Human exposure to diesel exhaust induces CYP1A1 expression and AhR activation without a coordinated antioxidant response.

BACKGROUND: Diesel exhaust (DE) induces neutrophilia and lymphocytosis in experimentally exposed humans. These responses occur in parallel to nuclear migration of NF-κB and c-Jun, activation of mitogen activated protein kinases and increased production of inflammatory mediators. There remains uncertainty regarding the impact of DE on endogenous antioxidant and xenobiotic defences, mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) and the aryl hydrocarbon receptor (AhR) respectively, and the extent to which cellular antioxidant adaptations protect against the adverse effects of DE. METHODS: Using immunohistochemistry we investigated the nuclear localization of Nrf2 and AhR in the epithelium of endobronchial mucosal biopsies from healthy subjects six-hours post exposure to DE (PM10, 300 µg/m3) versus post-filtered air in a randomized double blind study, as a marker of activation. Cytoplasmic expression of cytochrome P450s, family 1, subfamily A, polypeptide 1 (CYP1A1) and subfamily B, Polypeptide 1 (CYP1B1) were examined to confirm AhR activation; with the expression of aldo-keto reductases (AKR1A1, AKR1C1 and AKR1C3), epoxide hydrolase and NAD(P)H dehydrogenase quinone 1 (NQO1) also quantified. Inflammatory and oxidative stress markers were examined to contextualize the responses observed. RESULTS: DE exposure caused an influx of neutrophils to the bronchial airway surface (p = 0.013), as well as increased bronchial submucosal neutrophil (p < 0.001), lymphocyte (p = 0.007) and mast cell (p = 0.002) numbers. In addition, DE exposure enhanced the nuclear translocation of the AhR and increased the CYP1A1 expression in the bronchial epithelium (p = 0.001 and p = 0.028, respectively). Nuclear translocation of AhR was also increased in the submucosal leukocytes (p < 0.001). Epithelial nuclear AhR expression was negatively associated with bronchial submucosal CD3 numbers post DE (r = -0.706, p = 0.002). In contrast, DE did not increase nuclear translocation of Nrf2 and was associated with decreased NQO1 in bronchial epithelial cells (p = 0.02), without affecting CYP1B1, aldo-keto reductases, or epoxide hydrolase protein expression. CONCLUSION: These in vivo human data confirm earlier cell and animal-based observations of the induction of the AhR and CYP1A1 by diesel exhaust. The induction of phase I xenobiotic response occurred in the absence of the induction of antioxidant or phase II xenobiotic defences at the investigated time point 6 h post-exposures. This suggests DE-associated compounds, such as polycyclic aromatic hydrocarbons (PAHs), may induce acute inflammation and alter detoxification enzymes without concomitant protective cellular adaptations in human airways.

Improvement of clinical and immunological parameters after allergen-specific immunotherapy in atopic dermatitis.

BACKGROUND: Allergen immunotherapy (AIT) is considered a curative treatment in some atopic diseases, but in AD contradictory clinical results exist and the action of AIT has not been elucidated. In the literature, there is no evidence for parallel investigations of permeability barrier, cutaneous and blood immune responses after AIT in AD. OBJECTIVES: The objective was to investigate immune parameters in the blood and skin and to detect clinical and barrier changes after AIT in AD. METHODS: Mild-to-moderate AD patients (n = 14) with concomitant allergic rhinitis to house dust mites were selected. All patients received topical treatment, while eight patients were randomly selected for adjuvant AIT also. At baseline and after 6 months, clinical, barrier and immunological investigations (serum and skin tests) were performed. In selected patients, biopsies from atopy patch tests (APTs) were analysed by immunohistochemistry for AD-relevant immune cells and mediators. RESULTS: In the adjuvant AIT group, clinical parameters and barrier functions improved significantly. Blood immune parameters displayed no significant changes. Post-AIT APT became negative in all patients in the AIT group, but remained positive in the non-AIT group. Cutaneous dendritic cell and T-cell recruitment decreased significantly after allergen challenge in the AIT group, but no significant changes in skin or serum immunoglobulin E levels or prick test (SPT) reactivity were detected. CONCLUSIONS: Allergen immunotherapy is a beneficial adjuvant treatment for sensitized AD patients. AIT improves not only clinical symptoms, but also permeability barrier functions. The effect of AIT on sensitization should be detected by APT, not by SPT.

