Dynamics and proliferative capacities of CD8+CD28+TCRαß+CD62Lhigh T-cell subsets in healthy and asthmatic subjects.

Adhesion molecules, as such, play essential roles in T-cell transendothelial extravasation during inflammation. A better understanding of the mechanisms underlying this process may be of value in the management of asthma. The present study employed Magnetic-Activated Cell Sorting (MACS) to isolate human CD8+ T lymphocytes from peripheral blood of asthma patients and controls. The cells were flow cytometrically assessed to evaluate surface expression of an adhesion molecule, L-selectin (CD62L) on the surface of CD8FoxP3-/bright T cell subsets and its response to inflammatory cytokines. We showed that CD8+CD28+TCRαß+CD62LhighFoxP3bright T cells were deficient in blood of some asthma patients but abundant in others. After co-stimulation of CD8+ T cells with anti-CD3/CD28 in combination with IL-2 and IL-10 or TGF-ß, the frequencies of CD8+CD28+TCRα/ß+CD62Lhigh T cells in the group of patients were lower than at baseline. Our data indicate that L-selectin expression is regulated by inflammatory cytokines. Overall, these data reveal that asthma phenotypes may be further stratified into micro subtypes with distinct cellular and molecular characteristics, supporting the concept of asthma endotypes.

Interleukin-2-mediated stat-5 expression levels in CD8+CD25+ regulatory T cells: the relevance for asthma.

Preliminary studies demonstrated the potential role of CD8+CD25+ T regulatory cells (Tregs) in asthma. However, published data with regard to the relevance of signaling pathways that govern Tregs homeostasis are limited and conflicting. The first aim of this study was to characterize the phosphorylation of STAT-5 in CD8+CD25+ Tregs. The second aim was to investigate the ability of CD8+CD25+ Tregs from patients and controls to respond to interleukin (IL)-2 treatment in vitro. Twenty-five healthy subjects (NC) and 50 patients with either severe (SA) or mild-moderate (MA) asthma were enrolled in the study. STAT-5 phosphorylation was detected in purified CD8+CD25+ Tregs from healthy and asthma subjects, indicating that STAT-5 has a role in their pathobiology. At baseline, asthmas had either significantly lower [(SA=4.5±5% vs NC=26±25%, P < 0.001) and (MA=10±7.5% vs NC=26±25%, P < 0.001)] or higher [(SA=54±58.5% vs 26±25%, P < 0.01) and (MA=71±74.5% vs 26±25%, P < 0.01) proportion of Tregs expressing pSTAT-5 than controls. In contrast to healthy subjects, CD8+CD25+ Tregs from asthma subjects had either increased (pSTAT-5high) or decreased (pSTAT-5low) phosphorylated STAT-5 levels within individual cells. These data suggest that the alteration in STAT5 phosphorylation level might be associated with asthma and is a potential molecular basis of skewed CD8+CD25+ Treg differentiation. IL-2 treatment of cells from severe asthma subjects increased the proportion of cells expressing high level of pSTAT-5 while it decreased the proportion of those expressing low level of pSTAT-5. Strikingly, IL-2 increased the proportions of both subsets from mild-moderate asthma subjects. These findings demonstrate that altered IL-2-mediated STAT-5 phosphorylation within individual circulatory CD8+CD25+ Tregs may be associated with asthma and disease severity.

The relative values of CD8+CD25+Foxp3brigh Treg cells correlate with selected lung function parameters in asthma.

