Unlocking the Uterine Code: Microbiota, Immune Cells, and Therapy for Recurrent Reproductive Failure.

The uterine microbiota has been the subject of increasing study, but its interaction with the local immune system remains unclear. Successful embryo implantation relies on endometrial receptivity, which is pivotal for immunological tolerance to fetal antigens and precise regulation of inflammatory mediators. Emerging data suggest a dynamic interplay between endometrial microflora and the immune system, making dysbiosis a potential determinant of pregnancy outcomes. Imbalances in the regulation of immune cells in the endometrium and decidua have been associated with infertility, miscarriage, and obstetric complications. A thorough comprehension of the immune system in the female reproductive tract shows potential for improving women's health and pregnancy outcomes. The objective of this study was to evaluate the patterns of endometrial microbiota in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL) and to explore their implications for endometrial immune cells and chronic endometritis (CE). Immune cells in biopsies from 107 RIF and 93 RPL patients were examined using flow cytometry. The endometrial microbial composition was analyzed using real-time polymerase chain reaction (RT-PCR). The research uncovered disrupted endometrial microbiota in most women with RIF and RPL, which was often associated with significant effects on lymphocytes, T cells, and uNK cells.

Early Diagnostic Markers of Late-Onset Neonatal Sepsis.

OBJECTIVE: Early diagnosis of nosocomial infections in newborns is a great challenge, because in the initial phase of systemic infection, clinical symptoms are often non-specific, and routinely used hematological markers are not sufficiently informative. The aim of this study was to determine the potential of early inflammatory markers to diagnose late-onset neonatal sepsis-procalcitonin (PCT), interleukin 6 (IL-6), interleukin 8 (IL-8) and endocan (ESM-1). MATERIAL AND METHODS: A prospective clinical-epidemiological study was conducted in a third-level NICU in Pleven, Bulgaria. Patients with suspected late-onset sepsis and healthy controls were tested. A sandwich ELISA method was used to measure the serum concentrations of biomarkers. RESULTS: Sixty newborns were included, of which 35% symptomatic and infected, 33.3% symptomatic but uninfected and 31.7% asymptomatic controls. The mean values of PCT, IL-6, I/T index and PLT differ significantly in the three groups. For ESM-1, IL-8 and CRP, the difference was statistically insignificant. The best sensitivity (78%) and negative predictive value (84%) was found for IL-6. The combinations of PCT + IL-6 and PCT + IL-6+ I/T+ PLT showed very good diagnostic potential. CONCLUSION: The introduction into the routine practice of indicators such as PCT and IL-6 may provide an opportunity to promptly optimize the diagnostic and therapeutic approach to LOS.

Anaplasma and Ehrlichia Species in Ixodidae Ticks Collected from Two Regions of Bulgaria.

The aim of the study was to determine prevalence of Anaplasmataceae-infected ticks in the Black Sea Coast and the Pleven regions of Bulgaria. A total of 350 ticks from different tick species were collected. Two hundred fifty-five ticks were removed from dogs in the Black Sea Coast region, and 95 Ixodes ricinus ticks were collected by flagging vegetation with a white flannel cloth in two areas in the region of Pleven. After the DNA isolation of the ticks, a genus-specific polymerase chain reaction (PCR) was performed to identify Anaplasmataceae. Second PCRs were performed with species-specific primers to identify Ehrlichia canis (E. canis) and Anaplasma phagocytophilum (A. phagocytophilum). The results showed that 26.9% of the Ixodes ricinus ticks were infected with Anaplasmataceae in the Black Sea Coast region and 36.8% in the Pleven region. The infection with E. canis was detected in 35.7% and A. phagocytophilum in 25.0% of positive ticks from the Black Sea Coast region. In the Pleven region, 22.9% of ticks were positive for E. canis, while 42.9% were positive for A. phagocytophilum. The molecular identification of E. canis in ticks collected from Bulgaria was performed for the first time. In conclusion, the present study revealed a higher prevalence of ticks infected with Anaplasmataceae, particularly A. phagocytophilum, in the Pleven region than in the Black Sea Coast region.

