Analysis of prognostic factors in critically ill patients with COVID-19.

The Coronavirus Disease 2019 (COVID-19) has caused a global health crisis. Mortality predictors in critically ill patients remain under investigation. A retrospective cohort study included 201 patients admitted to the intensive care unit (ICU) due to COVID-19. Data on demographic characteristics, laboratory findings, and mortality were collected. Logistic regression analysis was conducted with various independent variables, including demographic characteristics, clinical factors, and treatment methods. The study aimed to identify key risk factors associated with mortality in an ICU. In an investigation of 201 patients comprising non-survivors (n = 80, 40%) and Survivors (n = 121, 60%), we identified several markers significantly associated with ICU mortality. Lower Interleukin 6 and White Blood Cells levels at both 24- and 48-hours post-ICU admission emerged as significant indicators of survival. The study employed logistic regression analysis to evaluate risk factors for in-ICU mortality. Analysis results revealed that demographic and clinical factors, including gender, age, and comorbidities, were not significant predictors of in-ICU mortality. Ventilator-associated pneumonia was significantly higher in Survivors, and the use of antibiotics showed a significant association with increased mortality risk in the multivariate model (OR: 11.2, p = 0.031). Our study underscores the significance of monitoring Il-6 and WBC levels within 48 hours of ICU admission, potentially influencing COVID-19 patient outcomes. These insights may reshape therapeutic strategies and ICU protocols for critically ill patients.

Immune response variation in mild and severe COVID-19 patients.

Sixty patients with COVID-19 infection were categorized into mild and severe groups, and their immune response was analyzed using flow cytometry and complete blood count. An observed increase in immune activation parameters, notably a higher percentage of CD4 lymphocytes co-expressing CD69 and CD25 molecules, and enhanced activity of the macrophage-monocyte cell line was noted in the mild group. Although Group 2 (severe COVID) had fewer CD4 cells, significant migration and proliferation were evident, with increased CD4CD69, CD8 HLA-DR+, and CD8CD69 lymphocytes. The CD4 to CD8 ratio in Group 1 suggested potential autoimmune reactions, while Group 2 indicated potential immunosuppression from severe infection and employing immunosuppressive drugs. Additionally, Group 2 exhibited an increased neutrophil count, hinting at possible bacterial co-infection. Group 1 showed differences in CD4RO and CD8RA lymphocyte populations, implying that cellular immunity plays a role in developing efficient postinfectious immunity. This intimation suggests that vaccination might mitigate the severity of the coronavirus infection and prevent complications, including long-term COVID-19.

The COVID-19 inflammation and high mortality mechanism trigger.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lasted from March 2020 to May 2023, infecting over 689 million and causing 6.9 million deaths globally. SARS-CoV-2 enters human cells via the spike protein binding to ACE2 receptors, leading to viral replication and an exaggerated immune response characterized by a "cytokine storm." This review analyzes the COVID-19 pathogenesis, strains, risk factors for severe disease, and vaccine types and effectiveness. A systematic literature search for 2020-2023 was conducted. Results show the cytokine storm underlies COVID-19 pathogenesis, causing multiorgan damage. Key viral strains include Alpha, Beta, Gamma, Delta, and Omicron, differing in transmissibility, disease severity, and vaccine escape. Risk factors for severe COVID-19 include older age, obesity, and comorbidities. mRNA, viral vector, and inactivated vaccines effectively prevent hospitalization and death, although new variants exhibit some vaccine escape. Ongoing monitoring of emerging strains and vaccine effectiveness is warranted. This review provides updated information on COVID-19 pathogenesis, viral variants, risk factors, and vaccines to inform public health strategies for containment and treatment.

The Impact of Pathogen Reduction on Total IgG and IgG Subclass Profiles of Convalescent Plasma.