Regulatory T Cells in the Context of New-Onset Diabetes After Renal Transplant: A Single-Center Experience.

BACKGROUND: New-onset diabetes mellitus after transplant (NODAT) is a well-known complication of renal transplant that severely affects graft and patient survival. It is necessary to explore further risk factors and reveal the underlying pathomechanism. METHODS: Renal transplants performed between January 2010 and June 2018 were involved. Exclusion criteria were the recipient age younger than 18 years, follow-up period less than 6 months, and patients with diabetes at the time of transplant. Only primary kidney transplants were involved in our study, which totaled 223 cases. Besides donor and recipient demographic data, the type of immunosuppression, the average fasting glucose level, and T-subset profiles were compared. RESULTS: Of 223 cases there were 33 patients (14.8%) with NODAT (17 female; mean age, 54.2 [SD, 10.3] years; mean body mass index [calculated as weight in kilograms divided by height in meters squared], 27.8 [SD, 5.1]; mean follow-up, 43.3 [SD, 25.5] months). The control group consisted of 190 patients. The average fasting blood glucose level was higher in the NODAT group vs the control group (P < .001). The average fasting blood glucose level above diabetic threshold (≥7 mmol/L) was in association with a 6-fold higher risk of NODAT (odds ratio, 5.86; 95% CI, 2.46-13.97; P < .001). Absolute value of CD4+CD25brightCD127dim regulatory T cells was lower in the NODAT group at the first month after transplant (P = .048) Immunosuppressive protocol and survival data did not differ. CONCLUSIONS: Intensive management of the carbohydrate excursions during the early post-transplant period may decrease the incidence of NODAT. Further investigations will be required to decide whether the reduced CD4+CD25brightCD127dim/regulatory T-cell count contributes the development of NODAT.

Efficacy of 2% mepivacaine and 2% lignocaine in the surgical extraction of mesioangular angulated bilaterally impacted third molars: A double-blind, randomized, clinical trial.

OBJECTIVE: The purpose of this prospective, randomized, double-blind study was to compare the anesthetic efficacy of 2% mepivacaine and 2% lidocaine (both with 1:80,000 epinephrine) for inferior alveolar nerve block in mesioangular bilaterally impacted third molar extraction. STUDY DESIGN: Forty patients with mesioangular bilaterally impacted third molars were taken for the study; either 2% mepivacaine or 2% lidocaine is given in a double-blind manner. Surgery started 5 min after solution deposition. Success was defined as no or mild discomfort (visual analog scale [VAS] recordings) during the surgical procedure. RESULTS: The mean time for onset period 4.2 min and 4.6 min (P = 0.018). The mean duration anesthesia 177.17 min 166.71 min (P = 0.085). No significant difference between the scores of pain reported by the patients by VAS and venovenous bypass treated with mepivacaine and lidocaine (P = 0.000). Slight increased postoperative analgesics required for mepivacaine group (4.000 tablets) and lidocaine group (4.170 tablets) (P = 0.335). The sharp increase of pulse rate with respect to both the solutions at 5 min after postinjection of local anesthetics. However, there was no statically significant difference in systolic and diastolic blood (P = 0.681) and (P = 0.270). CONCLUSION: Lidocaine and mepivacaine with the same vasoconstrictor have similar action and both solutions are effective in surgical procedures. There were also no significant differences between them in relation to the intensity of postoperative pain.

Periodontal risk calculator versus periodontal risk assessment.