Elevated IgG and IgM Autoantibodies to Advanced Glycation End Products of Vascular Elastin in Hypertensive Patients with Type 2 Diabetes: Relevance to Disease Initiation and Progression.

The increased glycation of elastin is an important factor in vascular changes in diabetes. Using the ELISA method, we determined serum levels of IgM and IgG autoantibodies to advanced glycation end products of vascular elastin (anti-AGE EL IgM and anti-AGE EL IgG) in 59 hypertensive patients with type 2 diabetes (T2D) and 20 healthy controls. Serum levels of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and the C-reactive protein (CRP) were also determined. The levels of anti-AGE EL IgM antibodies in the T2D group were similar to those in the control group, while those of anti-AGE EL IgG antibodies were significantly higher (p = 0.017). Significant positive correlations were found between the levels of anti-AGE EL IgM antibodies and MMP-2 (r = 0.322; p = 0.013) and between the levels of anti-AGE EL IgG antibodies and CRP (r = 0.265; p = 0.042). Our study showed that elevated anti-AGE EL IgG antibody levels may be an indicator of the enhanced AGE-modification and inflammatory-mediated destruction of vascular elastin in hypertensive patients with T2D. Anti-AGE EL IgM antibodies may reflect changes in vascular MMP-2 activity, and their elevated levels may be a sign of early vascular damage.

Prevalence of Borrelia burgdorferi Sensu Lato in Ixodes ricinus Ticks Collected from Kaylaka Park in Pleven, Bulgaria.

We aimed to determine the presence and distribution of Borrelia burgdorferi sensu lato (s.l.) in Ixodes ricinus ticks collected from urbanized and wild areas in Kaylaka Park (Bulgaria). A total of 546 ticks were collected over three years (2017-2019). The presence of Borrelia in 334 of the collected I. ricinus was detected by dark-field microscopy (DFM) and two nested PCRs (nPCR) targeting the borrelial 5S-23S rRNA intergenic spacer and Flagellin B (FlaB) gene. DFM was performed on a total of 215 ticks, of which 86 (40%) were positive. PCR was performed on 153 of the ticks. In total, 42.5% of the 5S-23S rRNA intergenic spacer and 49% of FlaB were positive. Considering as positive any single tick in which Borrelia sp. was detected regardless of the used method, the infection rate reached 37% (10/27) in the nymphs and 48.5% (149/307) in the adults (48.7% (77/158) females, 48.3% (72/149) males). The incidence of B. burgdorferi infection in I. ricinus did not differ statistically significantly between female, male, and nymph. This study provides evidence that Lyme disease spirochetes are present in various regions of Kaylaka Park with extremely high prevalence in their vectors.

Changes in Serum Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinases-1 in Patients with Essential Hypertension.

Hypertension (HTN) is a leading risk factor for cardiovascular (CV) disease. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are thought to be actively involved in the remodeling of the CV extracellular matrix (ECM) during hypertensive damage. Therefore, in this study, we aimed to investigate serum levels of MMP-1 and TIMP-1 in patients with essential HTN and compare them with those of normotensive individuals. We measured serum concentrations of MMP-1 and TIMP-1 in 60 patients with HTN and 20 healthy controls using an ELISA. The obtained results showed that in patients with HTN, the mean levels of MMP-1 (1.82 ± 0.9 ng/mL) were significantly higher (p = 0.03) than the mean levels in the control group (1.19 ± 0.7 ng/mL). The levels of TIMP-1 in patients with essential HTN (0.44 ± 0.1 ng/mL) were also significantly higher (p = 0.005) than those in the control group (0.33 ± 0.1 ng/mL). In HTN, elevated serum MMP-1 levels may be associated with increased collagen degradation in the CV ECM, whereas elevated TIMP-1 levels may favor its accumulation and the development of pathological remodeling and fibrosis of the heart and arterial vessels.

Estimation of Ixodes ricinus (Acari: Ixodidae) Populations of Kaylaka Park in the Town of Pleven, Bulgaria.

