Anna Stasiak-Barmuta

Low serum IgA and increased expression of CD23 on B lymphocytes in peripheral blood in children with regressive autism aged 3-6 years old.

Introduction Immune system dysfunction is considered to be one of many medical disorders found in children with autism. The primary objective of the study was to assess if blood tests reflecting humoral immunity (IgA, IgG, IgM, IgE) are useful in identifying children with regressive autism. The secondary objective was to evaluate a part of the cellular arm of immunity (CD4/CD25 Tregs, CD4/CD23 cells) in those children. Material and methods Using a clinical case-control design, the systemic levels of immunoglobulins and lymphocyte subpopulations analysed by flow cytometry were compared in children aged 3-6 years old with a new diagnosis of regressive autism (n = 24; mean age: 4.25 ±1.70 years; male 23/24) and in sex- and age-matched healthy children (n = 24; aged 4.25 ±2.20 years; male 23/24). Results The humoral immunity profile, described by three binary variables, IgA < 0.97 g/l, IgE > 36 IU/ml, and IgG > 6.3 g/l, with a sensitivity of 79% and a specificity of 83% (p < 0.0001), was able to identify children with autism. The highest risk of autism diagnosis was associated with IgA < 0.97g/l (OR – 23.0; p < 0.001). A higher number of CD19/CD23 was found in children diagnosed with autism than in the control group (36.82 ±6.72% vs. 18.20 ±3.95%; p < 0.02). No correlation between the number of CD23-positive cells and serum IgE levels was observed. Conclusions A subtle shift of serum immunoglobulins consisting of low-normal IgA and B cell activation expressed by an increase of CD23-positive cells may characterize children with regressive autism aged 3-6 years old.

Profile of Exoglycosidases in Synovial Cell Cultures Derived from Human Synovial Membrane

Objective Determining the activity of lysosomal exoglycosidases in tissue cultures of synoviocytes derived from the knee joints of patients with injured anterior cruciate ligaments (ACL), juvenile idiopathic arthritis (JIA), and rheumatoid arthritis (RA). Methods The following exoglycosidases in cultured synoviocytes were analyzed with p-nitrophenyl derivatives of appropriate sugars as substrates: hexosaminidase (HEX) and its isoenzyme A (HEX-A), β-glucuronidase (GluA), β-galactosidase (GAL), α-mannosidase (MAN), and α-fucosidase (FUC). Results In our cell cultures, fibroblast-like synovial cells (FLS) dominated. In the group of patients with ACL-injuries, and in the groups of patients with JIA and RA, the activity of the investigated exoglycosidases was significantly higher in the intra- rather than in the extracellular compartment. Hexosaminidase was the predominant exoglycosidase. Stimulation of synoviocytes by IL-1β in cell cultures significantly increased the activity of HEX, HEX-A, and GluA in both compartments, as well as of GAL, MAN, and FUC in the intracellular compartment. Stimulation by IL-1β rheumatoidal synoviocytes increased by 128–201% the activity of HEX and HEX A in intracellular compartments and 33–72% in extracellular compartment. Conclusions The profile of lysosomal exoglycosidases in a cell culture of human synoviocytes is similar, but not identical, to those in the knee joint. Hexosaminidase is the dominant glycosidase in cultured unstimulated and IL-1β-stimulated human synoviocytes. The HEX inhibitors may be new drugs for the treatment of inflamed knee joints.

Relationship between CTLA-4 and CD28 molecule expression on T lymphocytes and stimulating and blocking autoantibodies to the TSH-receptor in children with Graves' disease.