The study aimed to detect CD8(+)CD25(+)FoxP3(brigh) Tregs and investigate their possible association with selected lung function values. CD8(+)CD25(+)FoxP3(brigh) Tregs were detected by flow cytometry in the peripheral blood of 25 patients with severe asthma (SA), 25 patients with mild-to-moderate asthma (MA), and 25 age-matched healthy donors (NC). The percentages of CD8(+)CD25(+)FoxP3(brigh) Tregs of the patients with severe (3.4 ± 4.55), and mild-to-moderate asthma (7.5 ± 8.15), were markedly lower than those of controls (12.1 ± 13.2). The mean forced expiratory volume in 1 s (FEV1) % predicted value in severe asthma subpopulation was significantly lower (67.05 ± 15.98%) when compared with that of mild-to-moderate asthma subgroup (87.71 ± 16.12%). Interestingly, the percentages of CD8(+)CD25(+)FoxP3(brigh) Tregs correlate with mean peak expiratory flow (PEF)% predicted values in severe (r = 0.7, P <0.01) and mild-to-moderate (r = 0.73, P <0.01) asthma. In contrast, this parameter was positively correlated with FEV1% predicted values in the severe asthmatics only (r = 0.71, P <0.01). In summary, this study establishes a link between the percentage of CD8(+)CD25(+)FoxP3(brigh) Tregs and selected lung function parameters, suggesting that this parameter has potential as a marker for inflammation and airflow obstruction.

Allergy-related changes in levels of CD8+CD25+FoxP3(bright) Treg cells and FoxP3 mRNA expression in peripheral blood: the role of IL-10 or TGF-beta.

There is an increasing body of evidence that alterations of regulatory T (Treg) cell numbers and functions lead to immune disorders. Accordingly, understanding the regulatory mechanisms that maintain peripheral regulatory T (Treg) cell homeostasis is key to the development of effective targeted biologic therapies. We previously demonstrated the effects of IL-10 or TGF-beta on distinct CD8+CD28- T cell subsets in vitro. Allergy-related changes of CD8+CD25+FoxP3(bright)Treg cells and FoxP3 mRNA expression in peripheral blood were assessed by means of multicolor flow cytometry and real-time polymerase chain reaction (RT-PCR). Co-stimulation of CD8+CD25+ T cells with anti-CD3/CD28 in the presence of either IL-10 or TGF-beta increased the frequency of CD8+CD25+FoxP3(bright)Treg cells in patients with asthma and controls. Likewise, CD8+CD25+ T cell activation with anti-CD3/CD28 and TGF-beta increased FoxP3mRNA expression in all groups. Anti-CD3/CD28 and IL-10 appeared to regulate FoxP3 mRNA expression in a phenotype-dependent manner. Specifically, co-stimulation by anti-CD3/CD28 and IL-10 markedly increased FoxP3 mRNA expression in the severe asthma group whereas it had opposite effects on this value in other groups. Taken altogether, these data suggest that IL-10 and TGF-beta may modulate FoxP3 expressions at the protein and mRNA levels in respect to their need for peripheral tolerance.

The effects of interleukin-10 or TGF-beta on anti-CD3/CD28 induced activation of CD8+CD28- and CD8+CD28+ T cells in allergic asthma.

The CD8+CD28- and CD8+CD28+ T cells play a primordial role in peripheral tolerance, but little is known about their implication in allergic asthma. This study was designed to determine the changes in a proportion of human circulatory CD8+ subsets before and after short term culture in the presence of anti-CD3/CD28 and IL-10 or TGF-beta. Flow cytometry analysis revealed increased percentage of CD8+CD28- T cells but decreased percentage of CD8+CD28+ T cells enriched from peripheral blood of adult allergic asthma individuals compared to controls (baseline). In comparison to the baseline, co-stimulation with anti-CD3/CD28 and IL-10 decreased the proportion of CD8+CD28- T cells in severe allergic asthma subjects, whereas it increased this value in mild to moderate asthmatic subjects and controls. Adding TGF-beta decreased the proportion of CD8+CD28- T cells from allergic asthma subjects, whereas it has opposite effects on this subset from controls. IL-10 and TGF-beta had some plethoric effects on FoxP3 expression in anti-CD3/CD28 activated CD8+CD28- T cells. Thus, these findings indicate that a control mechanism involving IL-10 and TGF-beta might be defective in allergic asthma subjects.

Comparison of changes in the percentages of CD8+CD28-TCRalpha beta+ T cell subpopulations in allergic asthma subjects vs controls before and after anti-CD3/anti-CD28/IL-2 stimulation in vitro.