(1) Background: Ticks are vectors of a large number of pathogenic microorganisms, which cause serious diseases in both humans and animals. Kaylaka Park is located in northern Bulgaria close to the city of Pleven. Part of the park is urbanized and visited daily by many citizens. The aim of our study was to determine the presence and distribution of hard ticks in the park area by surveying and comparing four urbanized with four wild areas. (2) Methods: Ticks were collected by flagging from 2016 to 2020 during the spring-summer season (March-July). Air temperature, relative humidity, collection time and flagging area were measured during the campaign. (3) Results: A total of 622 ticks were collected: 285 females (46%), 272 (44%) males and 64 (10%) nymphs. All were identified as Ixodes ricinus. Wild areas showed statistically significant higher values of ticks collected per minute (p = 0.009) and nymph densities (p = 0.003) compared to urbanized sampling sites. Other densities indices did not have a significant difference between urban and wild areas. Highest numbers of Ixodes ticks were collected at a temperature of 20 °C and at 60% relative humidity. The active questing began in March, peaked in end of April and declined in June. (4) Conclusions: In the present study, we found that ecological factors in the Kaylaka Park area are favourable for the development and distribution of tick populations. The results give us reason to consider that there is a high risk to visitors from tick bites in the Kaylaka Park area.

Serum Anti-Collagen IV IgM and IgG Antibodies as Indicators of Low Vascular Turnover of Collagen IV in Patients with Long-Term Complications of Type 2 Diabetes.

Thickening of the vascular basement membrane (BM) is a fundamental structural change in the small blood vessels in diabetes. Collagen type IV (CIV) is a major component of the BMs, and monitoring the turnover of this protein in type 2 diabetes (T2D) can provide important information about the mechanisms of vascular damage. The aim of the study was through the use of non-invasive biomarkers of CIV (autoantibodies, derivative peptides, and immune complexes) to investigate vascular turnover of CIV in patients with long-term complications of T2D. We measured serum levels of these biomarkers in 59 T2D patients with micro- and/or macrovascular complications and 20 healthy controls using an ELISA. Matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) were also tested. In the T2D group, significantly lower levels of CIV markers and significantly higher levels of MMP-2 and MMP-9 were found compared to controls. A significant positive correlation was found between IgM antibody levels against CIV and MMP-2. These findings suggest that vascular metabolism of CIV is decreased in T2D with long-term complications and show that a positive linear relationship exists between MMP-2 levels and CIV turnover in the vascular wall.

Comparison of serum levels of collagen type I turnover markers in early-onset preeclampsia and healthy pregnant women.

INTRODUCTION: Collagen type I is a major structural component of human uterus. Recent studies have found that collagen type I turnover is altered in preeclampsia and imbalance occurs between collagen synthesis and degradation processes. This acts as a stimulus for abnormal changes in the concentrations of collagen type I derived products in circulating blood. AIM: Having in mind the pathological collagen type I turnover in preeclampisa, the aim of the present study was to determine the levels of N-terminal propeptide of collagen type I (PINP), C-terminal propeptide of collagen type I (PICP): both markers of collagen type I synthesis and levels of matrix metalloproteinase-1 (MMP-1): marker of collagen type I degradation in sera of women with early-onset preeclampsia. MATERIALS AND METHODS: Thirty-two patients with early-onset preeclampsia were examined. The mean age of the patients was 28.8±5.5 years; and the mean age of 22 age-matched healthy pregnant women was 28.5±6.0 years. The enzyme-linked immunosorbent assay (ELISA) was used for measuring serum levels of PINP, PICP and MMP-1. RESULTS: There were no statistically significant differences between levels of PINP, PICP and MMP-1 in the sera of women with preeclampsia and healthy pregnancies: 16.991 (15.41÷21.143) vs. 17.189 (15.805÷20.747) µg/L (KW=0.02; p>0.05), 10.929 (8.7÷13.937) vs. 11.474 (7.369÷11.932) µg/L (KW=0.026; p>0.05), and 2.097 (1.384÷2.923) vs. 2.589 (1.41÷4.533) µg/L (KW=0.238; p>0.05), respectively. There were statistically significantly higher number of patients from the preeclampsia group than healthy pregnant women with BMI≥25 (23/32 vs. 4/22) and abnormal UAD (18/32 vs. 2/22) (p.

