Prevention of EAE by tolerogenic vaccination with PEGylated antigenic peptides.

BACKGROUND: Therapeutic treatment options for chronic autoimmune disorders such as multiple sclerosis (MS) rely largely on the use of non-specific immunosuppressive drugs, which are not able to cure the disease. Presently, approaches to induce antigen-specific tolerance as a therapeutic approach; for example, by peptide-based tolerogenic 'inverse' vaccines have regained great interest. We have previously shown that coupling of peptides to carriers can enhance their capacity to induce regulatory T cells in vivo. METHOD: In this present study, we investigated whether the tolerogenic potential of immunodominant myelin T-cell epitopes can be improved by conjugation to the synthetic carrier polyethylene glycol (PEG) in an experimental autoimmune encephalomyelitis (EAE) mouse model for chronic MS (MOG C57BL/6). RESULTS: Preventive administration of the PEGylated antigenic peptide could strongly suppress the development of EAE, accompanied by reduced immune cell infiltration in the central nervous system (CNS). Depletion of regulatory T cells (Tregs) abrogated the protective effect indicating that Tregs play a crucial role in induction of antigen-specific tolerance in EAE. Treatment during the acute phase of disease was safe and did not induce immune activation. However, treatment at the peak of disease did not affect the disease course, suggesting that either induction of Tregs does not occur in the highly inflamed situation, or that the immune system is refractory to regulation in this condition. CONCLUSION: PEGylation of antigenic peptides is an effective and feasible strategy to improve tolerogenic (Treg-inducing) peptide-based vaccines, but application for immunotherapy of overt disease might require modifications or combination therapies that simultaneously suppress effector mechanisms.

Analysis of selected pro- and anti-inflammatory cytokines in patients with multiple injuries in the early period after trauma.

INTRODUCTION: Severe trauma causes damage to the protective barriers of the organism, and thus activates immunological reaction. Among substances secreted during this process pro-inflammatory cytokines are of high importance. THE AIM OF THE STUDY: Severe trauma causing multiple injuries is more likely to lead to particularly intensive inflammatory reaction, which can sometimes lead to serious complications, even life-threatening. The aim of the study is to determine those parameters which may serve as predictors of infectious complications and to enable estimation of the patient's immunological status before the decision to introduce elective procedures. MATERIAL AND METHODS: The study population included patients with multiple trauma treated in the Department of Trauma Surgery of the Medical University of Gdansk. The severity of injuries was evaluated with commonly used numerical scales (Revised Trauma Score - RTS, Injury Severity Score - ISS, Glasgow Coma Scale - GCS). Blood samples were collected on the first, second, and fifth day after injury. Evaluated parameters: C-reactive protein (CRP), the level of cytokines: IL-8, IL-1ß, IL-6, TNF, IL-12p70, and IL-10. Control population: individuals without injury. RESULTS: Evaluation of IL-6, IL-8, and CRP levels in patients with multiple trauma in the early period after injury (2-3 days) could be considered as a predictor of delayed infection (5-10 days). CRP level, being cheap and commonly accessible, can be used in clinical practice enabling identification of patients at higher risk of infectious complications and introduction of appropriate treatment and prevention. The analysis of the mentioned parameters may contribute to choosing an appropriate management strategy, including "timing" depending on the patient's biological status.

Associations between single-nucleotide polymorphisms of RFC-1, GGH, MTHFR , TYMS, and TCII genes and the efficacy and toxicity of methotrexate treatment in patients with rheumatoid arthritis.

