CCR5-Δ32 polymorphism is a genetic risk factor associated with dyslipidemia in patients with type 1 diabetes.

AIM: In the currently available literature there are no works investigating the correlation between CCR5-Δ32 polymorphism and dyslipidemia in children with type 1 diabetes mellitus (T1D). Therefore, we have decided to explore the potential role played by this polymorphic locus in the incidence of dyslipidemia as an important risk factor for cardiovascular disease (CVD) in patients with T1D. METHODS: A total of 380 patients with T1D were selected. Patients were divided into two groups: 180 patients with diabetic dyslipidemia and 200 controls without dyslipidemia. Characterization of CCR5-Δ32 genotypes was analyzed by polymerase chain reaction. Logistic regression model was used to examine the association between CCR5-Δ32 polymorphism and dyslipidemia. RESULTS: When participants were analyzed according to CCR5-Δ32 polymorphism, Δ32 carriers presented higher levels of: HbA1c (p < 0.001), fasting plasma glucose (p < 0.001), LDL (p = 0.02) as well as TG (p = 0.01) and lower levels of HDL (p = 0.01) than noncarriers. Moreover, the minor allele Δ32 was more frequent in dyslipidemic subjects than controls (p < 0.001) and conferred an increased individual risk for dyslipidemia (OR = 2.327; 95% CI = 11.241-4.365; p = 0.009). CONCLUSIONS: The findings of our study suggest that the CCR5-Δ32 polymorphism is associated with elevated plasma lipid levels and the Δ32 allele increases the risk of dyslipidemia in patients with T1D. Identification of the functional variant underlying these associations may potentially lead to the development of a novel and adjunctive approach for the treatment of dyslipidemia and CVD.

The KL-VS polymorphism of KLOTHO gene is protective against retinopathy incidence in patients with type 1 diabetes.

BACKGROUND AND AIMS: KLOTHO is an anti-ageing circulating hormone involved in insulin signaling, inflammation and vascular homeostasis through its protective effects on the endothelium and antioxidant actions. The common functional "KL-VS" variant of the KLOTHO gene is reproducibly associated with longevity in humans. Large number of studies have evaluated close relationship between KLOTHO protein and diabetes but the association between KL-VS variant and retinopathy in type 1 diabetes mellitus (T1D) is unknown. Therefore, in the present study we examined the association between the KL-VS polymorphism and the risk of diabetic retinopathy (DR) in patients with T1D. METHODS: We examined 400 patients with T1D and 350 healthy age-matched controls. The analysis concerned KL-VS polymorphism along with the levels of serum inflammatory (CRP, TNF-α) and anti-inflammatory (IL-10) markers, pro-angiogenic (angiogenin) and anti-angiogenic interferon gamma-induced protein 10 (IP-10) factors as well as adhesion molecules (ICAM-1, ICAM-3). RESULTS: We did not find significant association between T1D and KL-VS alleles. However, we observed that the incidence of KL-VS genotype is lower in a group with retinopathy in comparison to diabetic patients without this complication. Moreover, we established that KL-VS carriers had the lowest levels of inflammatory markers, pro-angiogenic factors and adhesion molecules. Simultaneously, the KL-VS carriers had increased serum levels of anti-inflammatory and anti-angiogenic cytokines than holders bearing wild type genotype. CONCLUSIONS: In conclusion, the findings of our studies suggest that the functional KL-VS variant of the KLOTHO gene protects against the development of retinopathy in patients with T1D.

Estrogen receptor α gene polymorphism and vascular complications in girls with type 1 diabetes mellitus.