Circulating Levels of Endothelin-1 and Big Endothelin-1 in Patients with Essential Hypertension.

The role of endothelin-1 (ET-1) in the pathogenesis of hypertension (HTN) is not clearly established. There is evidence that its circulating levels are elevated in some forms of experimental and human HTN, but this was not a consistent finding. Based on these controversial data, we tested serum levels of ET-1 and Big ET-1 (the precursor of ET-1) in patients with essential HTN, comparing the results with those of healthy normotensive controls. The levels of ET-1 and Big ET-1 were measured by ELISA. Our results in patients with essential HTN showed that the mean levels of ET-1 (5.01 ± 2.1 pg/mL) were significantly higher (F = 6.34, p = 0.0144) than the mean levels in the control group (3.2 ± 1.0 pg/mL). The levels of Big ET-1 in patients with essential HTN (0.377 ± 0.1 pmol/L) were similar to those in the control group (0.378 ± 0.07 pmol/L) and did not differ significantly (F = 0.00, p = 0.9531). These data suggest that ET-1, but not Big ET-1, may play an important role in the pathogenesis of primary HTN.

Serum Galectin-3 Levels Are Unlikely to Be a Useful Predictive Marker for Early-onset Preeclampsia Development.

Galectin-3 (gal-3) is lectin which is presumed to interact with extracellular matrix proteins and cell surface glycoproteins in normal and pathophysiological conditions. The expression of gal-3 at the fetal-maternal interface partially overlaps that of gal-1, suggesting that an interplay between them might be important for hypertensive disorders in pregnancy like preeclampsia. The aim of our study was to test the hypothesis whether galectin-3 could be used as a predictive marker for early-onset preeclampsia development. 32 patients with early-onset preeclampsia were examined, mean age 28.8 ± 5.5; and 22 age matched normal pregnancies mean age 28.5 ± 6.0. The enzyme-linked immunosorbent assay (ELISA) was used for measuring serum galectin-3 levels. There were no significant differences between serum levels of galectin-3 in sera of preeclampsia patients compared to normal pregnant women - 14.1 ± 4.77 vs. 15.7 ± 5.95 ng/ml (p>0.05). Serum galectin-3 levels correlated with maternal age (r=0.33; p=0.03) and BMI (body mass index) (r=0.52; p=0.01). Our data suggest that determination of serum galectin-3 levels may not be a useful method for prediction of early-onset preeclampsia. Studies should be aimed to other categories of biomarkers.

Serum Levels of Antibodies to Advanced Glycation End Products in Patients with Type 2 Diabetes Mellitus and Hypertension.

BACKGROUND AND AIMS: Proteins containing advanced glycation end products are highly immunogenic and anti-advanced glycation end products antibodies (anti-AGEs antibodies) are found in the sera of diabetics. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay (ELISA) was used for measuring levels of anti-advanced glycation end products antibodies in sera of 93 patients with type 2 diabetes mellitus and arterial hypertension (mean age 61.4±11.3 years, diabetes duration 9.88±3.12 years; hypertension duration 9.28±4.98). These values were compared to serum anti-AGEs antibodies in 42 age and sex matched controls. Diabetics were divided in two groups according to presence or absence of microangiopathy, group 1 (n=67) and group 2 (n=26), respectively. RESULTS: Serum levels of anti-AGEs antibodies in patients with type 2 diabetes mellitus and arterial hypertension were statistically significantly higher than those in the control group (1.39±0.39 vs. 1.05±0.32), (p<0.05). Group 1 showed significantly higher levels of anti-AGEs antibodies than those of healthy controls (1.53±0.14 vs. 1.05±0.32), (p<0.01). Anti-AGEs antibodies levels were higher in patients with microvascular complications than these in patients without complications. Anti-AGEs antibodies correlate with diastolic blood pressure (r=0.26, p=0.05) and body mass index (r=0.37, p=0.03). We found significantly higher percentage of positive patients for anti-AGEs antibodies (mean+2SD) in group 1 than in group 2. CONCLUSION: Determining the levels of serum anti-AGEs antibodies can help physicians make early diagnosis and prognosis of the severity of late diabetic complications in hypertensive patients.