INTRODUCTION: The purpose of study was twofold: To determine the extent of inter valuator and inter group variation in risk scores assigned to study subjects by PRC and PRA. To explore the relationship between risk scores assigned by PRC and using the PRA. MATERIALS AND METHODS: 57 patients (33 male patients and 24 Female patients between 20 and 65 years age group) were assessed with PRC and PRA tools during their first visit. RESULTS AND CONCLUSION: We entered the resulting information in to the PRC and PRA to obtained a riskscore for each subject at first visit. The chi-square test significance between PRC and PRA is < 0.05 indicatesthe accuracy of the both tools.

Derailed B cell homeostasis in patients with mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disorder, characterized by the presence of antibodies to U1-RNP protein. We aimed to determine phenotypic abnormalities of peripheral B cell subsets in MCTD. Blood samples were obtained from 46 MCTD patients, and 20 controls. Using anti-CD19, anti-CD27, anti-IgD and anti-CD38 monoclonal antibodies, the following B cell subsets were identified by flow cytometry: (1) transitional B cells (CD19+CD27-IgD+CD38(high)); (2) naive B cells (CD19+CD27-IgD+CD38(low)); (3) non-switched memory B cells (CD19+CD27+IgD+); (4) switched memory B cells (CD19+CD27+IgD-); (5) double negative (DN) memory B cells (CD19+CD27-IgD-) and (6) plasma cells (CD19+CD27(high)IgD-). The proportion of transitional B cells, naive B cells and DN B lymphocytes was higher in MCTD than in controls. The DN B cells were positive for CD95 surface marker. This memory B cells population showed a close correlation with disease activity. The number of plasma cells was also increased, and there was an association between the number of plasma cells and the anti-U1RNP levels. Cyclophosphamide, methotrexate, and corticosteroid treatment decreased the number of DN and CD27(high) B cells. In conclusion, several abnormalities were found in the peripheral B-cell subsets in MCTD, which reinforces the role of derailed humoral autoimmune processes in the pathogenesis.

The effects of various doses of bacterial lipopolysaccharide on the expression of CD63 and the release of histamine by basophils of atopic and non-atopic patients.

OBJECTIVE: We tested the effect of various doses of bacterial lipopolysaccharide (LPS, endotoxin) on the expression of CD63 and the in vitro release of histamine by basophils stimulated with ragweed allergen in patients with or without ragweed and mite allergies. METHODS: The peripheral blood of 11 patients with ragweed allergy, 10 patients with mite allergy and 14 control patients was incubated with ragweed allergen extract following pretreatment with varying doses of LPS. The expression of CD63 in basophils was measured by flow cytometry, and the release of histamine was determined by ELISA. RESULTS: In the samples of patients with ragweed allergy that were exposed to specific allergen, only high doses of LPS significantly elevated the expression of CD63 (200 ng/ml; 1,000 EU/ml) and the release of histamine (2,000 ng/ml; 10,000 EU/ml). There was no effect of LPS in any other cases. CONCLUSIONS: Bacterial LPS (endotoxin) concentrations higher than 200 ng/ml (1,000 EU/ml), which rarely occurs in nature, could only activate the basophils from atopic patients whilst in the presence of the specific allergen. Thus, the restoration of the urban, "microbe-poor" milieu with endotoxin (as LPS) can be a promising and harmless approach for allergy prevention.

Different effects of bortezomib on the expressions of various protein kinase C isoenzymes in T cells of patients with systemic lupus erythematosus and in Jurkat cells.