The effect of estrogens is mediated by activation of estrogen receptors (ERs). Because ER-α gene polymorphisms may exert different effects in childhood, we analyzed the associations between the IVS1 -397T>C (PvuII) polymorphism and systemic inflammatory state, proangiogenic factors, frequency of monocyte subsets, lipid profile, blood pressure, and vascular complications in girls with type 1 diabetes mellitus (DM1). We examined 180 young girls with DM1 and 120 healthy age-matched controls. The analysis concerned PvuII polymorphism of the ER-α gene as well as the levels of serum inflammatory markers (CRP, IL-6, TNF-α), proangiogenic factors (VEGF, angiogenin), 17ß-estradiol, values of monocyte subsets (CD14++CD16- and CD14+CD16+), lipid profile, and blood pressure. In our study, girls with CC genotype had lower level of inflammatory and angiogenic factors and lower frequencies of CD14+CD16+ monocytes in comparison to CT or TT carriers. Simultaneously, the CC carriers had a greater population of CD14++CD16- monocytes, increased blood pressure, and serum levels of: estrogen, total cholesterol, triglycerides, and low-density lipoprotein cholesterol than girls bearing CT or TT genotype. Our study suggests a pleiotropic effect of PvuII polymorphic CC variant on diabetic vasculopathies. Although the CC genotype carriers demonstrate less inflammatory and angiogenic activity, they seem to display less favorable cardiometabolic features. Based on our study, we cannot distinguish PvuII ER-α genotype that could be useful in identification of DM1 girls that are more prone to develop of late vascular complications, before the occurrence of first clinical symptoms.

The GA genotype of the -1154 G/A (rs1570360) vascular endothelial growth factor (VEGF) is protective against hypertension-related chronic kidney disease incidence.

Vascular endothelial growth factor (VEGF) is a highly specific mitogen with angiogenic and vascular permeability activities for endothelial cells. VEGF participates in maintaining the renal vasculature integrity. There is no doubt that hypertension accelerates progression to renal dysfunction, resulting in chronic kidney disease (CKD). The purpose of our study was to examine the VEGF -1154 G/A (rs1570360) polymorphism among hypertensive patients with CKD. Additionally markers of endothelial damage have been related to the advancement of CKD. There were 96 consecutively admitted hypertensive patients referred to our Institution by their general practitioner. The patients were treated with an anti-hypertensive polytherapy. Ninety-nine healthy volunteers were enrolled as the control group. Our study revealed that both healthy and hypertensive groups frequencies of the genotypes varied significantly (p = 0.030, χ (2) test). The GA genotype frequency was significantly lower among patients in comparison with healthy. The presence of GA genotype was connected with a decreased risk of hypertension disease (OR = 0.48, p = 0.01). The VEGF -1154 GA carriers have been associated with the lowest values of Endostatin (p = 0.020), Angiogenin (p = 0.040) as well as vWf (p = 0.005). The GA genotype has been characterized by the highest values of eGFR (p = 0.024) and the lowest values of creatinine (p = 0.028) and BUN (p = 0.012). It is evident that the GA genotype of VEGF polymorphism localized at -1154 position is a genetic protective factor for development arterial hypertension and is associated with less progressed CKD.

The GG genotype of the -152 G/A vascular endothelial growth factor (VEGF) polymorphism predisposes to hypertension-related chronic kidney disease.

Our purpose was to determine whether the VEGF -152 G/A polymorphism could be associated with chronic kidney disease and endothelial dysfunction in hypertensive patients. There were 100 healthy volunteers enrolled into the control group. The group of patients was constituted by 99 consecutively admitted hypertensive patients referred to our Institution by their general practitioner. All patients were treated with anti-hypertensive polytherapy. Presented study revealed that the hypertensive patients bearing the GG genotype were characterized by the highest values of diastolic blood pressure and markers of endothelial damage such as Angiogenin, Endostatin, CRP as well as von Willebrandt factor. In addition, higher number of immature endothelial progenitor cells with CD34(+)CD133(+), CD34(+)CD133(-) markers was observed in GG hypertensive carriers while in normotensive individuals no differences were found. Such phenomenon may indicate an increased mobilization of bone-marrow derived endothelial progenitors. It may testify to the preserved compensatory mechanism in chronic kidney disease (CKD) patients until the G3a stage of the disease. Moreover, patients with higher estimated glomerular filtration rate (eGFR) level had lower of vWf and Endostatin values, and higher level of VEGF. Taken together our findings clearly indicate the -152 GG hypertensive carriers as more prone to develop CKD. We can suspect that the VEGF -152 GG genotype is strongly associated with hypertension-dependent CKD.

