Helena Mareckova

High-dose immunosuppressive therapy with PBPC support in the treatment of poor risk multiple sclerosis.

High-dose immunoablative chemotherapy with autologous haematopoietic cell support might be beneficial in the treatment of intractable forms of MS. We mobilised PBPC in 11 patients with secondary progressive MS and finally eight patients were grafted after high-dose BEAM chemotherapy with either in vitro or in vivo T cell depletion. Median EDSS and SNRS scores at the time of inclusion were 6.5 (6.5–7.5) and 56 (44–65), respectively. PBPC mobilisation was safe with no serious adverse effects, and without significant aggravation of disability. One patient improved significantly (by 1.0 point on EDSS) after the mobilisation. Two mobilisation failures were observed. No life-threatening events occurred during the transplantation. All grafted patients, except one, at least stabilised their disability status. One patient improved significantly (by 1.5 points on EDSS), two patients improved slightly (by 0.5 points on EDSS), one patient worsened by 1.0 point on the EDSS in 10 months. Improvement occurred with a delay of 2–4 months. Median EDSS and SNRS of grafted patients at the last follow up were 6.5 (5.5–8.5) and 64 (39–73), respectively with median follow-up of 8.5 months. Further follow-up is needed to determine the disease course after complete immune reconstitution. Bone Marrow Transplantation (2000) 25, 525–531.

Cercarial Dermatitis, a Neglected Allergic Disease

Cercarial dermatitis (swimmers itch) is a common non-communicable water-borne disease. It is caused by penetration of the skin by larvae (cercariae) of schistosomatid flukes and develops as a maculopapular skin eruption after repeated contacts with the parasites. The number of outbreaks of the disease is increasing, and cercarial dermatitis can therefore be considered as an emerging problem. Swimmers itch is mostly associated with larvae of the bird schistosomes of Trichobilharzia spp. Recent results have shown that mammalian infections (including man) manifest themselves as an allergic reaction which is able to trap and eliminate parasites in the skin. Studies on mammals experimentally infected by bird schistosome cercariae revealed, however, that during primary infection, parasites are able to escape from the skin to the lungs or central nervous system. This review covers basic information on detection of the infectious agents in the field and the clinical course of the disease, including other pathologies which may develop after infection by cercariae, and diagnosis of the disease.

Production of interleukins 10 and 12 by activated peripheral blood monocytes/macrophages in patients suffering from chronic hepatitis C virus infection with respect to the response to interferon and ribavirin treatment.

Circulating monocytes/macrophages are important for the initiation of immune responses to hepatitis C virus (HCV). Their presentation capacities and production of immunoregulatory cytokines enable them to activate cellular immune responses which is critical in determining the outcome of infection. We used flow cytometry to examine the expression of a CD80 costimulatory molecule on the surface of peripheral blood CD14+ monocytes/macrophages and to analyse the production of IL10 and IL12 by these cells. Forty-three individuals (6 asymptomatic HCV carriers, 37 patients with chronic hepatitis C (CHC)) were enrolled in this study. Thirty-seven patients with CHC (23 responders and 14 non-responders, NR) received combination (interferon+ribavirin) treatment for 52 weeks. The baseline percentage of CD14+CD80+ peripheral blood monocytes/macrophages was high in patients with CHC (P<0.001) and returned to normal after the treatment. All patients with CHC showed significantly high production of IL10 (P<0.001). In asymptomatic HCV carriers production level of this cytokine tended to be higher than in patients with CHC (P<0.001). A baseline production of IL12 was higher in asymptomatic HCV carriers and patients with CHC compared to healthy controls (P<0.001). The level of IL12 production was increased in treatment responders whereas in NR returned to normal value. Our data argue against functional impairment of circulating monocytes/macrophages during HCV infection. Furthermore, the positive therapeutic outcome following combination treatment might associate with increased production of IL12 by these cells.

Intracellular cytokine production in ANCA-associated vasculitis: low levels of interleukin-10 in remission are associated with a higher relapse rate in the long-term follow-up.