The effects of proteosome inhibitor Bortezomib (BZ) were studied in vitro for 24 h on the protein kinase C (PKC) profiles, rates of proliferation and apoptosis in Jurkat cells and lymphocytes of 10 patients with systemic lupus erythematosus (SLE) and nine healthy subjects. The expressions of PKC proteins, the rates of proliferation and apoptosis were determined. The effects of BZ were different in the Jurkat and lupus T cells. Whereas BZ elevated the expression of PKC θ, δ and ξ isoenzymes in the Jurkat cells, it was unable to do that in the lupus T cells. BZ induced a dose-dependent increase in the apoptosis of Jurkat cells, while decreased the proliferation. The same effect of BZ was observed on the apoptosis of lymphocytes both in SLE and healthy subjects at concentrations higher than the therapeutic dose. We conclude that BZ treatment in vitro was not able to restore the SLE-specific defect (decrease) in the expression of PKC isoenzymes in the T cells as it was expected. This can be a limiting factor in the positive clinical effects of BZ in lupus.

Immunological reconstitution after autologous stem cell transplantation in patients with refractory systemic autoimmune diseases.

OBJECTIVE: High-dose chemotherapy followed by autologous haematopoietic stem cell transplantation (AHSCT) can be a salvage therapy for patients with severe, refractory systemic autoimmune diseases. The function of the newly rebuilt immune system is important, but little is known about immune reconstitution after AHSCT in autoimmune disorders. Our aim was to investigate the repopulation of different lymphocyte subsets in patients with systemic autoimmune diseases after AHSCT. METHODS: Twelve patients with severe refractory, autoimmune diseases were enrolled in the study: four with rheumatoid arthritis (RA), four with systemic sclerosis (SSc), three with systemic lupus erythematosus (SLE), and one with autoimmune overlap syndrome (myositis and RA). After stem-cell mobilization, CD34+ apheresis was carried out, followed by conditioning and AHSCT. After transplantation, peripheral lymphocyte subsets were regularly assessed by flow cytometry. RESULTS: The follow-up time was 24 months. The overall transplantation-related mortality (TRM) was 16.7% and the transplant-related toxicity was 33% 2 years after AHSCT. Regarding the immune reconstitution, CD56+ natural killer (NK) cells appeared in the earliest phase after transplantation, followed by CD8+ T cells. B cells and CD4+ T cells became normal within 150 days. The ratio of naive cells was low 30 days after AHSCT; however, naive B cells regenerated within 2 months whereas the repopulation of naive T cells took longer. After a short increase, the ratio of memory cells decreased 2 months after transplantation. Regulatory T (Treg) cells did not change significantly in the peritransplant period. Altogether approximately 5-6 months were required for the reconstitution of the peripheral immune network. CONCLUSIONS: AHSCT can be a salvage therapeutic modality in autoimmune patients who are refractory to other conventional therapies.

Altered T-cell and regulatory cell repertoire in patients with diffuse cutaneous systemic sclerosis.

OBJECTIVES: The aim of this study was to evaluate a wide spectrum of peripheral immune-competent cell types, reflecting overall disturbances in immune homeostasis, characteristic of systemic sclerosis (SSc). We also assessed visceral organ involvement and evaluated the relationship between cell proportions and clinical symptoms of the disease. METHODS: Twenty-one patients with diffuse cutaneous SSc (dcSSc) and 15 healthy individuals participated in the study. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed by enzyme-linked immunosorbent assay (ELISA), serum complement levels were measured by nephelometry, and autoantibodies were determined by indirect immunofluorescence staining and ELISA technique. Functional tests of regulatory T (Treg) cells were also carried out. RESULTS: Patients with SSc had higher percentages of activated CD3+/HLA-DR+ T cells. Comparing naive vs. memory subsets of CD4+ and CD8+ T cells, a shift towards central memory phenotype was observed in SSc. Natural killer (NK) and T-helper (Th)17 cell percentages were increased, while NKT, Th1, Treg type 1 (Tr1), and CD4+CD25+ Treg cell percentages were decreased in patients. Moreover, the suppressor activity of CD4+CD25+ Treg cells was lower in SSc. Negative correlations occurred between modified Rodnan skin score (MRSS) and Tr1 cell percentages and between complement levels and CD4+CD25+ Treg cells. We also found decreased interleukin (IL)-10 levels in SSc. CONCLUSIONS: Our data suggest that the increased Th17/CD4+CD25+ Treg ratio and the altered regulatory function of CD4+CD25+ Treg cells play an important role in the development of SSc. Moreover, our study reveals the potential role of the decreased profile of IL-10-producing Tr1 cells in the progression of disproportionate immune responses in SSc.

