The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-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.

Toxocara canis larvae reinfecting BALB/c mice exhibit accelerated speed of migration to the host CNS.

Using a small animal imaging system, migratory activity of Toxocara canis larvae stained by carboxyfluorescein succinimidyl ester (CFSE) was observed post primary infection (PPI) and post reinfection (PR) of BALB/c mice. Each infection was performed with 1,000 larvae per mouse. Primary infections were performed with labeled larvae, while for challenge infections the reinfecting larvae were stained by CFSE. The worm burden in mouse organs was determined during a period from 6 h to 21 days and 4 months PPI and PR. In comparison with primary infections that led to the first larvae appearance in the brain after 60 h, greatly accelerated migration of the parasites administered 3 weeks PPI to the CNS and eyes of challenged mice was noted-in both organs the larvae appeared 6 h PR. In all challenged mice, reinfecting larvae prevailed in the resident parasite population. Preliminary experiments with Toxocara cati larvae also revealed early brain involvement in primarily infected mice. Staining of T. canis larvae by CFSE had no effect on the development of a humoral antibody response against T. canis excretory-secretory antigens. In ELISA, elevated levels of specific IgG and IgG1 were noted on day 14 PPI and the levels of antibodies increased till the end of experiment. Reinfection induced an increase in the levels of both antibodies. In terms of optical density, IgG1 antibodies gave higher values in all sera examined. In ELISA for IgG antibodies, an increase in the avidity index of around 50% was detected 1 month PPI; higher-avidity antibodies were also detected in sera of reinfected animals.

Plasma calprotectin in chronically dialyzed end-stage renal disease patients.

OBJECTIVE: The current study aimed to evaluate plasma calprotectin levels and clearance end-stage renal disease (ESRD) patients with and without acute infection undergoing chronic hemodialysis (HD). MATERIALS AND METHODS: Blood samples from 54 HD patients were obtained before and after the HD and 42 healthy blood donors were examined as controls. The blood levels of calprotectin, procalcitonin, C-reactive protein (CRP), and intracellular production of interleukins 10 and 12 in monocytes were determined in both groups. RESULTS: The concentrations of plasma calprotectin in ESRD patients were significantly higher than in healthy controls (p < 0.05). No differences between pre- and post-HD calprotectin plasma levels were observed (p = 0.07 for two-tailed test). Plasma calprotectin levels were not significantly influenced by the presence of acute infection (p = 0.19) or diabetes (p = 0.42). A significant positive correlation of plasma calprotectin to plasma beta-2 microglobulin was proven (p < 0.05). Procalcitonin (PCT), CRP, IL-10, and IL-12 were not correlated with plasma calprotectin before or after HD. The elevation of plasma calprotectin was correlated strongly to the hemodialysis vintage (r = 0.55, p < 0.01). CONCLUSIONS: Significantly elevated levels of plasma calprotectin in ESRD patients occur without an acute infectious cause and are not affected by the presence of diabetes. By analogy to plasma beta-2 microglobulin, a close relation of plasma calprotectin to HD vintage was shown.

Natalizumab in the treatment of patients with multiple sclerosis: first experience.

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.

Occupational asthma after withdrawal from the occupational allergen exposure.

Occupational asthma is characterised by airway inflammation, variable airflow limitation and airway hyperresponsiveness related causally to work. The aim of the study was to ascertain whether in patients with occupational asthma findings persist after withdrawal from occupational allergen exposure. A group of 37 patients with occupational asthma and a control group of 19 persons were examined. Results in asthmatics obtained during the first visit when occupational asthma was acknowledged, were compared with recent results about 6.5 yr on average after elimination of occupational allergen exposure. Recent findings in occupational asthma patients were compared with the control group. In patients with occupational asthma, no significant differences were found between results obtained at the first and recent visit. Symptoms of asthma persisted in as much as 86.5% of patients. During recent examination there were more positive results in asthmatic patients comparing with the control group in histamine challenge (61.3 vs. 5.3%, p<0.01), eosinophile cationic protein (41.7 vs. 5.3%, p<0.05), prick tests (45.9 vs. 10.5%, p<0.05). Positive results of the present histamine challenge test and elevated eosinophils in sputum were more frequent (p<0.05) in patients with occupational asthma due to high molecular weight allergens than to low molecular weight allergens.

CXCR3, CCR5, and CRTH2 Chemokine Receptor Expression in Lymphocytes Infiltrating Thyroid Nodules with Coincident Hashimoto's Thyroiditis Obtained by Fine Needle Aspiration Biopsy.