IL-33 Effect on Quantitative Changes of CD4+CD25highFOXP3+ Regulatory T Cells in Children with Type 1 Diabetes.

IL-33 is an IL-1 cytokine family member, with ability to induce both Th1 and Th2 immune responses. It binds to ST2 receptor, whose deficiency is associated with enhanced inflammatory response. The most recent studies have shown the immunoregulatory effect of IL-33 on Tregs in animal models. As type 1 diabetes is an autoimmune, inflammatory disease, where Treg defects have been described, we aimed to analyze the in vitro influence of recombinant IL-33 on quantitative properties of regulatory CD4+CD25highFOXP3+ T cells. CD4+CD25highFOXP3+ as well as CD4+CD25highFOXP3+ST2+ Tregs were analyzed by flow cytometry. In a group of patients with type 1 diabetes in vitro IL-33 treatment induced regulatory CD4+CD25highFOXP3+ cell frequencies as well as upregulating the surface expression of ST2 molecule. In addition, the number of CD4+CD25highFOXP3+ cells carrying ST2 receptor increased significantly. Similar effect was observed in case of the FOXP3 expression. We did not observe any significant changes in IL-33 treated cells of healthy controls. The level of ST2 was higher in serum of patients with type 1 diabetes in comparison to their healthy counterparts. We propose that IL-33 becomes an additional immunostimulatory factor used to induce Treg expansion in future clinical trials of adoptive therapy in type 1 diabetes.

Vascular endothelial growth factor polymorphism (-460 T/C) is related to hypertension-associated chronic kidney disease.

Our aim was to characterize the endothelial progenitor cells (EPCs) in normotensive controls and treated hypertensive individuals within the vascular endothelial growth factor (VEGF) -460 C/T polymorphism as well as to investigate whether this polymorphism predisposes to hypertension-related chronic kidney disease. The hypertensive patients bearing the TT genotype had the highest levels of immature EPC with the following phenotypes: CD34(+), CD34(+)CD45(dim), CD34(+)CD133(+)CD45(dim). The study showed the estimated glomerular filtration rate values significantly lower and creatinine and BUN parameters higher among the TT hypertensive patients. We presume that the highest mobilization of EPCs from bone marrow may signalize more severe renal hypertension-related complications in the VEGF -460 TT genotype.

Cytokine profiles during delivery affect cord blood hematopoietic stem and progenitors cells.

The study was aimed to determine the correlations between serum levels of cytokines (GM-CSF, IL-4, IL-10 and TNF) in maternal (MB) and cord blood (CB) and some features of cord blood hematopoietic stem and progenitor cells (CB HSPCs). Study material was MB and concomitant CB samples collected from 98 volunteers at the moment of delivery. The IL-4, IL-10, TNF and GM-CSF concentrations in serum and in supernatants from PMA-stimulated mononuclear cells isolated from both blood types were measured using BD Cytometric Bead Array Flex Set System. CB HSPCs (CD34(+)CD45(low)) proportion was also estimated by flow cytometry. The most relevant results concerned the tendency to down regulation of CB HSPCs number with an increase of IL-4, IL-10 and GM-CSF levels, only the TNF concentration seems to have no influence on HSPCs pole size. The strongest positive correlations were found between CD34(+)CD45(low) HSPCs number and IL-10 and GM-CSF in MB serum and GM-CSF and TNF from CB supernatants. The strongest negative correlations were found between CD34(+)CD45(low) HSPCs number and IL-4 and GM-CSF in CB serum and IL-10 in MB supernatants. Interestingly, we observed 'opposite correlation' between serum and supernatant from CB and MB. We concluded that elevated serum levels of IL-4, IL-10 and GM-CSF in CB are indicative of enhanced differentiation of HSPCs and characterize a normal perinatal development. Elevated levels of cytokines seem to stimulate differentiation of HSPCs what is advantageous for neonates during perinatal period.

Different pathways of macrophage activation and polarization.