Introduction: In case of newly emerging pathogens, convalescent plasma (CP) is often the only early available treatment option. It has been shown that different IgG subclasses contribute differently to CP neutralizing activity. As CP donors often have a risk profile like first-time donors, especially with respect to window-period viral transmission, pathogen reduction (PR) could mitigate that risk. The aim of our study, especially in the light of potential future pandemics, was to evaluate the impact of commercially available PR technologies on total IgG and IgG subclasses quantity and distribution in CP using COVID-19 CP (CCP) as surrogate for CP in a side-by-side comparison approach. Methods: 36 apheresis CCP donations were allocated to three study groups and a side-by-side assessment of the potential impact of amotosalen (AS)/UVA treatment compared to a riboflavin (RB)/UVB treatment, AS against methylene blue (MB) treatment, and RB against MB treatment on the quantity of IgG and IgG subclasses with a nephelometric analyzer was performed. Results: IgG subclass distributions were not significantly changed post PR treatment with all three technologies. There was also no significant difference in the median loss of concentration for IgG1 and IgG2 between the three technologies. We recognized a non-significant trend of a higher IgG4 median loss post RB treatment compared to post AS and MB treatment, respectively. Conclusion: Although the three commercially available PR systems do not significantly alter the distribution of IgG subclasses, we detected a non-significant trend of higher IgG4 loss after RB treatment. The potential impact of that finding needs further investigation.

Osteopontin and Regulatory T Cells in Effector Phase of Allergic Contact Dermatitis.

Studies have shown that osteopontin (OPN) and regulatory T cells play a role in allergic contact dermatitis (ACD), but the mechanisms responsible for their function are poorly understood. The study aimed to determine CD4 T lymphocytes producing intracellular osteopontin (iOPN T cells) and assess the selected T lymphocyte subsets including regulatory T cells in the blood of patients with ACD. Twenty-six patients with a disseminated form of allergic contact dermatitis and 21 healthy controls were enrolled in the study. Blood samples were taken twice: in the acute phase of the disease and during remission. The samples were analyzed by the flow cytometry method. Patients with acute ACD showed significantly higher percentage of iOPN T cells compared with healthy controls which persisted during remission. An increase in the percentage of CD4CD25 and a reduced percentage of regulatory T lymphocytes (CD4CD25highCD127low) were also found in the patients with acute stage of ACD. The percentage of CD4CD25 T lymphocytes showed a positive correlation with the EASI index. The increase in the iOPN T cells can indicate their participation in acute ACD. The decreased percentage of regulatory T lymphocytes in the acute stage of ACD may be related to the transformation of Tregs into CD4CD25 T cells. It may also indicate their increased recruitment to the skin. The positive correlation between the percentage of CD4CD25 lymphocytes and the EASI index may be indirect evidence for the importance of activated lymphocytes-CD4CD25 in addition to CD8 lymphocytes as effector cells in ACD.

Analysis of the IgG subclass profile and IgG sum-total discrepancy in COVID-19 convalescent plasma donors: A single-centre prospective cohort study.

INTRODUCTION: Although IgG1 and IgG3 have been shown to be the dominant subclasses in the acute phase of SARS-CoV-2 infection, little is known about the distribution of IgG subclasses during the recovery phase of COVID-19. The aim of the study was to analyze the profile of IgG subclasses in COVID-19 convalescent plasma donors. METHODS: A total of 36 convalescent plasma donors were included in the analysis. IgG and IgG subclass levels were measured using a nephelometric assay in plasma samples obtained directly from the plasma container. RESULTS: Although there was no significant difference in the concentration of IgG subclasses between the study and control groups, the contribution of IgG1 to the total IgG pool between the study and control groups was statistically significant (p = 0.0478). In addition, there was a discrepancy between the total IgG and IgG sum values in the study group, exceeding 15 % in 19,4 % of samples (n = 7), while in the control group no samples with a sum/ total IgG difference > 15 % were observed. CONCLUSIONS: The selective affinity of the IgG1 subclass for the polyclonal anti-IgG reagent may interfere with the determination of total IgG and should be considered when interpreting the results of enzyme immunoassays DATA AVAILABILITY: The data that support the findings of this study are available on request from the corresponding author.

Innate and Acquired Cellular Immunity in Children with Familial Hypercholesterolemia Treated with Simvastatin.