INTRODUCTION: The differences in drug efficacy and adverse reactions may be caused by genetic variations in drug metabolism between individuals. OBJECTIVES: The aim of the study was to evaluate the effect of gene polymorphisms on the efficacy of therapy and side effects in patients with rheumatoid arthrit s (RA) treated with methotrexate (MTX). PATIENTS AND METHODS: A total of 273 Caucasian patients with RA were treated with MTX for at least 6 months or stopped MTX because of adverse effects. Seven polymorphisms (RFC-1 c.80G>A, GGH c.-401C>T, MTHFR c.1298A>C and c.677C>T, TYMS 2R/3R, TYMS 6-bp deletion, and TCII c.593T>C) were examined for their effects on MTX efficacy and toxicity. Genomic DNA was obtained from peripheral blood leukocytes. RESULTS: Of all patients, 53% reported some adverse effects during at least 1 visit, which led to MTX withdrawal in 17% of the patients. Adverse effects were more frequent in patients with the MTHFR 677T allele than in those with the 677CC genotype (odds ratio [OR], 1.97; P = 0.01) and in those with the GGH 401CC genotype than in those with the GGH 401CT and TT genotypes (OR, 3.8; P = 0.05). Furthermore, the MTHFR 677T allele was associated with increased activity of aminotransferases (OR, 3.4; P = 0.02). MTX-related hepatotoxicity and alopecia were more common in patients with the RFC-1 80AA genotype (OR, 3.5, P = 0.01; OR, 2.4, P = 0.04; respectively). A more rapid positive response to MTX therapy was demonstrated in MTHFR 677CC homozygotes (OR, 3.4; P = 0.001). There were no other associations between single -nucleotide polymorphisms and the efficacy of MTX treatment. CONCLUSIONS: The MTHFR 677CC and GGH 401TT and CT genotypes were associated with a reduction in the number of MTX-related adverse events. Future allele and genotype analyses may help identify the subsets of RA patients with an increased risk of adverse effects.

Different pattern of T-cell subpopulations in peripheral blood of patients with rheumatoid arthritis at various stages of disease development.

INTRODUCTION:  The comparison of changes in peripheral T-cell subpopulations at different stages of rheumatoid arthritis (RA) development may be important to understand the pathomechanism and to elucidate the course of RA. So far, there have been no comprehensive studies regarding the proportions of T cells in early and long­lasting RA. OBJECTIVES:  The aim of this study was to assess the proportion of the main peripheral T-cell subpopulations in patients at various stages of RA development. PATIENTS AND METHODS:  We enrolled 75 patients who were divided into 4 subgroups depending on the diagnosis: undifferentiated arthritis (UA), which later developed into RA (UA­RA) and other diseases (UA­non­RA); clinically confirmed untreated RA; long-term treated RA; and the control group. Flow cytometry was used to assess T-cell subpopulations. RESULTS:  Patients with clinically confirmed untreated RA differed (P <0.05) in the proportion of CD4+ T-cell subpopulations expressing activation markers compared with controls (CD69, CD25, HLA­DR, CD95) and UA patients (CD95). Untreated RA patients had the highest proportion of regulatory CD4+ T cells compared with control and other groups. The percentage of CD28- T cells was higher only in the group with clinically confirmed RA but not in those with early RA (at the UA stage). CONCLUSIONS:  The peripheral T lymphocyte phenotype in very early RA is not similar to that observed in clinically­confirmed RA. Patients with a confirmed diagnosis of RA can be easily differentiated based on the absolute numbers of the main T-cell subpopulations; however, the percentage of the main T-cell subpopulations do not discriminate those patients in the UA cohort who will develop RA.

Isolation of xanthone and benzophenone derivatives from Cyclopia genistoides (L.) Vent. (honeybush) and their pro-apoptotic activity on synoviocytes from patients with rheumatoid arthritis.

A fast and efficient method for the isolation of the C-glucosidated xanthones mangiferin and isomangiferin from the South-African plant Cyclopia genistoides was developed for the first time. The procedure involved extraction, liquid-liquid partitioning with ethyl acetate and subsequent precipitation of mangiferin and isomangiferin from methanol and acetonitrile-water fractions, respectively. Additionally, two benzophenone derivatives: 3-C-ß-glucosides of maclurin and iriflophenone, were isolated from C. genistoides extracts using semi-preparative HPLC. Apart from the above, the isolation procedure also yielded hesperidin and small amounts of luteolin. The structures of the compounds were determined by 1D and 2D NMR experiments and/or LC-DAD-ESI-MS. The selected Cyclopia constituents were screened for pro-apoptotic activity on TNF-α-stimulated synovial cells isolated from rheumatoid arthritis patients. The strongest effect, measured as percent of apoptotic cells, was recorded for isomangiferin (75%), followed by iriflophenone 3-C-ß-glucoside (71%), hesperidin (67%) and mangiferin (65%). The results are encouraging for further studies on the use of the above compounds in the treatment of rheumatoid arthritis.