The present study was performed to elucidate the relationship between CTLA-4/CD28 molecules and stimulating (TSAb) and blocking (TBAb) antibodies to the TSH-receptor (TSH-R) in Graves’ disease. CD28 and CD152 (CTLA-4) are glycoprotein molecules which provide a potent costimulatory signal for T-cell activation and proliferation via interactions with their ligands, B7.1/B7.2 molecules, which are present on the surface of antigen-presenting cells. The aim of the study was to estimate the expression of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4, CD152), CD28, B7.1 (CD80), and B7.2 (CD86) molecules on peripheral blood cells in patients with Graves’ disease (GD) (n = 55, mean age 15.5 ± 5.1 years) and nontoxic nodular goiter (NTNG) (n = 55, mean age 15.2 ± 4.5 years), in comparison with sex and age-matched healthy control subjects (n = 55, mean age 15.2 ± 3.9 years). The expression of the costimulatory molecules on mononuclear cells was analyzed by three-color flow cytometry using a Coulter EPICS XL cytometer. Detection of TSAb and TBAb to the TSH-R using JPO9 CHO cells in unfractionated serum was measured by a highly sensitive commercial radioimmunoassay. When compared with healthy control subjects and euthyroid patients with GD, untreated patients with GD showed a significant increase of CD152+ (p < 0.001, p < 0.001) and CD28+ (p < 0.01, NS) T lymphocytes, respectively. After 6–12 months of methimazole therapy, the percentage of these cells in the peripheral blood of hyperthyroid patients returned to normal values. In addition, patients with GD showed an increase in the percentage of both B7.1 (3.8%) and B7.2 (18.4%) molecules on activated monocytes, compared to patients with NTNG (0.5% p < 0.05, 2.5% p < 0.01, respectively) and healthy control subjects (0.2% p < 0.05, 0.8% p < 0.003, respectively). In patients with untreated GD there was a statistically significant positive correlation between the expression of CTLA-4 on the surface of peripheral blood T cells and the index of TSAb antibodies (R = 0.54, p < 0.001) as well as a negative correlation with TBAb antibody titer (R = –0.58, p < 0.001). However, no such correlations were noted with regard to CD28 and anti-TPO, anti-TG, and TRAb antibodies. We conclude that changes in the expression of costimulatory molecules on the surface of peripheral blood T cells and their significant relationship with the level of antithyroid antibodies indicate an involvement of these molecules in the pathogenesis of GD.

Generation of Functional T-Regulatory Cells in Children with Metabolic Syndrome

Recent research implies a role of decreased number and/or function of T-regulatory cells (Tregs) in low-grade inflammation associated with obesity and atherosclerosis. The enhancement of atheroprotective immunity by the expansion of Tregs could serve as a therapeutic strategy in obesity-related immunological disturbances. The aim of our study was an attempt to generate Treg cells in children with risk factors for the development of cardiovascular disease and to compare the results to those obtained in healthy subjects. The study group consisted of 30 children with metabolic syndrome (MS) and 30 controls. Conventional CD4+CD25− cells separated from the peripheral blood were converted into Treg cells with the use of CD3/CD28 antibodies and interleukin (IL)-2/transforming growth factor (TGF)-β stimulation. The expression of critical Treg molecules and cytokines was assessed at mRNA and protein levels. The percentages of Treg cells in the peripheral blood were significantly lower in the children with MS compared to the healthy subjects. After the culture with CD3/CD28 and IL-2/TGF-β we detected a significant increase in the expression of Tregs marker transcription factor FoxP3. The Tregs induced from the children with MS varied from the ones obtained in the controls in the expression of some molecules at mRNA level (e.g. IL-27, LGAL, KLF10 and NRP1) yet not in proliferation studies. For the first time, we have demonstrated the possibility of generating functional Treg cells in children with MS. The results of our study could be used in the design of therapeutic interventions in obesity associated immunologic disturbances.