The aim of this cross-sectional study was to assess the influence of simvastatin treatment in children with familial hypercholesterolemia (FH) on parameters of cellular immunity. Twenty-six children with FH were included, of which thirteen were treated with 10 mg simvastatin for at least 26 weeks, and thirteen were age- and sex-matched with a low-cholesterol diet only. Total WBC count and lipid profile were measured. Flow cytometry was used to identify lymphocyte subsets and determine the expression of adhesion molecules (AM) and toll-like receptors (TLRs) on leukocytes. No differences were found in the basic values of peripheral blood count and subpopulations of lymphocytes between groups. The percentage of granulocytes with the expression of AM was higher in those treated with statins. The TLR-2 expression on granulocytes and monocytes showed higher values, whereas the TLR-4 expression was lower on lymphocytes and granulocytes in simvastatin-treated children. Treatment with simvastatin in children with FH is not associated with alterations in the amounts of granulocytes and monocytes. There is no association between statin treatment and the pattern of peripheral blood lymphocyte subpopulations. The role of AM and TLRs needs further investigation, given the effect of statins on the innate immunity may be important for their efficacy and safety during growth.

Systemic COVID-19 Vaccination Enhances the Humoral Immune Response after SARS-CoV-2 Infection: A Population Study from a Hospital in Poland Criteria for COVID-19 Reimmunization Are Needed.

Systemic vaccination with the BNT162b2 mRNA vaccine stimulates the humoral response. Our study aimed to compare the intensity of the humoral immune response, measured by SARS-CoV-2 IgG, SARS-CoV-2 IgM, and S-RBD-neutralizing IgG antibody levels after COVID-19 vaccination versus after SARS-CoV-2 infection. We analyzed 1060 people in the following groups: convalescents; healthy unvaccinated individuals; individuals vaccinated with Comirnaty, AstraZeneca, Moderna, or Johnson & Johnson; and vaccinated SARS-CoV-2 convalescents. The concentrations of SARS-CoV-2 IgG, SARS-CoV-2 IgM, and S-RBD-neutralizing antibodies were estimated in an oncology hospital laboratory by chemiluminescent immunoassay (CLIA; MAGLUMI). Results: (1) We observed a rise in antibody response in both the SARS-CoV-2 convalescent and COVID-19-vaccinated groups. (2) The levels of all antibody concentrations in vaccinated COVID-19 convalescents were significantly higher. (3) We differentiated asymptomatic SARS-CoV-2 convalescents from the control group. Our analysis suggests that monitoring SARS-CoV-2 IgG antibody concentrations is essential as an indicator of asymptomatic COVID-19 and as a measure of the effectiveness of the humoral response in convalescents and vaccinated people. Considering the time-limited effects of post-SARS-CoV-2 infection recovery or vaccination and the physiological half-life, among other factors, we suggest monitoring IgG antibody levels as a criterion for future vaccination.

Cardiac Surgery is Associated with Biomarker Evidence of Neuronal Damage.

BACKGROUND: Anesthesia and surgery is commonly associated with central nervous system sequelae and cognitive symptoms, which may be caused by neuronal injury. Neuronal injury can be monitored by plasma concentrations of the neuronal biomarkers tau and neurofilament light protein (NFL). Currently, there are no studies examining whether neuronal injury varies between surgical procedures. OBJECTIVE: Our aim was to investigate if neuronal damage is more frequent after cardiac than after otolaryngeal surgery, as estimated by tau and NFL concentrations in plasma. METHODS: Blood samples were drawn before, during, and after surgery and concentrations of tau, NFL, Aß40, and Aß42 were measured in 25 patients undergoing cardiac surgery (9 off-pump and 16 on-pump) and 26 patients undergoing otolaryngeal surgery. RESULTS: Tau increased during surgery (1752%, p = 0.0001) and NFL rose seven days post-surgery (1090%, p < 0.0001) in patients undergoing cardiac surgery; even more in patients on-pump than off-pump. No changes were observed in patients undergoing otolaryngeal surgery and only minor fluctuations were observed for Aß40 and Aß42. CONCLUSION: Cardiac surgery is associated with neuronal injury, which is aggravated by extracorporeal circulation. Analyses of NFL and tau in blood may guide development of surgical procedures to minimize neuronal damage, and may also be used in longitudinal clinical studies to assess the relationship of surgery with future neurocognitive impairment or dementia.

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.