Disease activity in patients with long-lasting rheumatoid arthritis is associated with changes in peripheral blood lymphocyte subpopulations.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease and it is known that lymphocytes play a major role in its pathogenesis. However, there have been no comprehensive studies on the changes in peripheral blood lymphocyte (PBL) subpopulations expressing different clusters of differentiation (CD) in patients with long-lasting RA. OBJECTIVES: The aim of our study was to measure the main subpopulations of PBL, expression of costimulatory marker CD28, and activation status of CD4+ T cells depending on clinical disease activity in long-lasting RA. PATIENTS AND METHODS: The study comprised 60 patients with RA and 19 healthy volunteers. Disease activity, the proportion and number of the main PBL subpopulations (T, B, natural killer [NK], and NK T cells [NKT]), the expression of costimulatory marker CD28, and the activation status of CD4+ T cells were evaluated on the same day. A multicolor flow cytometry with marked monoclonal antibodies was used for the assessment of lymphocyte subpopulations. RESULTS: The percentage of CD3+CD4+, NKT, CD4+CD28-, CD8+CD28-, CD4+CD69+, CD4+CD25+, and CD4+HLA-DR+ was significantly higher in RA compared with the control group. A higher proportion of CD4+CD28- was associated with more active disease, while an inverse correlation was observed for B cells. The proportion of CD4+CD28- was not associated with disease activity. The number of CD4+CD69+ cells in RA patients increased with increasing DAS28, while the number of CD4+HLA-DR+ T cells showed no such association. CONCLUSIONS: Our results have shown for the first time an association between the phenotype patterns of PBL T, B, and NKT and RA activity in patients with long-lasting disease, which reinforces the hypothesis that PBL play an important role in modifying or maintaining the disease activity.

Increased percentage of CD8+CD28- T cells correlates with clinical activity in primary Sjögren's syndrome.

The role of CD28- T cell subpopulations in primary Sjögren's syndrome (pSS) has become controversial. Changes in the number of CD28- T cells have been demonstrated in autoimmune diseases in co-existence with Sjögren's syndrome. The study aimed to indicate differences in the number of CD4+CD28- and CD8+CD28- T cells in patients with sicca syndrome and suspected pSS. Thirty patients with sicca syndrome at baseline were studied and followed up for 5 months. After final diagnosis, comparison was made of the previously recorded lymphocyte subpopulations in patients with pSS and those in other defined subgroups. Notably high percentages of CD8+CD28- T cells were indicated in pSS patients, which correlated with the severity of the sicca symptoms and cutaneous and muscular systemic disease activity. Changes in CD8+CD28- T cell percentages may thus assist in the early differential diagnosis of pSS patients from those with similar clinical symptoms.

Changes in proliferation kinetics of T cells: a new predictive cellular biomarkers for early rheumatoid arthritis?

OBJECTIVE: It has been demonstrated that early treatment of rheumatoid arthritis (RA) patients prevents further joint damage and disability, but biomarkers enabling early RA to be distinguished within the undifferentiated arthritis (UA) cohort are still being sought. PURPOSE: The aim of the research was to study the pathomechanism of initiation and progression of UA→RA and to find such new predictive biomarkers on the basis of functional studies of the immune system. METHODS: 55 patients with UA were enrolled into the study and followed up for 2 years. The dynamic parameters of proliferation of the peripheral blood CD4+ T cells were recorded at the UA stage. During the follow-up study, standard diagnostic procedures were performed to make the final diagnosis. Comparison of the CD4+ T cell proliferation parameters in the UA-RA and UA-non-RA patients was conducted after the final diagnosis was established. RESULTS: Our studies showed that the G0-G1 transition time, the cell cycle duration, the number of cell divisions per dividing CD4+ cells and the percentage of dividing CD4+ T cells differed significantly between UA-RA and UA-non-RA patients. Moreover, these proliferation parameters achieved higher specificity and sensitivity in the detection of early RA within UA patients compared to the routine serological tests available. CONCLUSION: The proliferation parameters of CD4+ T cells reflect central pathophysiological changes in RA and can be used as new biomarkers for early RA diagnosis, which would enable the international rheumatology recommendation to be achieved concerning the early diagnosis and treatment of RA patients.