Upregulation of antigen-processing machinery components at mRNA level in acute lymphoblastic leukemia cells after CD40 stimulation

The development of immunotherapy in hematologic malignancies has been observed in the last few years. One of the approaches is the use of cancer vaccines based on leukemia-derived dendritic cells (DC). Recent studies from our laboratory and other laboratories have shown that CD40 stimulation improves leukemia cells immunogenicity and generates an antitumor immune response. The design of future cancer vaccines requires the knowledge concerning the function of dendritic cells including antigen processing. The aim of our present study was the assessment of antigen-processing machinery (APM) components in acute lymphoblastic leukemia (ALL) cells before and after CD40 stimulation at messenger RNA (mRNA) level. Twenty-five children with ALL were enrolled into the study. Leukemic cells were stimulated (or not) with CD40L and IL-4. Elements of the antigen-processing machinery (MB1, LMP2, LMP7, LMP10, TAP1, TAP2, calnexin, calreticulin, tapasin, ERp57, zeta, delta) were determined by real-time PCR technique. The expression of important costimulatory and adhesion molecules considered as DC markers (CD40, CD54, CD80, CD83, CD86) were determined at the mRNA (PCR) and protein (flow cytometry) levels. The following are the results of our study: (1) We noted an upregulation of all costimulatory and adhesion molecules at the mRNA and protein levels in ALL cells after the culture; (2) the significant rise in expression of nearly all APM components after CD40 stimulation was observed. This confirms specific stimulation of the antigen-processing system in ALL cells by CD40L. Future work should focus on the clinical significance of these findings for immunotherapy in leukemias.

The mRNA expression of pro- and anti-inflammatory cytokines in T regulatory cells in children with type 1 diabetes

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T regulatory cells (Tregs) represent an active mechanism of suppressing autoreactive T cells that escape central tolerance. The aim of our study was to test the hypothesis that T regulatory cells express pro- and anti-inflammatory cytokines, elements of cytotoxicity and OX40/4-1BB molecules. The examined group consisted of 50 children with T1DM. Fifty two healthy individuals (control group) were enrolled into the study. A flow cytometric analysis of T-cell subpopulations was performed using the following markers: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD134 and anti-CD137. Concurrently with the flow cytometric assessment of Tregs we separated CD4+CD25+CD127dim/- cells for further mRNA analysis. mRNA levels for transcription factor FoxP3, pro- and anti-inflammatory cytokines (interferon gamma, interleukin-2, interleukin-4, interleukin-10, transforming growth factor beta1 and tumor necrosis factor alpha), activatory molecules (OX40, 4-1BB) and elements of cytotoxicity (granzyme B, perforin 1) were determined by real-time PCR technique. We found no alterations in the frequency of CD4+CD25highCD127low cells between diabetic and control children. Treg cells expressed mRNA for pro- and anti-inflammatory cytokines. Lower OX40 and higher 4-1BB mRNA but not protein levels in Treg cells in diabetic patients compared to the healthy children were noted. Our observations confirm the presence of mRNA for pro- and anti-inflammatory cytokines in CD4+CD25+CD127dim/- cells in the peripheral blood of children with T1DM. Further studies with the goal of developing new strategies to potentiate Treg function in autoimmune diseases are warranted.

Disturbances in some Gene Expression in T Regulatory Cells Separated from Children with Metabolic Syndrome

The metabolic syndrome (MS) is defined as a cluster of risk factors, including abdominal obesity, dyslipidaemia, glucose intolerance and hypertension, which increase the risk for coronary heart disease. The immunological aspects of obesity and MS, including the role of T regulatory cells, have been intensively investigated. The aim of this study was to determine whether there is any disturbance in T regulatory cells number and/or function in children with MS. The percentages of T regulatory cells in the peripheral blood of children fulfilling the International Diabetes Federation criteria of the disease (n = 47) were assessed. Treg cells were also separated for further analysis of multiple genes important in their function with the use of real‐time RT‐PCR. We did not observe any difference in Treg percentages between study and control group but there was lower expression of some molecules including transforming growth factor‐β and interleukin‐12 family members in Treg cells separated from children with MS compared to the healthy subjects. Our study is the first to report significant disturbances in some gene expression in T regulatory cells separated from children with MS. The results should be useful for further research in this field, including immunotherapeutic interventions.