Asthma is a chronic inflammatory disease characterized by the migration of activated T cells into the bronchial mucosa. TGF-beta and IL-10 have proved to regulate airway hyper-responsiveness and leukocytes recruitment to the airways of ovalbumin (OVA) sensitized mice. We examined relative changes in CD8+T cell subpopulations between fifty allergic asthma subjects and twenty five aged-matched healthy adults before and after anti-CD3/CD28 and IL-2 stimulation in the presence of IL-10 or TGF-beta, focusing on CD62L and FoxP3 expressing TCR alpha beta + cells. Severe asthma group had a significantly higher percentage of CD8+ CD28-and CD8+ CD28-TCR alpha beta + CD62L highFoxP3 bright T cells than other groups after enrichment. Compared to the baseline, co-stimulation with either IL-10 or TGF-beta increased the percentage of CD8+CD28-but decrease the percentage of CD8+CD28+T cells within anti-CD3/anti-CD28/IL-2 activated CD8+T cells in all groups. Co-stimulation with anti-CD3/anti-CD28/IL-2 in presence of either IL-10 or TGF-beta decreased the frequencies of CD8+CD28-TCR alpha beta +CD62Lhigh FoxP3 bright T cells in severe asthma subgroup but increased this parameter in other groups. We suggest that altered high level expression of CD62L and FoxP3 on CD8+ CD28-TCR alpha beta + T cell is relevant to allergic asthma. These data have implications for further characterization of CD8+ CD28-TCR alpha beta+ T cell subsets, with special emphasis on their implication in healthy or allergic immune response.

Phenotypic characterization of ex vivo CD4+CD25highCD127low immune regulatory T cells in allergic asthma: pathogenesis relevance of their FoxP3, GITR, CTLA-4 and FAS expressions.

BACKGROUND: CD4+CD25high regulatory T (Treg) cells are crucial for immune homeostasis and peripheral tolerance, but their relevance to allergic asthma has not been fully elucidated. OBJECTIVE: To assess peripheral blood CD4+ T cells, and CD4+CD25highCD127low Treg cells expressing phenotypic markers (FoxP3, GITR, CTLA-4, and FAS) in allergic asthma subjects. MATERIALS AND METHODS: Peripheral blood mononuclear cells were isolated from 60 allergic asthma (AA) subjects and 30 healthy controls (HC). We examined by flow cytometry, the proportion of CD4+ T cells and CD4+CD25highCD127low Treg cells as well as the expression of FoxP3, GITR, CTLA-4, and FAS by CD4+CD25highCD127low Treg cells. Moreover, FOXP3 mRNA expression was measured by quantitative real time polymerase chain reaction (real-time RT-PCR). RESULTS: The absolute number of CD4+CD25highCD127low Treg cells and the percentages of CD4+CD25highCD127low Treg cells expressing one of the four selected markers were significantly lower in allergic asthma subjects compared with controls. We observed no significant decreased absolute CD4+ T cell count in the examined groups compared to the control group. Except for GITR, circulatory CD4+CD25highCD127low Treg cells of severe allergic asthma (SA) subjects showed significantly lower expressions of FoxP3, CTLA-4, and CD95 than did those isolated from mild to moderate asthma (MA) patients. There was no statistically significant difference in the level of mRNA FoxP3 expression in CD4+CD25+ Treg cells between allergic asthma subjects and healthy controls groups, and within the examined groups (p>0.05). CONCLUSIONS: These findings suggest that allergic asthma and the use of glucocorticosteroids are associated with decreased absolute number of circulatory CD4+CD25highCD127low Treg cells and the decreased frequencies of CD4+CD25highCD127low Treg cells expressing one of the four selected markers.

Low frequency of CD8+CD25+FOXP3(BRIGHT) T cells and FOXP3 mRNA expression in the peripheral blood of allergic asthma patients.