Impaired regulatory T-cell homeostasis due to vitamin D deficiency in undifferentiated connective tissue disease.

OBJECTIVE: The aim of this study was to perform a quantitative and functional analysis of natural CD4+CD25(high)Foxp3+ regulatory T cells (nTregs) and CD4+IL-17+ T cells, and to assess the serum levels of proinflammatory cytokines in patients with undifferentiated connective tissue disease (UCTD) before and after 5 weeks of 0.5 µg/day alfacalcidol supplementation. METHODS: Twenty-five patients with UCTD were enrolled in an open-label trial of alfacalcidol. Plasma levels of 25-hydroxyvitamin D [25(OH)D] were assessed by a high-performance liquid chromatography (HPLC) method. Flow cytometry was used for the quantification of nTregs and the IL-17 expression of T-helper (Th)17 cells. The serum concentrations of cytokines interleukin (IL)-12, interferon (IFN)-γ, IL-23, IL-17, IL-6, and IL-10 were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Treatment with alfacalcidol raised 25(OH)D levels from a mean of 23.5 ± 5.6 to 34.5 ± 7.4 ng/mL (p = 0.059; NS). Alfacalcidol treatment decreased both Th1- (IL-12 and IFN-γ) and Th17-related (IL-23, IL-17, IL-6) cytokine levels in UCTD patients, while the soluble IL-10 level increased (IL-12: 156.7 ± 75.2 vs. 87.5 ± 42.1 pg/mL, p < 0.001; IFN-γ: 41.5 ± 12.0 vs. 21.7 ± 9.9 pg/mL, p < 0.001; IL-23: 385.2 ± 82.2 vs. 210.0 ± 69.3 pg/mL, p < 0.001; IL-17: 37.8 ± 9.6 vs. 17.8 ± 4.5 pg/mL, p = 0.009; IL-6: 39.4 ± 11.3 vs. 23.5 ± 6.3 pg/mL, p < 0.001, IL-10: 8.4 ± 3.0 vs. 21.4 ± 9.7 pg/mL, p < 0.001). Alfacalcidol improved the Th17/nTreg imbalance, as it inhibited the IL-17 expression of Th17 cells, and increased the number of nTregs. The alfacalcidol might increase the capacity of nTreg cells to suppress the proliferation of autologous CD4+CD25⁻ cells. CONCLUSION: Our findings support the idea that vitamin D influences the Th17/nTreg imbalance in vitamin D-insufficient patients with UCTD and could be beneficial in the management of the disease.

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome.

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)-10 producing T regulatory type 1 (Tr1) cells and CD4(+)CD25(+) regulatory T cells (T(reg)) cells were determined by flow cytometry and serum cytokine levels of IL-4, IL-6, IL-10, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma were evaluated by enzyme-linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T(reg) cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4(+)CD25(+) T(reg) cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4(+)CD25(+) T(reg) cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4(+)CD25(+) T(reg) cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4(+)CD25(+) T(reg) cells in patients compared to controls. Serum IL-6 and TNF-alpha levels were elevated, while IL-10 was decreased in patients compared to controls. Negative correlation was found between IL-10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.

Regulatory T cells in atopic dermatitis: epidermal dendritic cell clusters may contribute to their local expansion.