Study of the protective effects of nootropic agents against neuronal damage induced by amyloid-beta (fragment 25-35) in cultured hippocampal neurons.

BACKGROUND: Alzheimer's disease (AD) is a common neurodegenerative disorder, in which progressive neuron loss, mainly in the hippocampus, is observed. The critical events in the pathogenesis of AD are associated with accumulation of ß-amyloid (Aß) peptides in the brain. Deposits of Aß initiate a neurotoxic "cascade" leading to apoptotic death of neurons. Aim of this study was to assess a putative neuroprotective effects of two nootropic drugs: piracetam (PIR) and levetiracetam (LEV) on Aß-injured hippocampal neurons in culture. METHODS: Primary cultures of rat's hippocampal neurons at 7 day in vitro were exposed to Aß(25-35) in the presence or absence of nootropics in varied concentrations. Flow cytometry with Annexin V/PI staining was used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. RESULTS: Aß(25-35) caused concentration-dependent death of about one third number of hippocampal neurons, mainly through an apoptotic pathway. In drugs-containing cultures, number of neurons injured with 20 µM Aß(25-35) was about one-third lesser for PIR and almost two-fold lesser for LEV. When 40 µM Aß(25-35) was used, only LEV exerted beneficial neuroprotective action, while PIR was ineffective. CONCLUSIONS: Our results suggest the protective potential of both studied nootropics against Aß-induced death of cultured hippocampal neurons with more powerful neuroprotective effects of LEV.

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)-ß 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-ß 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.

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.

CD4+CD25+ and CD4+CD2+high regulatory T cells in disseminated and localized forms of allergic contact dermatitis: relation to specific cytokines.

The aim of this study was to evaluate regulatory T lymphocytes (Tregs) in the course of allergic contact dermatitis (ACD) and to elucidate the role of IL-10 and TGF-ß in Tregs activity. Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) cells were determined by flow cytometry in patients with acute disseminated ACD ('ad', n = 36), acute localized ACD ('al', n = 26), and disseminated ACD during remission ('rd', n = 27) as well as in controls (n = 22). Serum levels of cytokines were measured using ELISA. The mean percentage of CD4(+)CD25(+) and CD4(+)CD25(high) cells in patients with ad ACD was significantly higher than in controls (p < 0.01) and the remaining patients (p < 0.05). Both cell populations were significantly elevated in persons with widespread skin lesions (p < 0.05). In ad patients the CD4(+)CD25(+) increased during three weeks of disease, although the significant increase of CD4(+)CD25(high) was noted only in the third week. Patients with ad ACD showed a significantly decreased serum level of TGF-ß1 as compared with controls and the remaining ACD patients. IL-10 level did not differ between all groups. The elevated population of CD4(+)CD25(high) cells in ad ACD patients, and its dependence on the extension of skin lesions, suggest a role of Tregs in regulating the course of ACD. The growing Tregs percentages may indicate their peripheral generation during ACD. The development of lesions despite an increased population of Tregs suggests their functional defect. The role of TGF-ß1 in the suppressive activity of Tregs cannot be excluded.

[Generation of T regulatory cells in children with type 1 diabetes mellitus]. / Generacja komórek T regulatorowych u dzieci z cukrzyca typu 1.

INTRODUCTION: In spite of intensive research the pathogenesis of type 1 diabetes mellitus is not thoroughly understood. One of the ideas which currently has a great number of supporters is the theory of the participation of T regulatory cells in the mechanism of insufficient suppression of the immune response against pancreatic self -antigens. According to some authors, the infusion of T regulatory cells in autoimmune diseases could lead to long -term remission or even a complete cure. AIM OF THE STUDY: The aim of our present study was to achieve T regulatory cells from conventional T lymphocytes isolated from a small amount of peripheral blood in children with type 1 diabetes mellitus and their comparison to the Tregs generated from the blood of control children. Additionally, we assessed the changes in the expression of selected genes essential for the function of these cells during Tregs generation. MATERIAL AND METHODS: The examined group consisted of 20 children with type 1 diabetes mellitus, the control group consisted of 20 non -diabetic children. From the peripheral blood CD4+CD25 - cells were separated and cultured with T -reg expander and interleukin (IL) 2. Before and after the culture the cells were analysed according to the expression of transcription factor FoxP3 and other molecules/cytokines: OX40, 4 -1BB, GITR, ICOS -1, CTLA -4 and IFN -γ, IL -10 and TGF -ß. RESULTS: We observed a significantly higher percentage of T regulatory cells after the culture (with no difference between diabetic and control children). Moreover, we observed a lower expression of mRNA for GITR molecule and a higher IL -10 expression in the cultures of diabetic children compared to the control ones. The cells cultured from the blood of control children were characterised by a higher increase in the expression of mRNA for ICOS -1 and a lower expression of mRNA for TGF -ß in comparison to the cultures from diabetic children. CONCLUSIONS: The results of our investigations confirm the possibility of generating T regulatory cells from conventional T lymphocytes from peripheral blood of children with newly recognised type 1 diabetes mellitus.