The aim of this study is to determine whether frequencies of CD8+CD25+ T cells and FoxP3 messenger RNA (mRNA) expression levels in CD8+ T cells isolated from peripheral blood are related to allergic asthma and disease severity. We enrolled 50 patients with allergic asthma (AA) and 25 healthy control subjects (NC) in our study. The frequencies of CD8+CD25+FoxP3 -/+ T cells were assessed with flow cytometry, and mRNA FoxP3 level in CD8+ T cells was determined with real time polymerase chain reaction (RT-PCR). Asthma patients had fewer CD8+CD25+FoxP3bright T cells [SA (median = 3.4%, IQR = 3.1) vs MA (median = 7.5%, IQR = 4.7)] than controls NC [median = 12.1 %, IQR = 8, P < 0.0001] but more CD8+CD25+FoxP3- T cells [SA (median = 96 %, IQR = 3.1) vs MA (median = 92.5%, IQR = 4.7)] than controls NC [median = 87.9%, IQR = 9.2, P < 0.0001]. FoxP3 mRNA level was significantly decreased in CD8+ T cells of severe asthma patients (median = 0.82, IQR = 0.54) than that of patients with mild to moderate asthma and controls [(median = 2.29, IQR = 4.40) vs (median = 2.11, IQR = 3.2)]. The percentage of FoxP3+ T cells was correlated positively with the percentage of forced expiratory volume in 1 second (FEV1) (r = 0.71, p< 0.01) in patients with severe asthma. The proportion of CD8+CD25+FoxP3bright T cells and the level of FOXP3 gene expression in CD8+ T cells are relevant to allergic asthma and disease severity. The manipulation of FoxP3+CD25+CD8+ T cells may prevent chronic allergic inflammation and improve lung function during an acute allergic asthma exacerbation.

Adipohormones as prognostric markers in patients with nonalcoholic steatohepatitis (NASH).

Nonalcoholic steatohepatitis (NASH) represents one of the most common liver diseases. It is strongly associated with obesity and insulin resistance and is thought to be part of the metabolic syndrome. NASH can progress to cirrhosis and liver failure. Adipohormones, synthesized in adipose tissue, are involved in the pathophysiology of many acute and chronic liver diseases. The aim of this study was to evaluate the plasma concentrations of adiponectin, resistin, leptin, TNF-alpha and Il-6 in patients with NASH, as well as their correlation with the pathologic parameters. Serum concentration of leptin, adiponectin, resistin, insulin, TNF-alpha, IL-6 were measured with ELISA method. Liver biopsies were obtained from 18 (age 42.55+/-21 years) patients. NASH has been classified according to Dixon score. The control group was represented by 16 non-obese subjects. Mean serum concentration of adiponectin in patients with NASH was significantly lower than in healthy subjects (4.87+/-1.96 vs. 8.33+/-4.56 ng/ml; p<0.05). Mean serum levels of TNF-alpha in patients with NASH were significantly higher than in controls (34.2+/-19.7 vs. 20.7+/-15.5 ng/ml; p<0.05). In patients with more advanced inflammation (grade 2-3) and fibrosis (stage 2) in pathology, serum concentration of leptin was significantly higher than in patients with steatosis and less advanced inflammation (grade 1) and fibrosis (stage 1) (median 8.94 vs. 16.2 ng/ml; p<0.05). No significant differences of serum concentration of others adipohormones between these two groups of patients were stated. Moreover, we observed the correlation in serum levels (examined group vs controls) between: resistin and TNF-alpha (r = 0.62; p<0.05), adiponectin and IL-6 (r = -0.60; p<0.05) and leptin and insulin (r = -0.51; p<0.05). In conclusion, based on our study we speculate that changes of adipohormones levels may be markers of NASH and the serum level of leptin can be associated with more advanced form of NASH.

Serum levels of interleukin-18 (IL-18), interleukin-1beta (IL-1beta), its soluble receptor sIL-1RII and C-reactive protein (CRP) in patients with Lyme arthritis.

BACKGROUND: In recent years, the role of pro- and antiinflammatory cytokines in the development of Lyme arthritis (LA) has been widely discussed. The purpose of the present study was to determine the concentration of interleukin-18 (IL-18), interleukin-1beta (IL-beta) and its soluble receptor sIL-1RII in serum of patients with LA as well as the usefulness of serum C-reactive protein (CRP) determination in LA diagnosis and monitoring of its treatment. PATIENTS AND METHODS: The study group consisted of 20 patients with LA. Before and after antibiotic treatment, the serum levels of IL-18, IL-1beta and sIL-1RII were measured immunoenzymatically using standard kits and the CRP level was measured by immunoturbidimetric method. RESULTS: Before treatment, the serum levels of IL-18, IL-1beta and sIL-1RII were significantly higher than in control group and after treatment the concentrations of IL-18, IL-1beta and sIL-1RII decreased significantly, but the level of IL-18 and sIL-1RII still remained higher than in control group. The elevated serum level of CRP was detected only in 6 of 20 patients and in 5 of them it returned to the baseline after treatment. CONCLUSION: The results of our study suggest that IL-18, IL-1beta and sIL-1RII might be involved in the development of LA. CRP may be useful in differential diagnosis in patients with suspicion of Lyme arthritis.