BACKGROUND: Regulatory T cells (Tregs) have an essential role in tolerance and immune regulation. However, few and controversial data have been published to date on the role and number of these cells in atopic dermatitis (AD). OBJECTIVES: To investigate the number of CD4+CD25+FOXP3+ Tregs and interleukin 10-producing T regulatory type 1 (Tr1) cells in patients with AD. METHODS: Peripheral blood and skin biopsy samples from atopy patch test (APT)-positive patients with acute- and chronic-phase AD were investigated. Immunohistochemistry was applied to identify CD4+CD25+FOXP3+ Tregs in the skin, while flow cytometry was used to detect CD4+CD25highFOXP3+ Tregs and Tr1 cells in the peripheral blood. RESULTS: In the peripheral blood samples of patients with AD significantly elevated numbers of Tr1 cells were found. Although neither the absolute number nor the percentage of CD4+CD25highFOXP3+ Tregs showed significant alteration in the peripheral blood of patients, increased numbers of FOXP3+ Tregs were detected in skin biopsy specimens. All of the APT-positive skin samples showed epidermal dendritic cell aggregates, morphologically consistent with so-called Langerhans cell microgranulomas, which also contained intermingled FOXP3+ Tregs. CONCLUSIONS: Tr1 cell numbers were elevated in the peripheral blood and increased numbers of CD4+CD25highFOXP3+ Tregs were detected in the skin of patients with AD. The epidermal dendritic cell clusters in APT-positive lesional skin showed a close connection to the FOXP3+ Tregs.

Immunological alterations in newly diagnosed primary Sjögren's syndrome characterized by skewed peripheral T-cell subsets and inflammatory cytokines.

OBJECTIVES: To describe how certain peripheral immune parameters reflect the inflammatory alterations in patients with primary Sjögren's syndrome (pSS). We determined lymphocyte subpopulations and their state of activation from peripheral blood, evaluating both soluble serum T-helper (Th)1/Th2-type cytokines and intracytoplasmic cytokines. METHODS: Forty-nine patients with newly diagnosed pSS and 40 healthy individuals, all free from immunomodulant or immunosuppressive medication, were studied. Peripheral blood lymphocyte subgroups were quantified by flow cytometry, soluble cytokines were assessed using enzyme-linked immunosorbent assay (ELISA), and intracellular cytokine levels were measured after phorbol myristate acetate (PMA) stimulation by flow cytometry after staining of intracellular cytokines. RESULTS: Patients with primary SS had higher percentages of activated CD3+/CD69+ T cells than controls. When comparing naïve vs. memory subsets of CD4+ and CD8+ T cells, a shift towards the memory phenotype was observed for both. Natural killer (NK) cell and NK T-cell (NKT) percentages and Th0 and Th1 cell numbers were increased in patients compared to controls. Among circulating cytokines, interferon (IFN)-gamma was high, whereas interleukin (IL)-10 was decreased in SS when compared to controls. CONCLUSIONS: SS, considered as a systemic autoimmune disease, is characterized by a complex interplay of various cytokines and immune cells. The skewed T-cell subsets and cytokine imbalance play important roles in an orchestrated proinflammatory cascade.

Diesel exhaust exposure enhances the ozone-induced airway inflammation in healthy humans.

Exposure to particulate matter and ozone cause adverse airway reactions. Individual pollutant effects are often addressed separately, despite coexisting in ambient air. The present investigation was performed to study the effects of sequential exposures to diesel exhaust (DE) and ozone on airway inflammation in human subjects. Healthy subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) and bronchial wash (BW) sampling on two occasions. Once following a DE exposure (with 300 mug.m(-3) particles with a 50% cut-off aerodynamic diameter of 10 mum) with subsequent exposure to O(3) (0.2 ppm) 5 h later. The other bronchoscopy was performed after a filtered air exposure followed by an ozone exposure, using an identical protocol. Bronchoscopy was performed 24 h after the start of the initial exposure. Significant increases in neutrophil and macrophage numbers were found in BW after DE followed by ozone exposure versus air followed by ozone exposure. DE pre-exposure also raised eosinophil protein X levels in BAL compared with air. The present study indicates additive effects of diesel exhaust on the ozone-induced airway inflammation. Together with similar results from a recent study with sequential diesel exhaust and ozone exposures, the present data stress a need to consider the interaction and cumulative effects of different air pollutants.