BACKGROUND AND AIMS Dysregulation of cell-mediated immune response likely plays a role in the pathogenesis of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), but it has not yet been fully established. The aim of this study was to assess the intracellular cytokine production in patients with AAV at different stages of the disease, in particular, in relation to the long-term prognosis. METHODS We included 69 patients with AAV and 24 healthy controls. Using flow cytometry, the following intracellular cytokines (IC) were measured in all patients: interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-2 and interleukin-4 in CD3+T cells and interleukin-10 (IL-10) and interleukin 12 (IL-12) in monocytes. Patients were then prospectively followed for a median of 43 months and cytokine production was related to the long-term prognosis. RESULTS When compared to healthy controls, increased IL-12 production was observed in AAV patients, both active (p<0.01) and in remission (p<0.05). In remission, increased IFN-gamma production was also found (p<0.01). IL-10 production was higher in active patients than in patients in remission (p<0.05) but did not differ from controls. Patients in remission who developed a relapse during follow-up had significantly lower IL-10 production than those without relapse (p<0.01). Results of this prospective study of IC production in AAV confirm findings of previous studies measuring circulating cytokine levels. CONCLUSIONS Activation of the immune system in AAV patients is noticeable even in remission. Patients with AAV display increased IL-12 production, which seems to be counterbalanced by IL-10. Low IL-10 levels in remission are associated with a higher relapse rate in the long-term follow-up.

Dipeptidyl peptidase-IV in synovial fluid and in synovial fluid mononuclear cells of patients with rheumatoid arthritis

BACKGROUND Dipeptidyl peptidase-IV (DPP-IV) enzymatic activity controls biological halftime of multiple local mediators. Its deregulation is associated with pathogenesis of several autoimmune diseases, including rheumatoid arthritis (RA). Although DPP-IV is the canonical representative of the group, a number of other proteins have been shown to have similar enzymatic activity. This study was aimed to identify the molecular source of DPP-IV activity in synovial fluid (SF) and fluid mononuclear cells (FMNC) in patients with RA and osteoarthritis (OA). In addition, the association of DPP-IV and the concentration of stromal cell-derived factor-1alpha (SDF), DPP-IV substrate, were evaluated. METHODS DPP-IV activity was measured by the kinetic fluorimetric method. The expression of studied molecules in FMNC and their concentrations in SF were assayed using flow cytometry and ELISA respectively. RESULTS DPP-IV activity in SF, dominantly derived from the canonical DPP-IV, does not significantly differ between RA and OA. However, a significantly lower DPP-IV activity and expression in FMNC was found in RA as opposed to OA patients. Negative correlation between SDF concentration in SF and the relative amount of CD3+CD26+ cells was observed. CONCLUSIONS We report decreased presence of DPP-IV/CD26 in CD3+ FMNC in RA, which also may participate on impaired balance of SDF concentration in SF.

Immunological predictors of different responses to combination therapy with interferon α and ribavirin in patients with chronic hepatitis C

Background: This study aimed to investigate peripheral blood CD4+ T-helper (Th) and CD8+ cytotoxic T-lymphocyte (CTL) responses to combination treatment with interferon (IFN) α and ribavirin in 59 patients with chronic hepatitis C, and to correlate the results with the therapy outcome. Methods: The expression of activation molecules on the surface of CD8+ T cells and cytokine production by in-vitro activated CTLs and Th lymphocytes were examined before and at the end of the therapy, using flow cytometry. Results: There were 36 complete responders to the treatment and 23 transient responders who relapsed after withdrawal of the therapy. A significant increase in the production of Th1-type cytokines [IFNγ, interleukin 2 (IL2), and tumor necrosis factor-α (TNFα)] was found at the end of the treatment in complete responders compared with baseline values (P < 0.001). In contrast, transient responders had a marked decrease in the percentage of activated CD8+ T cells expressing CD28 or HLA-DR costimulatory molecules in peripheral blood, and a lower production of TNFα by CTLs and Th cells at the end of the therapy with respect to pretreatment values (P < 0.001). Conclusions: The efficacy of IFNα and ribavirin combination therapy for chronic hepatitis C is associated with a vigorous response of peripheral blood Th1 cells, whereas weak CTL responses at the end of the therapy might predict a further relapse of the disease.