Monocytes are short-lived cells and undergo spontaneous apoptosis every day. Inflammatory responses may induce dramatic up-regulation of monocyte survival and differentiation. When monocytes are recruited to an area of infection they may differentiate into macrophages. In different microenvironments macrophages polarize into two types. The M1 or classically activated macrophages are characterized by the high ability to produce pro-inflammatory cytokines and the production of NO through the induced synthesis of iNOS. The M2 or alternatively activated macrophages are divided into 3 subtypes, M2 a, b and c, and they have anti-inflammatory properties. Mediators of M1 macrophage TLR-dependent polarization include transcription factors such as NF-κB, AP-1, PU.1, CCAAT/enhancer-binding protein α (C/EBP-α), STAT1 as well as interferon regulatory factor 5 (IRF5), while the transcription factors which promote M2 activation include IRF4, C/EBP-ß, Krüppel-like factor 4 (KLF4), STAT6 and PPARγ receptor.

IVS1 -397T>C estrogen receptor α polymorphism is associated with low-grade systemic inflammatory response in type 1 diabetic girls.

PURPOSE: The study aimed to investigate the influence of estrogen receptor α (ER-α) genotypes on inflammatory response and development of microvascular complications in girls with type 1 diabetes. METHODS: 152 young regularly menstruating girls with diagnosed type 1 diabetes and 84 young, healthy menstruating girls were recruited. ER-α genotyping was carried out by PCR. Serum concentrations of 17ß-estradiol, as well as IL-6, TNF-α, VEGF, and IL-10, were measured. CD4(+)Foxp3(+) TH17 cells were isolated and analyzed by flow cytometry. RESULTS: Type 1 diabetic girls carrying TT genotype were characterized by the lowest serum estradiol level and IL-10 and highest IL-6, TNF-α, and VEGF. The association between the level of certain cytokine and the genetic variant of estrogen receptor α polymorphism was analyzed. Frequencies of CD4(+)Foxp3(+) TH17 cells were also enhanced in TT bearing girls with type 1 diabetes and correlated with the level of analyzed cytokines. In addition, the correlation between serum estradiol level and cytokine concentrations was observed. CONCLUSIONS: We propose that TT variant of estrogen receptor α polymorphism may be associated with enhanced inflammatory response, which in turn may lead to acceleration of diabetic retino- and nephropathy in girls with type 1 diabetes. This finding may help the physicians to predict the onset and progression of diabetic microvascular complications.

Enhanced apoptosis of monocytes from complication-free juvenile-onset diabetes mellitus type 1 may be ameliorated by TNF-α inhibitors.

Diabetes mellitus type 1 is associated with an enhanced apoptosis of different cells and tissues, accelerating occurrence of diabetic microvascular complications. The aim of our study was to determine spontaneous apoptotic potential of the monocyte subsets in juvenile-onset complication-free diabetes mellitus type 1 and to compare them with the corresponding values of the healthy. Moreover, we wanted to assess effects of TNF-R1 blocking agents and those of general TNF-α blocker (Infliximab) on spontaneous apoptosis of monocytes. Sixty randomly selected DM1 patients (14.5 ± 3.2 years) and 30 healthy (13.5 ± 2.8 years) volunteers were enrolled in the study. Our results indicate that three monocyte subsets are distinguishable in the groups of young diabetic patients and the healthy, similarly to in the blood of adults. DM1 patients were characterized by higher values of apoptotic monocytes than the healthy. The manipulation with drugs inhibiting TNF-R1 expression diminished the pool of CD16(+) apoptotic monocytes. Infliximab reduced the apoptotic CD16(-) cells. In conclusion, diabetes mellitus type 1 is associated with greater apoptosis of three monocyte subsets which may contribute to the development of microvascular complications. TNF-α modifiers appear to ameliorate monocyte apoptosis. They may be useful for controlling excessive monocyte apoptosis in diabetic patients.

The serum IL-6 profile and Treg/Th17 peripheral cell populations in patients with type 1 diabetes.