Osteochondral defects of the talus treated by mesenchymal stem cell implantation--early results.

The purpose of this study was to present early results of talus cartilage defects treatment with autologous mesenchymal stem cells CD34+ implantation technique. Nine (9) patients were treated, due to IV degree chondromalation (by ICRS). The applied standard procedure included: clinical examination, AP and lateral x-ray, MRI, preoperative, as well as during control examination. The surgical procedure consisted of the defect's debridement, harvesting and fixation of the periosteal flap and CD34+ implantation. Clinical results were assessed after 6 months to 3 years by clinical examination, Magee score and MRI. Good and very good clinical results were obtained and confirmed by MRI in 8 cases. In one case, cartilage hypertrophy was noted. There were no delamination and infection signs.

The rate of apoptosis and expression of Bcl-2 and Bax in leukocytes of acute myeloblastic leukemia patients.

The rate of apoptosis as well as expression of Bcl-2 and Bax was evaluated before and after induction therapy in leukocytes of 70 patients with acute myeloblastic leukemia (AML), retrospectively divided into group A (with longer survival) and group B (with shorter survival). We found, that leukocytes of untreated AML patients showed susceptibility to apoptosis similar to control cells. Marked increase in percentage of apoptotic leukocytes was observed after induction therapy exclusively in patients with longer survival, which was accompanied by better normalization of routine hematological parameters. In this group, the Bcl-2/Bax ratio was similar to the control and remained unchanged after treatment. In AML patients with shorter survival, a twofold increase in this ratio was observed both before and after the completion of induction therapy. In both groups of untreated patients, western blot analysis revealed the presence of prominent additional bands reacting with anti-Bcl-2 or anti-Bax antibody, which were undetectable in control leukocytes. After the therapy, these bands disappeared, especially in patients from group A. In conclusion, the lack of therapy-induced enhancement in leukocyte apoptosis, an increased ratio of Bcl-2/Bax as well as persistent presence of abnormal Bcl-2 and Bax protein bands after induction therapy in AML patients may be considered as factors associated with unfavorable clinical outcome.

Does renal carcinoma affect the expression of P-selectin on platelets?

We studied changes in the expression of P-selectin on the blood platelet plasma membrane. The aim of the study was to determine the influence of renal carcinoma on P-selectin expression associated with changes in platelet morphology. Venous blood was collected from 30 patients with renal carcinoma and from 24 control subjects for cytometric analysis and to evaluate platelet morphology. P-selectin being the CD62P receptor on blood platelets was marked by anti-CD61/62P MoAb, and the results were presented as the percentage of CD62P-positive cells. Changes in the expression of the CD62P on the platelet plasma membrane during activation were investigated by flow cytometry in a comparative study of in vivo activation and in vitro platelet reactivity. Platelet activation reflected by P-selectin expression was higher in the group of patients (4.45 +/-1.96), compared to control (2.48 +/-1.66) (p < 0.05). However, adenosine diphosphate [ADP] -stimulated platelet reactivity in renal cancer patients increased only by 0.24% (p > 0.05), while following activation by thrombin by 0.54% (p < 0.05). Moreover, a higher (4.72 +/-2.02), statistically significant percentage of platelets with P-selectin expression was found in patients with disseminated neoplastic changes in renal parenchyma, compared to patients with a single localized neoplastic lesion (4.17 +/-1.89) (p < 0.05). A statistically significant difference was noted in the platelet count and anisocytosis in renal cancer patients. Renal cancer enhances P-selectin expression. It is due to the presence of intensified thrombinogenesis and other platelet agonists in the blood.