Longitudinal observation of children with enhanced total serum IgE.

BACKGROUND Long-term studies on the evolution of elevated total IgE (tIgE) concentration are in demand. OBJECTIVE To investigate the prevalence of allergic diseases and influential factors in children with high tIgE levels during a 5-year period. METHODS Children with high tIgE levels (>100 IU/mL) were study subjects. After the 5-year follow-up, an interview with the parents, clinical examination, and evaluation of tIgE and specific IgE (sIgE) to selected food and inhalant allergens were performed. RESULTS The mean tIgE decreased significantly after 5 years in girls and boys regardless of the place of residence. Monosymptomatic patients accounted for most cases throughout the study, with the highest tIgE level at the beginning. After follow-up, the percentage of polysymptomatic patients increased. Their mean tIgE level was significantly higher than in the other groups. After follow-up, 11.7% of participants remained asymptomatic, and another 11.7% reported relief from symptoms. Allergy symptoms persisted in most children with normal tIgE levels. The 2-allergen sensitization was the most common through the study. Only patients sensitized to 4 allergens had unchanged levels of mean tIgE after follow-up and those with the highest mean tIgE level had a newly diagnosed sensitization to at least 1 allergen. A significant decrease of sIgE level was observed for food allergens. The values of sIgE to inhalant allergens even increased after the 5-year follow-up, despite decreased tIgE levels. CONCLUSION In children with allergy and an elevated concentration of tIgE, the increasing or stable value of tIgE could be a useful parameter for the prediction of the development of polysymptomatic allergy.

Generation of T regulatory cells in children with newly diagnosed type 1 diabetes mellitus.

UNLABELLED There is increasing evidence that T-regulatory (Treg) cells could be used to prevent or cure autoimmune diseases including type 1 diabetes mellitus (T1DM). The aim of the present study was to verify the hypothesis that functional Treg cells can be generated from conventional T-cells separated from a small amount of peripheral blood of children with newly diagnosed T1DM (N=25). METHODS CD4(+)CD25(-) cells were cultured with Treg expander (CD3/CD28) and IL-2 for generating de novo Treg cells. The assessment of the expression of selected genes and proteins critical to Treg function and the proliferation assays were performed with the use of real-time RT-PCR and flow cytometry. RESULTS After a 4-week stimulation with Treg expander and IL-2, the percentage of T-regulatory cells was significantly higher compared to the cells treated with medium alone (with no difference between diabetic and control children). However, we found some disturbances in the gene expression at mRNA level for molecules crucial for T-reg function. The induced Tregs from diabetic and control children were fully functional as assessed in proliferation assays. IN SUMMARY Despite some disturbances at mRNA level in the critical gene expression, the suppressive properties of induced Treg cells from diabetic and control children were effective.

Alterations of lymphocyte subpopulations and TGF-β in children with transient or persistent cow’s milk allergy

ABSTRACT The aim of the study was to evaluate changes in surface receptor expression of B and T lymphocytes and concentration of TGF-β in children who either developed tolerance to cow’s milk protein (CMP) or manifested persistent cow’s milk allergy (CMA). The study involved 30 patients with CMA who underwent an open food challenge after 12 months of milk-free diet. After the milk challenge, decreased concentration of CD19+CD23+ was observed in children who acquired tolerance to CMP, in comparison with the test before cow’s milk (CM) challenge (42.2% vs. 29.1%, p = .006). The same group demonstrated lower concentration of TGF-β than patients with persistent allergy (median 37.9 pg/ml vs. 52.8 pg/ml, p = .003, respectively). Moreover, before CM challenge, higher percentage of CD3+CD8+CD28+CD152+ cells (median 2.88% vs. 1.2%, p = .03) and CD3+CD4+CD25+CD62L+ (median 42.3% vs. 13.4%, p = .032) was noted in children who acquired tolerance to CMP, in comparison with subjects who remained allergic to CMP.