IL-6 is a pleiotropic cytokine involved in the regulation of the immune response, inflammation, and hematopoeisis. Its elevated levels are found in a range of autoimmune and chronic inflammatory diseases. IL-6 is also involved in regulation of the balance between two T cell subsets: Tregs and Th17, which have contradictory functions in the control of inflammation. The present study provides a quantitative analysis regarding the Th17/Treg cell balance in peripheral blood of children with type 1 diabetes and its association with serum IL-6 level.

Expansion of CD14+CD16+ monocytes producing TNF-α in complication-free diabetes type 1 juvenile onset patients.

We concentrated on the complication-free phase of juvenile onset type 1 diabetes mellitus (T1DM) searching for associations between concentration of inflammatory factors TNF-α, CRP and VEGF and two monocyte subsets the CD14(++)CD16(-) and CD14(+)CD16(+). We analysed a randomly selected group of 150 patients without complications (disease duration 2.74 ± 2.51 years) at the start of the project and 5 years later. They were compared with 24 patients with retinopathy (6.53 ± 3.39 years of disease) and 30 healthy volunteers. Our results indicate that in the complication-free period the concentration of TNF-α significantly increased and continued to increase after retinopathy was established. After 5 years the percentage and absolute number of CD14(+)CD16(+) monocytes doubled in complication-free patients. Our study indicates that the size of CD14(+)CD16(+) monocyte subset may be used alternatively to CRP values as an indicator of inflammation grade. Our results imply the necessity of trials using anti-TNF-α therapy in the complication-free phase of the disease.

Anti-TNF rescue CD4+Foxp3+ regulatory T cells in patients with type 1 diabetes from effects mediated by TNF.

The presence of low-grade chronic inflammation is a known feature of long standing diabetes type 1. The association between serum level of several markers of inflammation and severity of DM1 was proven. Serum concentrations of TNF were reported to be elevated in diabetic patients, especially those who developed diabetic complications. Lately, it has been also shown that TNF may impair the subset of naturally arising regulatory T cells, which control autoimmunity. The presented study, for the first time, shows the regulatory T cells in the context of an inflammatory environment that is present in patients with type 1 diabetes. It indicates that TNF reduces the number and frequency of regulatory CD4(+)Foxp3(+) T cells in children with diabetes type 1 and that in vitro treatment with anti-TNF antibody seems to rescue this cell subset from its defective effects.

Decreased angiogenin concentration in vitreous and serum in proliferative diabetic retinopathy.

Diabetic retinopathy is the most common cause of vision loss in young adults in developed countries. The disease therapy with anti-vascular endothelial growth factor (VEGF) agents gives some positive results, but is associated with retinal ischemia and vasoconstriction. Therefore, determination of factors involved in the physiological and pathological angiogenesis in the diabetic eye is of great importance for understanding of the pathogenesis of diabetic retinopathy and its effective treatment. Previously, we found that diabetic patients were characterized by increased serum concentration of VEGF, but decreased levels of other proangiogenic factor-angiogenin. The involvement of VEGF in pathogenesis of diabetic retinopathy is well established, but there is lack of data regarding angiogenin in retinopathy. Therefore, in the present study we measured angiogenin concentration in vitreous and serum samples of the patients with type 1 diabetes to determine its role in diabetic retinopathy. In addition, in each time, we compared the level of angiogenin with level of VEGF as a known factor involved in the pathogenesis of the disease. Angiogenin was found to be significantly more abundant in serum than in vitreous in both diabetic groups. In addition, patients with retinopathy had twofold lower vitreous angiogenin levels than diabetic individuals without complications. On the contrary, vitreous concentration of VEGF was dramatically increased only in participants with retinopathy. Patients without diabetic complications had significantly lower VEGF levels in vitreous than in serum and were characterized by high local and systemic concentration of angiogenin. These data suggest a local imbalance between two proangiogenic factors-VEGF and angiogenin in retinopathy. Low vitreous concentration of angiogenin in diabetic patients suggests that this factor is not responsible for pathological neovascularization in diabetic eye. Further studies will elucidate if angiogenin can be used to improve the insufficient angiogenesis in diabetes and prevent retinal ischemia after retinopathy treatment with anti-VEGF agents.