Objective. To determine the expression of chemokine receptors in lymphocytes from thyroid nodules and peripheral blood in patients with and without Hashimoto's thyroiditis (HT). Patients and Methods. The study included 46 women with thyroid nodules and HT and 60 women with thyroid nodules without HT (controls) who underwent a fine needle aspiration biopsy (FNAB). Expression of chemokine receptors CXCR3, CCR5, and CRTH2 was assessed by flow cytometry in lymphocytes from FNAB samples and from peripheral blood. Results. The percentage of CRTH2+ lymphocytes was higher in nodules with HT in comparison with controls, both in FNAB samples (13.95 versus 6.7%, p = 0.008) and in peripheral blood (6.7 versus 5.13%, p = 0.047), and positively correlated with serum antibodies to thyroid peroxidase (r = 0.243; p = 0.026) and negatively correlated with thyroid volume (r = -0.346; p = 0.008). Lymphocytes from neoplastic nodules showed a higher expression of both CXCR3 and CCR5 than those from hyperplastic ones. Conclusion. Flow cytometry performed in FNAB samples may serve as a good tool in investigation of intrathyroidal expression of immunological parameters. In our study, the CRTH2 expression on thyroid-infiltrating lymphocytes as well as on lymphocytes from peripheral blood was increased in HT as compared to controls.

Two-year outcome of thymectomy with or without immunosuppressive treatment in nonthymomatous myasthenia gravis and its effect on regulatory T cells.

BACKGROUND: Myasthenia gravis (MG) is the autoimmune disorder in which the thymus is considered the pathogenic organ. Thymectomy (TE) is a therapeutic option for MG and often ameliorates clinical symptoms. METHODS: We evaluated clinical features and outcomes after TE in patients without thymoma and the influence of TE with or without concomitant immunotherapy on the CD4(+)CD25(+) regulatory T cell subpopulation of lymphocytes in peripheral blood in defined followed groups of nonthymomatous MG patients. RESULTS: A total of 46 patients with generalized MG who underwent transsternal TE were identified. Neurologic outcomes after TE were favorable for the majority of patients mainly from the group treated with corticosteroids or combined immunosuppressive treatment. TEs with immunosuppressive treatment in MG patients were associated with increased percentages of CD4(+)CD25(+) cells (p<0.001). No significant change in the postoperative levels of CD4(+)CD25(+) cells was observed in thymectomized patients who preoperatively only received pyridostigmine. Also their clinical response to TE after 2 years of follow-up was worst of all followed groups. CONCLUSIONS: The exact mechanism by which TE ameliorates symptoms of MG is yet not clear. These observations indicate that increased percentages of CD4(+)CD25(+) T cells in MG may be related to disease stability and that TE and synergistic effect with concomitant, continuing immunotherapy augmented the proportion of CD4(+)CD25(+) T cells. On the basis of our observations TE alone is not enough to increase the number of circulating CD4(+)CD25(+) regulatory T cells and to establish complete stable remission.

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.

Immunological predictors of different responses to combination therapy with interferon alpha 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) alpha 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 [IFNgamma, interleukin 2 (IL2), and tumor necrosis factor-alpha (TNFalpha)] 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 TNFalpha by CTLs and Th cells at the end of the therapy with respect to pretreatment values (P < 0.001). CONCLUSIONS: The efficacy of IFNalpha 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.

Cercarial dermatitis, a neglected allergic disease.

Cercarial dermatitis (swimmer's 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. Swimmer's 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.

Intracellular cytokine production in peripheral blood lymphocytes: a comparison of values in infertile and fertile women.

PROBLEM: To analyze the relation of the fertility and pregnancy of women of childbearing age to the intracellular (IC) production of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukins 2 and 4 (IL-2 and IL-4). METHOD OF STUDY: Intracellular cytokine production in peripheral blood CD3(+) CD4(+) lymphocytes was analyzed by flow cytometry in 185 women being treated for infertility and 50 fertile women of childbearing age. RESULTS: Infertile women have a significantly higher IC production of TNF-α, IFN-γ, IL-2, and IL-4 and higher ratios of TNF-α/IL-2, TNF-α/IL-4, and TNF-α/IFN-γ compared to the fertile women. CONCLUSION: Cytokines produced by Th lymphocytes are important in orchestrating the immune response during conception, and Th-cell dysregulation could be a reason for infertility.

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.

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.