The severity of systemic lupus erythematosus negatively correlates with the increasing number of CD4+CD25(high)FoxP3+ regulatory T cells during repeated plasmapheresis treatments of patients.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by increased pathologic autoantibody production. A decrease in the number of CD4+CD25(high)FoxP3+ regulatory T cells can play a key role in the loss of tolerance to self antigens. Our aim was to determine the absolute number of peripheral CD4+CD25(high)FoxP3+ T cells in 44 patients with SLE, furthermore, to measure the changes in the number of CD+CD25(high)FoxP3+ T cells in 5 patients with severe SLE treated with repeated plasmapheresis for 4-6 days in comparison to the changes in the activity of disease (SLEDAI). Percent of CD4+CD25(high)FoxP3+ T cells were measured by flow cytometry. The absolute number of peripheral CD4+CD25(high)FoxP3+ T cells was significantly decreased in the 44 patients with SLE compared to the healthy controls n = 32 (0.012 +/- 0.006 vs. 0.038 +/- 0.017 G/L, p < 0.05). In the 5 patients with severe SLE the repeated plasmapheresis treatments increased the peripheral number of CD4+CD25(high)FoxP3+ T cells. As the number of CD4+CD25(high)FoxP3+ T cells increased during the treatment, the activity of disease (the value of SLE activity index) decreased. In the peripheral blood of SLE patients not only the ratio was decreased (as it was published earlier) but also the absolute number of these regulatory T cells. The repeated plasmapheresis treatments of SLE patients induced a significant increase in the number of peripheral CD4+CD25(high)FoxP3+ T cells in parallel to the decrease in the values of SLEDAI (the activity of disease). This phenomenon is, among others, possibly due to the elimination of interpheron-alpha and lymphocytotoxic antibodies during plasmapheresis.

Measurement of natural (CD4+CD25high) and inducible (CD4+IL-10+) regulatory T cells in patients with systemic lupus erythematosus.

Abnormalities of regulatory T cells may play an important role in the loss of self-tolerance, which is a major characteristic of lupus. The objective of this study was to determine the ratio and the number of natural CD4+CD25highFoxp3+ and inducible CD4+IL-10+ regulatory T cells in lupus patients and to search correlation with disease activity. Seventy-two Hungarian lupus patients were enrolled in the study. Fourty-one age- and sex matched healthy donors served as controls. Flow cytometry was used for the quantification of CD4+CD25high Foxp3+ (nTreg) and CD4+IL-10+ (iTreg) cells. The ratio (3.06 +/- 1.45%) and the number (0.019 +/- 0.012 x 10(9)/L) of nTreg cells decreased in lupus significantly (P < 0.001 in both) as compared to normal controls (4.26 +/- 1.01% and 0.039 +/- 0.017 x 10(9)/L). The ratio of iTreg cells were significantly higher in patients than in controls (20.92 +/- 14.02% versus 15.49 +/- 11.65%, P < 0.03), but the number of these cell type did not differ in significant manner (0.314 +/- 0.236 x 10(9)/L versus 0.259 +/- 0.183 x 10(9)/L). The 19 active patients were characterised by significantly higher disease activity index (SLEDAI 8.63 +/- 2.95 versus 1.74 +/- 1.68, P < 0.001) and anti-DNA concentration (117.85 +/- 145.89 versus 37.36 +/- 68.85 IU/mL, P = 0.001) as compered to the 52 inactive patients. Furthermore, active patients required higher dose of methylprednisolon than inactive ones (14.8 +/- 10.6 versus 4.8 +/- 3.4 mg/day, P < 0.001). However, we did not find statistical significant difference in the number and ratio of the examined cell populations regarding to disease activity. Altered ratio and number of both natural and inducible regulatory T cells may play a role in the pathogenesis of lupus. There are small but appreciable difference in the number of regulatory T cells between inactive patients and healthy controls. It suggests that immunoregulatory deficiencies are present in the inactive stage of the disease also.