Lower frequency of CD62L(high) and higher frequency of TNFR2(+) Tregs are associated with inflammatory conditions in type 1 diabetic patients.

Diabetes type 1 is a chronic autoimmune disease in which insulin-producing cells are gradually destroyed by autoreactive T cells. Human regulatory cells play important role in controlling autoimmunity, and their qualitative or quantitative dysfunctions may result in ineffective suppression of autoreactive T cells. CD62L is a surface molecule that plays role in homing capabilities of Tregs, and only cells with high expression of CD62L have high suppressive potential. Tregs are also characterized by the constant expression of TNFR2. The frequency of Tregs carrying TNFR2 is higher in inflammatory conditions. We investigated blood regulatory T cells with CD62L expression and regulatory T cells expressing TNFR2 in type 1 diabetic patients. We found differences in these populations when comparing to healthy individuals. We propose that these may be associated with inflammatory conditions that are present in patients with type 1 diabetes. The lower percentage of Tregs and Treg CD62L(high) may contribute to ineffective suppression of proinflammatory cytokines production during type 1 diabetes.

Increased spontaneous production of VEGF by CD4+ T cells in type 1 diabetes.

In the present study we report that CD4(+) T cells from patients with type 1 diabetes produce significantly higher amounts of VEGF than respective cells from the healthy individuals. Among CD4(+) T cells memory subsets were the main source of VEGF. In addition, memory CD4(+) T subsets were the most numerous in patients with diabetic retinopathy (DR). DR was also characterized by significant increase of VEGF concentration in serum and culture supernatants. Hence, these data indicate that there is a sustained spontaneous production of VEGF by CD4(+) T cells in type 1 diabetes, which is additionally exacerbated in DR. In our opinion alterations in the proportions of CD4(+) T cell subsets and their VEGF production may be a useful tool for early assessment of the risk of DR onset and progression.

Inflammatory response of coronary artery disease postmenopausal women is associated with the IVS1-397T > C estrogen receptor alpha polymorphism.

We analyzed the relationship between the IVS1-397T > C polymorphism of estrogen receptor alpha gene and inflammatory response of eighty postmenopausal women with an established coronary artery disease (CAD). We found that the IVS1-397T allele carriers exhibited an enhanced inflammatory response in vivo and in vitro. These patients had a higher number of total monocytes and their CD14(+)CD16(+) inflammatory subset in relation to CC homozygotes. The CT and TT women's LPS-stimulated whole blood cell cultures produced more IL6 and TNFalpha than did the cultures of CC women. Moreover, significantly more of the T allele patients had major post-coronary artery bypass grafting complications and less of them experienced subjective angina pectoris improvement. Summarizing, the IVS1-397T > C polymorphism allows us to identify a group of postmenopausal women with the strong inflammatory response which is associated with a higher incidence of the major post-CABG adverse cardiovascular complications.

First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells.

Here, we describe a procedure and first-in-man clinical effects of adoptive transfer of ex vivo expanded CD4+CD25+CD127- T regulatory cells (Tregs) in the treatment of graft versus host disease (GvHD). The cells were sorted from buffy coats taken from two family donors, expanded ex vivo and transferred to respective recipients who suffered from either acute or chronic GvHD. The therapy allowed for significant alleviation of the symptoms and reduction of pharmacologic immunosuppression in the case of chronic GvHD, while in the case of grade IV acute GvHD it only transiently improved the condition, for the longest time within all immunosuppressants used nonetheless.

Ex vivo expansion of CD4(+)CD25(+) T regulatory cells for immunosuppressive therapy.

Immunosuppressants are powerful drugs, capable of triggering severe adverse effects. Hence, there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models, they have been found to quench autoimmune diseases, maintain allogeneic transplants, and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery, not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles, and has opened the doors to the clinical application of Tregs. In the review, we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory, as well as some published reports, Treg adoptive therapy is a promising tool in immunosuppressive therapy, and should be considered for clinical trials.