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of 3H-thymidine and differential gating method

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, (3)H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.

Natalizumab in the treatment of patients with multiple sclerosis : First experience

Abstract:  Multiple sclerosis (MS) usually develops in young adults with a complex predisposing genetic background. Polymorphisms in the gene for chemokine receptor CCR5 have been proposed to confer susceptibility to or protection from MS. Study of molecules participating in the inflammatory process contributed to the development of a new humanized monoclonal antibody, natalizumab, aimed at the adhesive molecule VLA‐4. Natalizumab (Biogen Idec/Elan) went through successful clinical studies and its clinical testing was also carried out in the Czech Republic. Twenty‐one patients with MS were included in the AFFIRM study (2‐year, placebo‐controlled study and consecutive 7‐month unblinded natalizumab treatment); immunophenotyping of the cerebrospinal fluid (CSF)– CD4+CCR5+CXCR3+ lymphocytes, using flow cytometer FACSCalibur and monoclonal antibodies (BD Biosciences), was done at the end of natalizumab treatment and 1 year after the therapy withdrawal. Compared to MS patients receiving other therapy, the patients treated with natalizumab had statistically significantly (P < 0.0001) higher levels of CCR5+ and lower levels of CD4+ T lymphocytes in CSF, whereas the levels of CXCR3+ lymphocytes were almost the same as in other patients. CCR5‐positive CSF lymphocytes decreased 1 year after treatment withdrawal. Natalizumab treatment alters the percentage of CCR5+ and CD4+ cells in CSF. In view of the excellent temporary clinical results of the therapy, which are yet to be assessed in the course of a longer time period, our results show a possible explanation for the therapeutic success of this drug as well as for the development of progressive multifocal leukoencephalopathy.

T helper, cytotoxic T lymphocyte, NK cell and NK-T cell subpopulations in patients with chronic hepatitis C.

The phenotype of intrahepatic (IHL) and peripheral blood lymphocytes (PBL) was determined, and the production of cytokines by T lymphocytes analyzed in patients with chronic hepatitis C (CHC). Three-color fluorescence-activated cytometric analysis was done for 36 patients with untreated CHC. The percentage of peripheral blood memory T cells was higher in patients with CHC than in healthy controls (all data in %, significant atp<0.001; 74.6±2.7vs. 58.3±4.5), and a greater proportion of them were observed in the intrahepatic compartment (IHL—94.2±2.8vs. PBL—74.6±2.7). There was a higher percentage of peripheral blood T helper 1 lymphocytes expressing IFN-γ (IFN-γ/IL-4) in these patients (4.6±0.7vs. control—2.2±0.5). The expression of CXCR3 chemokine receptors on peripheral blood T helper cells was also high compared with the control (39.8±4.8vs. 26.8±2.5) and a large percentage of T cells expressing CXCR3 or CCR5 chemokine receptors was observed in hepatitis C virus (HCV)-infected liver (CXCR3: IHLvs. PBL—74.9±5.7vs. 39.8±4.8; CCR5: IHLvs. PBL—65.9±5.9vs. 19.1±2.1). The intrahepatic compartment contains a greater proportion of activated cytotoxic T lymphocytes (CTL) and natural killer-T (NK-T) cells than peripheral blood (CTL: IHLvs. PBL—69.5±3.2vs. 59.9±3.1; NK-T: IHLvs. PBL— 10.6±2.5vs. PBL: 3.99±0.5). The data suggest that in HCV-infected subjects, memory TH1 lymphocytes, activated CTL and NK-T cells compartmentalize in liver tissue and could play an important role in pathogenesis of chronic hepatitis.

Intracellular Cytokine Production in Peripheral Blood Lymphocytes: A Comparison of Values in Infertile and Fertile Women

Citation 
Horká P, Jarošová R, Malíčková K, Janatková I, Marečková H, Zima T, Kalousová M. Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women. Am J Reprod Immunol 2011; 65: 466–469