Systemic sclerosis-rheumatoid arthritis overlap syndrome: a unique combination of features suggests a distinct genetic, serological and clinical entity.

OBJECTIVE: To determine the genetic, clinical and serological characteristics of systemic sclerosis (SSc)-rheumatoid arthritis (RA) overlap syndrome. METHODS: Clinical manifestations and immunolaboratory features of 22 SSc-RA patients were assessed. The HLA-DR genotype of the 22 SSc-RA patients determined by SSP-PCR was compared with that of 38 SSc patients, 100 RA patients and 50 healthy controls. RESULTS: All overlap patients fulfilled the American College of Rheumatology (ACR) criteria for SSc and RA. Five of the 22 patients (23%) had diffuse cutaneous SSc (dcSSc) and 17 patients (77%) had limited cutaneous SSc (lcSSc). Antinuclear antibody, anti-Scl70, IgM rheumatoid factor and anti-CCP antibody positivity were detected in 22 (100%), 5 (23%), 16 (73%) and 18 patients (82%), respectively. Seventeen patients (77%) had pulmonary fibrosis, 12 (55%) had oesophageal dismotility, 11 (50%) had cardiac and five (23%) had renal involvement. Hand joint destruction was observed in 18 patients (82%). Significantly increased frequencies of HLA-DR3 (36% vs 5%), HLA-DR7 (9% vs 4%), HLA-DR11 (36% vs 7%) and HLA-DRw53 (23% vs 5%) were observed in SSc-RA compared with RA patients (P < 0.05). Allele frequencies of the 'shared epitope' (HLA-DR1 and -DR4) were significantly increased in SSc-RA (32% and 27%, respectively) and RA patients (46% and 31%, respectively) in comparison with SSc patients (10.5% and 16%, respectively) or healthy controls (16% and 14%, respectively) (P < 0.05). CONCLUSIONS: To date this is the largest SSc-RA overlap cohort. Genetics, clinical and immunolaboratory features suggest a mixed phenotype. Our data suggest that SSc-RA overlap syndrome may be a distinct genetic, immunological and clinical entity.

Regulatory T cells in peripheral blood of patients with mixed connective tissue disease.

OBJECTIVE: To determine the percentage and absolute number of CD4+ regulatory T-cells (Treg) in 48 patients with mixed connective tissue disease (MCTD). METHODS: Data were classified on the basis of the stage of the disease: 17 patients were in the active stage and 31 in the inactive stage. The absolute number of CD4+/intracellular interleukin-10+ (IL-10+) and CD4+CD25+high Treg cells was determined by flow cytometry. The percentage of CD4+CD25+high suppressor T-cells was determined on the basis of Foxp3 expression. RESULTS: The percentage and the absolute number of CD4+CD25+high Treg cells were lower in patients than in healthy controls (p<0.04), and were further decreased in patients with active MCTD and were lower than in the inactive stage (p<0.01). There was an increase in the percentage and absolute number of CD4+IL-10+ Treg cells in patients with MCTD compared to the healthy controls (p<0.02). The percentage of CD4+IL-10+ Treg cells was higher in the active stage of MCTD than in the inactive stage of the disease (p<0.005). However, we did not find any significant difference in the absolute number of CD4+IL-10+ Treg cells between the patients in the active and inactive stages of MCTD. CONCLUSION: Our results suggest that the decrease in the number of CD4+CD25+high Treg cells in an important factor in the immunoregulatory disturbance in patients with MCTD. We suggest that the increase in the number of CD4+IL-10+ Treg cells is a compensatory mechanism aiming to restore the balance between type 1 and type 2 cytokines in MCTD.