Use of Glycated Hemoglobin (A1c) as a Biomarker for Vascular Risk in Type 2 Diabetes: Its Relationship with Matrix Metalloproteinases-2, -9 and the Metabolism of Collagen IV and Elastin.

Background and objectives: HbA1c measurements may be useful not only in optimizing glycemic control but also as a tool for managing overall vascular risk in patients with diabetes. In the present study, we investigate the clinical significance of HbA1c as a biomarker for hyperglycemia-induced vascular damages in type 2 diabetes (T2D) based on the levels of matrix metalloproteinases-2, -9 (MMP-2, MMP-9), anti-collagen IV (ACIV), and anti-elastin (AE) antibodies (Abs) IgM, IgG, and IgA, and CIV-derived peptides (CIV-DP) reflecting collagen and elastin turnover in the vascular wall. The aim is to show the relationship of hyperglycemia with changes in the levels of vascular markers and the dynamics of this relationship at different degrees of glycemic control reported by HbA1c levels. Materials and Methods: To monitor elastin and collagen IV metabolism, we measured serum levels of these immunological markers in 59 patients with T2D and 20 healthy control subjects with an ELISA. Results: MMP-2, MMP-9, and the AEAbs IgA levels were significantly higher in diabetic patients than in control subjects, whereas those of the AEAbs IgM, ACIVAbs IgM, and CIV-DP were significantly lower. MMP-9 levels were significantly lower at HbA1c values >7.5%. Conclusions: A set of three tested markers (MMP-2, MMP-9, and AEAbs IgA) showed that vascular damages from preceding long-term hyperglycemia begin to dominate at HbA1c values ≥7.5%, which is the likely cut-point to predict increased vascular risk.

Relationship between Lipid Indices, Type IV Collagen Turnover and the Development of Microvascular Complications in Diabetic Patients with Arterial Hypertension.

BACKGROUND AND AIMS: An important factor in the development of vascular wall lesions is the degradation of the major protein of connective tissue - type IV collagen. Type IV collagen peptides (CIVDP) derived from this degradation are present in the circulation and are a stimulus for production of anti-collagen type IV antibodies (ACIVAbs) IgM, IgG and IgA. The aim of this study was to find a possible association between ACIVAbs, lipid indices and the development of microvascular complications. MATERIALS AND METHODS: Sera of 93 patients (mean age 61.4±11.3 yrs, diabetes duration 9.88±3.12 yrs; hypertension duration 9.28±4.98) with type 2 diabetes mellitus (T2DM) and arterial hypertension (AH) were investigated. ACIVAbs was determined using ELISA and then compared to serum ACIVAbs in 42 age- and sex-matched controls. Diabetics were divided into two groups according to presence (group 1, n=67) or absence (group 2, n=26) of microangiopathy. Lipid profile and lipid indices (log TG/HDL, LDL/HDL, TC/HDL and TG/HDL) were examined too. RESULTS: Patients with T2DM and AH showed statistically significant higher levels of serum ACIVAbs IgG than healthy controls [0.298 (0.237÷0.381) vs 0.210 (0.149÷0.262), KW=14.01, p<0.0001]. Group 1 had statistically significant higher levels of ACIVAbs IgG than patients without microangiopathy [0.323 (0.243÷0.391) vs 0.241 (0.207÷0.291), KW=7.66, p=0.006] and healthy controls [0.210 (0.149÷0.262), KW=17.52, p<0.0001). ACIVAbs IgG showed correlation with duration of diabetes (r=0.49, p=0.01), retinopathy (r=-0.20, p=0.04) and BMI (r=-0.24, p=0.05), HbA1c (r=0.21, p=0.04), SBP (r=0.16, p=0.05). ACIVAbs IgG correlated with log TG/HDL (r=0.21, p=0.01), LDL/HDL (r=0.19, p=0.02) TC/HDL (r=0.16, p=0.05) and with TG/HDL (r=0.15, p=0.05). CONCLUSION: Our study shows relationship between elevation of ACIVAbs IgG, high lipid indices and development of microvascular complications in patients with type 2 diabetes mellitus and arterial hypertension.