Potential role of beta 1 integrin and collagen biosynthesis in estrogen-dependent reduction of apoptosis in tamoxifen-treated breast cancer cells.

It was found that 10 microM tamoxifen induced apoptosis and a significant (approximately 50%) depletion of beta 1 integrin levels in human breast cancer cells. Estradiol-treated MCF-7 cells exhibited exceptional viability and adherence, high levels of beta 1 integrin and increased (by 100%) collagen biosynthesis. Pretreatment of MCF-7 cells with 1 nM estradiol prevented tamoxifen-induced cell death, loss of cell adherence and decrease in beta 1 integrin level. Tamoxifen and estradiol had an opposite effect on the beta 1 integrin level and adherence in breast cancer cells, suggesting that the decrease in the beta 1 integrin level may be an early event during tamoxifen-induced apoptosis in breast cancer cells.

Influence of estrogen, antiestrogen and UV-light on the balance between proliferation and apoptosis in MCF-7 breast adenocarcinoma cells culture.

Studies of the mechanism of actions of estrogen, antiestrogen and physical factors may provide clues to an understanding of breast cancer growth and/or regression regulation and thus identify novel targets for therapeutic intervention. Defective control of apoptosis appears to play a central role in the pathogenesis of neoplasia. Conversely, cancer therapy and ionizing radiation can induce cancer cell death by apoptosis and/or necrosis. bcl-2 gene and p-53 gene products have been both linked to programmed cell death pathways. We have analyzed the effect of estradiol, tamoxifen and UV exposure on the induction of apoptosis, expression of p53 and bcl-2 gene products as well as the proliferative activity (expressed as [3H]thymidine incorporation and PCNA and MPM2 antigens involvement) in MCF7. It has been found that estradiol increases the speed of cell cycle in MCF7 and acts as antiapoptotic factor. Tamoxifen has multiple influence on the rate of growth of cancer cells: depends on estrogen receptor (ER), conducts reduction of proliferation rate; depends on ER and other mechanisms conducts to suppressions of Bcl-2 protein expression and induction of cell death through apoptotic pathway. Estradiol prevents the apoptotic influence of tamoxifen probably by enhancement of Bcl-2 protein expression and does not prevent the inhibition of proliferation rate. The irradiation with UV induces apoptosis by over-expression of p53 and down-regulation of bcl-2 gene.

[The phenotype characteristics of cord blood lymphocytes in premature neonates]. / Ekspresja receptorów powierzchniowych na limfocytach krwi pepowinowej wczesniaków.

It has been observed high risk of infections in neonates as a result from lymphocytes immaturity. It is connected to phenotype differences of lymphocytes between neonates and adults. This high susceptibility to infections is especially high in premature neonates. With the use of flow cytometry we have evaluated the phenotype of cord blood lymphocytes in premature neonates. In comparison to results of healthy newborns we have observed significant decrease in CD7+, CD3+, CD4+, CD25+, CD57+ lymphocytes an increase in total number of CD8+ cells as well as alteration in CD4/CD8 ratio. Our results suggest deeply damage of cellular immunity in preterm infants.

An assessment of lymphocytic population in peripheral blood of patients with renal cell carcinoma before and after embolization.

The immunophenotype of peripheral blood lymphocytes was evaluated in 50 renal cell carcinoma (RCC) patients, before and after embolization. In RCC patients we have observed significant decrease in the total lymphocyte number as well as in T lymphocyte, their subpopulations and B lymphocyte number. Ten to twelve months after the treatment by embolization, significant increase in the total number of not only lymphocytes (p < 0.001), including T lymphocytes (p < 0.001), T-helper lymphocytes (p < 0.05), cytotoxic T lymphocytes (p < 0.05) and B lymphocytes (p < 0.05), but also an increase of NK cells number (p < 0.05) were found, compared to the values obtained before the treatment and reached the level observed in healthy subjects. Cytometric evaluation of peripheral blood lymphocytic phenotype in renal cell carcinoma patients can help evaluate the immune status of RCC patients as well as assess the embolization efficiency.

[Flow cytometry in hematologic diagnosis]. / Cytometria przeplywowa w diagnostyce hematologicznej.

