients develop secondary progressive multiple sclerosis. It is widely accepted that autoantibodies are important to the pathogenesis of multiple sclerosis, but hitherto it has been difficult to identify the target of such autoantibodies. As an alternative strategy, cell-based methods of detecting autoantibodies have been developed. The objective of this study was to explore differences in the binding of antibodies from sera and CSF of multiple sclerosis patients and controls to oligodendroglial and neuronal cell-lines, related to antibody type, immunoglobulin (IgG/IgM), matrix (serum/CSF) and disease course. The oligodendroglial and neuronal cell-lines were expanded in tissue culture flasks and transferred to 96-well plates at a concentration of 50 000 cells/well followed by fixation and blocking with bovine serum albumin. Sera and CSF samples, from healthy controls and multiple sclerosis patients, were incubated with the fixed cells. Epitope binding of immunoglobulins (IgG and IgM) in sera and CSF was detected using biotinylated anti-human IgM and IgG followed by avidin conjugated to horseradish peroxidase. Horseradish peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine substrate. Serum from 76 patients and 30 controls as well as CSF from 62 patients and 32 controls were investigated in the study. The binding was similar between clinically isolated syndrome patients and controls, whereas the largest differences were observed between secondary progressive multiple sclerosis patients and controls. Antibodies from multiple sclerosis patients (all disease course combined) bound more to all investigated cell-lines, irrespectively of matrix type, but binding of immunoglobulin G from CSF to human oligodendroglioma cell-line discriminated best between multiple sclerosis patients and controls with a sensitivity of 93% and a specificity of 96%. The cell-based enzyme linked immunosorbent assay (ELISA) was able to discriminate between multiple sclerosis patients and controls with a high degree of accuracy. The disease course was the major determinant for the antibody binding.

10.
JAMA Netw Open ; 5(5): e2211497, 2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35544139

RESUMO

Importance: B-cell-depleting monoclonal antibodies are widely used for treatment of multiple sclerosis but are associated with an impaired response to vaccines. Objective: To identify factors associated with a favorable vaccine response to tozinameran. Design, Setting, and Participants: This prospective cohort study was conducted in a specialized multiple sclerosis clinic at a university hospital from January 21 to December 1, 2021. Of 75 patients evaluated for participation who received a diagnosis of multiple sclerosis with planned or ongoing treatment with rituximab, 69 were included in the study, and data from 67 were analyzed. Exposures: Sex, age, number of previous rituximab infusions, accumulated dose of rituximab, previous COVID-19 infection, time since last rituximab treatment, CD19+ B-cell count before vaccination, CD4+ T-cell count, and CD8+ T-cell count were considered potential factors associated with the main outcome. Main Outcomes and Measures: Serological vaccine responses were measured by quantitation of anti-spike immunoglobulin G (IgG) antibodies, anti-receptor-binding domain (RBD) IgG antibodies, and their neutralizing capacities. Cellular responses to spike protein-derived SARS-CoV-2 peptide pools were assessed by counting interferon gamma spot-forming units in a FluoroSpot assay. Results: Among 60 patients with ongoing rituximab treatment (49 women [82%]; mean (SD) age, 43 [10] years), the median (range) disease duration was 9 (1-29) years, and the median (range) dose of rituximab was 2750 (500-10 000) mg during a median (range) time of 2.8 (0.5-8.3) years. The median (range) follow-up from the first vaccination dose was 7.3 (4.3-10.0) months. Vaccine responses were determined before vaccination with tozinameran and 6 weeks after vaccination. By using established cutoff values for anti-spike IgG (264 binding antibody units/mL) and anti-RBD IgG (506 binding antibody units/mL), the proportion of patients with a positive response increased with the number of B cells, which was the only factor associated with these outcomes. A cutoff for the B-cell count of at least 40/µL was associated with an optimal serological response. At this cutoff, 26 of 29 patients (90%) had positive test results for anti-spike IgG and 21 of 29 patients (72%) for anti-RBD IgG, and 27 of 29 patients (93%) developed antibodies with greater than 90% inhibition of angiotensin-converting enzyme 2. No factor associated with the cellular response was identified. Depending on the peptide pool, 21 of 25 patients (84%) to 22 of 25 patients (88%) developed a T-cell response with interferon gamma production at the B-cell count cutoff of at least 40/µL. Conclusions and Relevance: This cohort study found that for an optimal vaccine response from tozinameran, rituximab-treated patients with multiple sclerosis may be vaccinated as soon as possible, with rituximab treatment delayed until B-cell counts have reached at least 40/µL. An additional vaccination with tozinameran should be considered at that point.


Assuntos
COVID-19 , Esclerose Múltipla , Adulto , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19/uso terapêutico , Estudos de Coortes , Feminino , Humanos , Imunoglobulina G , Interferon gama , Masculino , Esclerose Múltipla/tratamento farmacológico , Estudos Prospectivos , Rituximab/uso terapêutico , SARS-CoV-2 , Vacinação
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