Serum Concentrations of Endothelin-1 and Matrix Metalloproteinases-2, -9 in Pre-Hypertensive and Hypertensive Patients with Type 2 Diabetes.

Endothelin-1 (ET-1) is one of the most potent vasoconstrictors known to date. While its plasma or serum concentrations are elevated in some forms of experimental and human hypertension, this is not a consistent finding in all forms of hypertension. Matrix metalloproteinases -2 and -9 (MMP-2 and MMP-9), which degrade collagen type IV of the vascular basement membrane, are responsible for vascular remodeling, inflammation, and atherosclerotic complications, including in type 2 diabetes (T2D). In our study, we compared concentrations of ET-1, MMP-2, and MMP-9 in pre-hypertensive (PHTN) and hypertensive (HTN) T2D patients with those of healthy normotensive controls (N). ET-1, MMP-2, and MMP-9 were measured by ELISA. Concentrations of ET-1 in PHTN and N were very similar, while those in HTN were significantly higher. Concentrations of MMP-2 and MMP-9 in PHTN and HTN were also significantly higher compared to N. An interesting result in our study is that concentrations of MMP-2 and MMP-9 in HTN were lower compared to PHTN. In conclusion, we showed that increased production of ET-1 in patients with T2D can lead to long-lasting increases in blood pressure (BP) and clinical manifestation of hypertension. We also demonstrated that increased levels of MMP-2 and MMP-9 in pre-hypertensive and hypertensive patients with T2D mainly reflect the early vascular changes in extracellular matrix (ECM) turnover.

Serum anti-collagen type IV IgM antibodies and development of diabetic nephropathy in diabetics with essential hypertension.

INTRODUCTION AND AIMS: Arterial hypertension and diabetic vascular complications are connected with an elevated degradation of elastic tissue. This process leads to an increased production of antibodies to collagen type IV (ACIV Abs). In the present investigation we studied whether the serum levels of antibodies (IgG, IgM and IgA) to collagen are related with microvascular complications. MATERIAL AND METHODS: Serum levels of antibodies to collagen type IV (ACIV) IgG, IgM and IgA were measured using an ELISA method in 93 patients with type 2 diabetes mellitus and arterial hypertension (AH) (mean age 61.4 ±11.3 years, diabetes duration 9.88 ±3.12 years; hypertension duration 9.28 ±4.98). These values were compared to serum antibodies to CIV in 42 age and sex matched controls. RESULTS: ACIV IgM antibodies levels in patients with AH and T2DM were statisticaly significantly higher than controls 0.178 (0.145÷0.220) vs. 0.142 (0.118÷0.173) (KW = 6.31; p = 0.01). Group 1 (patients with microvascular complications) showed significantly higher levels of ACIV IgM than controls 0.180 (0.136÷0.223) vs. 0.142 (0.118÷0.173) (KW = 5.03; p = 0.02). Patients from Group 2 showed statistically significantly higher levels of ACIV IgM than controls 0.176 (0.151÷0.202) vs. 0.142 (0.118÷0.173) (KW = 6.15; p = 0.01). ACIV IgM antibodies showed correlation with microalbuminuria (r = 0.21); (p = 0.04), BMI (r = 0.19); (p = 0.04), creatinine clearance (r = -0.36); (p = 0.01) and GFR (r = -0.34); (p = 0.02). CONCLUSIONS: Our study showed an association between elevation of serum levels of ACIV IgM and development of diabetic nephropathy. We suggest that levels of ACIV IgM can be useful method for identfying a high risk for development of diabetic nephropathy.