The paper presents flow cytometry method in haematology diagnostics. Beside to the estimation of blast cells phenotype in acute leukaemia, flow cytometry allows to evaluate the presence of minimal residual disease (MRD) and multi drug resistance protein (MDR). Flow cytometry plays a crucial role in the estimation of DNA profile in cancer cells and evaluation of stem cells. Repeatedly flow cytometry is used to estimate platelets and reticulocytes.

CD23 antigen expression on B lymphocytes and soluble CD23 levels in peripheral blood of high-risk type 1 diabetes subjects.

Soluble CD23 (sCD23), a recently discovered multifunctional cytokine, is a 25-kDa molecule released by autoproteolysis from the 45-kDa CD23 molecule which is found mainly on the surface of B lymphocytes. In the present study we aimed to evaluate, in association with humoral immune and metabolic markers, the changes in CD23 antigen expression on B lymphocytes and levels of sCD23 in the peripheral blood of subjects at high risk of type 1 diabetes. The study was carried out in 28 first-degree relatives of type 1 diabetes patients (versus a control group of 28 age- and sex-matched healthy volunteers) using antibodies against different B-cell antigens: ICA, GADA, IAA, IA-2. Flow cytometry was used to measure the percentage of CD20+ (B lymphocytes) and CD20+CD23+ lymphocyte subsets, and sCD23 levels in serum were determined by enzyme immunoassay. Prediabetic subjects had a significantly (P<0.01) lower percentage of CD20+CD23+ lymphocytes in comparison with healthy age- and sex-matched controls. Expression of CD23+ on B lymphocytes was similar in subjects with ICA only and with two or more antibodies against pancreatic antigens. In the prediabetic group, the median concentration of sCD23 was lower than in the control group and was statistically significant (P < 0.02) in the subgroup of subjects with the most impaired function of pancreatic beta-cells (the lowest values of first phase of insulin release). In conclusion, our study suggests that CD23 molecule expression on B lymphocytes and sCD23 levels in peripheral blood could be additional markers for monitoring the development of type 1 diabetes and play a role in determining the efficacy of prevention trials. However, further prospective studies are needed.

In vitro secretion of interleukin 2 and expression of IL-2 receptor in peripheral blood lymphocytes in high risk of insulin-dependent diabetes mellitus subjects.

Interleukin 2 (IL-2)--a Th1 lymphocyte-derived cytokine is at present considered to play an important role in the etiopathogenesis of insulin-dependent diabetes mellitus. In the previous studies increased, decreased and unchanged IL-2 levels in patients with recent onset of insulin-dependent diabetes mellitus (IDDM) were found. These differences could be a result of different metabolic status or/and a different stage of the autoimmune process. The aim of our study was to estimate in vitro secretion of IL-2 and CD25 antigen expression by the peripheral blood T lymphocytes in subjects at the preclinical stage of IDDM (prediabetes), but still without metabolic disturbances. In 27 first degree relatives of IDDM patients with antibodies against different pancreatic islet cell antigens (ICA, GADA, IAA, IA-2) CD25 antigen expression on peripheral blood lymphocytes T was measured by flow cytometry and IL-2 concentration in supernatants of 48 and 72 h cultures of peripheral whole blood with 10 microg/ml PHA was estimated by ELISA. The control group was comprised of 34 age and sex-matched healthy volunteers. In the studied high risk IDDM subjects the decreased CD25 expression in peripheral CD4+ lymphocytes T and a negative correlation between the percentage of CD25+ cells and islet cell antibodies (ICA) titres was observed. No differences in IL-2 levels in supernatants of 48 h and 72 h blood cultures was found in subjects with single antibody (ICA+) in comparison to healthy controls. A significant increase of IL-2 secretion at 72 h of PHA stimulation was shown in first degree relatives of IDDM patients with a combination of 3 or more antipancreatic-B cell antibodies. There were also a significant negative correlation between glutamic acid decarboxylase antibodies (GADA) titres and IL-2 levels in 72 h of culture. The present study suggests the involvement of IL-2 in the pathogenesis of IDDM. The estimation of CD25 antigen expression in the peripheral blood lymphocytes could be an additional immunological marker of identification of subjects in prediabetes.