[Aging of the immune system]. / Starzenie sie ukladu odpornosciowego.

The immune system theory of aging is that the rate of aging is largely controlled by the immune system. As we age, the numbers of critical cells in the immune system decrease and become less functional. Accumulating data are documenting an inverse relationship between immune status, response to health, and longevity, suggesting that the immune system becomes less effective with advancing age and that this is clinically relevant. The mechanisms and consequences of age-associated immune alterations are briefly reviewed here.

Transformation of conventional T cells into regulatory T cells in children with metabolic syndrome.

BACKGROUND: Much research has been done in the recent years to establish an association between obesity, metabolic syndrome and the immune system. Numerous data suggest that the decreased number and/or function of regulatory T cells (Treg cells) can lead to chronic minimal inflammation present in patients with obesity and trigger formation of atherosclerotic plaque. AIM: To generate Treg cells from the peripheral blood in children meeting the diagnostic criteria of metabolic syndrome. METHODS: A total of 25 children with metabolic syndrome and 25 controls were enrolled in the study. Peripheral blood was collected, CD4(+)/CD25(-) cells were separated and cultured for 4 weeks in the presence of a Treg expander (CD3/CD28) and interleukin-2. The expression of the transcription factor FoxP3 as a Treg marker was assessed before and after culture using reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry. RESULTS: Before the culture we observed a slightly lower percentage of Treg cells in children with metabolic syndrome vs controls. After the culture we noted a significant increase in mRNA expression and in the percentage of FoxP3-positive cells. We observed no differences in the results between the children with metabolic syndrome and the controls. CONCLUSIONS: Our study shows that it is possible to generate Treg cells from peripheral blood of children with metabolic syndrome. In future, these findings could be used to develop a model of immunotherapeutic intervention for patients at risk of cardiovascular disease.

[Higher percentages of T regulatory cells in children at risk for developing type 1 diabetes mellitus]. / Podwyzszone odsetki limfocytów T regulatorowych u dzieci z grupy ryzyka zachorowania na cukrzyce typu 1.

INTRODUCTION: The natural history of type 1 diabetes is concerned with the appearance of autoantibodies against antigens of pancreatic beta cells. The last decade revealed some evidence of the participation of T regulatory lymphocytes - cells which suppress immune response - in the pathogenesis of type 1 diabetes and prediabetes. AIM OF THE STUDY: was the assessment of T regulatory cells in the blood of children at risk for developing type 1 the diabetes mellitus. MATERIAL AND METHODS: 85 subjects, siblings of children with type 1 diabetes, were enrolled into the study. The presence of anti-GAD65 antibodies was assessed. With the use of flow cytometry the following cell subpopulations were noted: CD4+, CD4+CD25high and CD4+CD25highCD127low with the coexpression of: CD28, CD45RO, CD54, CD62L and CD134 molecules. RESULTS: We did not observe any differences in white blood cell count, lymphocyte (including CD4+) count and the percentage between the examined and control groups. We noted higher percentages of T regulatory cells: CD4+CD25high, CD4+CD127low and CD4+CD25highCD127low in children with the presence of anti-GAD65 antibodies as compared to the control children. CONCLUSION: Higher percentages of T regulatory cells in the blood of children with the presence of anti-GAD65 antibodies may suggest an intensive regulatory response present in patients at risk for developing type 1 diabetes.

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.