Abnormal levels of age-elastin derived peptides in sera of diabetic patients with arterial hypertension.

INTRODUCTION: An important factor in vascular wall alterations is degradation of elastic fiber major protein - elastin. As a result, elastin derived peptides (EDP) are found in circulation. Advanced glycation might also involve elastin, because it is a protein with slow metabolism. The aim of our study was to measure serum levels of glycated elastin derived peptides (AGE-EDP) of elastin in patients with type 2 diabetes mellitus (T2DM) and arterial hypertension (AH). MATERIAL AND METHODS: We adapted an ELISA technique for the determination of AGE-EDP. Sera of 93 patients with T2DM and AH (mean age 61.4 ±11.3 years, diabetes duration 9.88 ±3.12 years; hypertension duration 9.28 ±4.98) were tested. These values were compared to 42 age- and sex-matched controls. Diabetics were divided in two groups according to presence - Group 1 (n = 67) or absence - Group 2 (n = 26) of microangiopathy. RESULTS: Patients with T2DM and AH showed statistically significantly higher levels of AGEEDP in comparison with healthy controls 0.060 (0.053÷0.065) vs. 0.039 (0.031÷0.044) (KW = 35.2; p < 0.0001). Group 1 showed significantly higher levels of AGE-EDP than the control group 0.069 (0.051÷0.070) vs. 0.039 (0.031÷0.044) (KW = 33.0; p < 0.0001). Group 2 also showed significantly higher levels of AGE-EDP than controls 0.058 (0.049÷0.064) vs. 0.039 (0.031÷0.044) (KW = 22.1; p < 0.0001). AGE-EDP showed a correlation with an insulin dose (r = -0.28; p = 0.05), systolic blood pressure (r = 0.25; p = 0.01), BMI (r = 0.39; p = 0.01) and retinopathy (r = 0.18; p = 0.05). CONCLUSIONS: The measurement of non-invasive markers of elastin glycation may be useful in monitoring development of vascular wall alterations and therapeutic interventions.

Strain-dependent effects of long-term treatment with melatonin on kainic acid-induced status epilepticus, oxidative stress and the expression of heat shock proteins.

Oxidative stress is implicated in the pathogenesis of both hypertension and epileptogenesis, therefore it could be used as a tool for studying co-morbidity of hypertension and epilepsy. Clinical data suggest that melatonin is a potent antioxidant that is effective in the adjunctive therapy of hypertension and neurodegenerative diseases. The present study aimed to explore and compare the efficacy of chronic pretreatment with melatonin infused via subcutaneous osmotic mini-pumps for 14 days (10 mg/kg per day) on kainic acid (KA)-induced status epilepticus, oxidative stress and expression of heat shock protein (HSP) 72 in spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. SHRs showed higher lipid peroxidation (LP) in the frontal cortex and hippocampus and decreased cytosolic superoxide dismutase (SOD/CuZn) production in the frontal cortex compared to Wistar rats. Status epilepticus (SE) induced by KA (12 mg/kg, i.p.) was accompanied by increased LP and expression of HSP 72 in the hippocampus of the two strains and increased SOD/CuZn production in the frontal cortex of SHRs. Melatonin failed to suppress seizure incidence and intensity though the latency for seizure onset was significantly increased in SHRs. Melatonin attenuated the KA-induced increase in the level of LP in the hippocampus both in SHRs and Wistar rats. However, an increased activity in SOD/CuZn and mitochondrial SOD Mn as well as reduced expression of HSP 72 in the hippocampus was observed only in Wistar rats pretreated with melatonin. Taken together, the observed strain differences in the efficacy of chronic melatonin exposure before SE suggest a lack of a direct link between the seizure activity and the markers of oxidative stress and neurotoxicity.

Detection of free antielastin antibodies among diabetic children.

Antibodies to elastin breakdown products are found in the serum of all human subjects and correlate with their respective serum peptide levels. The presence of these antielastin antibodies (AEAbs) and the corresponding antigens in circulation leads to the formation of circulating immune complexes (CICs). The aim of this study was to determine if the serum levels of free AEAbs (not bound in CICs) correlate with the development of vascular complications in diabetic children. To this end, we used a method for detecting immune complexes (complement inhibition factor [CIF]-enzyme-linked immunosorbent assay [ELISA]) in combination with an ELISA for detection of AEAbs. The levels of free immunoglobulin G (IgG) AEAbs were studied in the sera of 54 diabetic children (mean age 12.3+/-4 years; diabetes duration 5.2+/-3.7 years). Thirty-two of the children had vascular complications (group 1), and 22 were without vascular complications (group 2). Twenty healthy children (mean age 13.6+/-4.2 years) were used as controls. The diabetics showed statistically significant higher levels of free AEAbs (0.490 E492+/-0.244 E492 vs 0.307 E492+/-0.081 E492; p = .02) compared with the control group. In group 1, free AEAbs showed statistically significant higher levels than controls (0.523+/-0.269 vs 0.307+/-0.081; p = .016). Eighteen of 54 (33%) patients were positive for free AEAbs (13 of 32 [41%] in group 1 and 5 of 22 [22%] in group 2). Free AEAb levels in all diabetics showed a correlation with systolic blood pressure (r = .44; p = .01), diastolic blood pressure (r = .46; p = .009), total cholesterol (r = .33; p = .05), triglycerides (r= .38; p = .03), high-density lipoprotein (r= -.46; p = .009), serum fructose (r= .43; p = .001), and microalbuminuria (r= .41; p = .002). Patients who had vascular pathology showed a correlation of free AEAbs with microalbuminuria (r= .434; p= .026), serum fructose (r= .63; p = .0004), hemoglobin A1c (r= .392; p = .043), and triglycerides (r= .456; p = .025). These findings suggest that elevated levels of free IgG AEAbs are associated with the development of diabetic vascular complications in children.

Circulating immune complexes among diabetic children.

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease associated with the presence of different types of autoantibodies. The presence of these antibodies and the corresponding antigens in the circulation leads to the formation of circulating immune complexes (CIC). CIC are known to persist in the blood for long periods of time. Such CIC following deposition in the small blood vessels have the potential to lead to microangiopathy with debilitating clinical consequences. The aim of our pilot study was to investigate whether a correlation exists between CIC and the development of microvascular complications in diabetic children. Isolation of a new glycoprotein complement inhibition factor (CIF) from the parasitic plant Cuscuta europea seed, which appears to bind specifically to complement component C3 has provided an unique tool for the measurement of immune complexes by means of ELISA-type techniques (CIF-ELISA). We studied the levels of CIC (IgG, IgM and IgA) in 58 diabetic children (mean age 12.28 +/- 4.04 years, diabetes duration 5.3 +/- 3.7 years), 29 of them had vascular complications (group 1) and the other 29 were without vascular complications (group 2). As controls, we studied sera samples from 21 healthy children (mean age 13.54 +/- 4.03 years). Sera from the diabetic patients showed statistically significant higher levels of CIC IgG (p = 0.03) than sera from the control group. In sera from group 1 values of CIC IgG showed statistically significant higher levels than controls (0.720 +/- 0.31 vs. 0.46 +/- 0.045; p = 0.011) Sera from 59% of the patients were positive for CIC IgG, 36% for CIC IgM and 9% for CIC IgA. Among 26 patients with microalbuminuria, sera from 17/26 (65%) were positive for CIC IgG, 8/26 (31%) for CIC IgM and 2/26 (8%) for CIC IgA. CIC IgG correlated with HbAlc (r = 0.51; p = 0.005) and microalbuminuria (r = 0.42, p = 0.033). CIC IgA correlated with age (r = 0.44, p = 0.03). CIC IgM correlated with the duration of diabetes (r = 0.63, p = 0.02). These findings suggest that elevated levels of CIC IgG are associated with the development of early diabetic nephropathy.