Down-regulation of neuronal form of Nitric oxide synthase in the Nurse cell of Trichinella spiralis.

The free radical nitric oxide (NO) and Ca2+ are critical regulators of skeletal muscle exercise performance and fatigue. The major source of NO in skeletal muscle cells is the neuronal form of the enzyme Nitric oxide synthase (nNOS). One of the most peculiar characteristics of the Nurse cell of Trichinella spiralis (T. spiralis) is the complete loss of the contractile capabilities of its derivative striated muscle fiber. The aim of the present study was to clarify the expression of nNOS protein and mRNA in striated muscles during the muscle phase of T. spiralis infection in mice. Muscle tissue samples were collected from mice at days 0, 14, 24, and 35 post infection (d.p.i.). The expression of nNOS was investigated by immunohistochemistry, and the expression levels of mRNA of mouse Nitric oxide synthase 1 (Nos1) by real-time PCR. The presence of nNOS protein was still well observable in the disintegrated sarcoplasm at the early stage of infection. The cytoplasm of the developing and mature Nurse cell showed the absence of this protein. At least at the beginning of the Nurse cell development, Trichinella uses the same repairing process of skeletal muscle cell, induced after any trauma and this corroborates very well our results concerning the nNOS expression on day 14 p.i. At a later stage, however, we could suggest that the down-regulation of nNOS in the Nurse cell of T. spiralis either serves a protective function or is an outcome of the genetic identity of the Nurse cell.

The Synthesis of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase (GNE), α-dystroglycan, and ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6) By Skeletal Muscle Cell As a Response To Infection with Trichinella Spiralis.

The Nurse cell of the parasitic nematode Trichinella spiralis is a unique structure established after genetic, morphological and functional modification of a small portion of invaded skeletal muscle fiber. Even if the newly developed cytoplasm of the Nurse cell is no longer contractile, this structure remains well integrated within the surrounding healthy tissue. Our previous reports suggested that this process is accompanied by an increased local biosynthesis of sialylated glycoproteins. In this work we examined the expressions of three proteins, functionally associated with the process of sialylation. The enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key initiator of the sialic acid biosynthetic pathway. The α-dystroglycan was the only identified sialylated glycoprotein in skeletal muscles by now, bearing sialyl-α-2,3-Gal-ß-1,4-Gl-cNAc-ß-1,2-Man-α-1-O-Ser/Thr glycan. The third protein of interest for this study was the enzyme ß-galactoside α-2,3-sialyltransferase 6 (ST3Gal6), which transfers sialic acid preferably onto Gal-ß-1,4-GlcNAc as an acceptor, and thus it was considered as a suitable candidate for the sialylation of the α-dystroglycan. The expressions of the three proteins were analyzed by real time-PCR and immunohistochemistry on modified methacarn fixed paraffin tissue sections of mouse skeletal muscle samples collected at days 0, 14 and 35 post infection. According to our findings, the up-regulation of GNE was a characteristic of the early and the late stage of the Nurse cell development. Additional features of this process were the elevated expressions of α-dystroglycan and the enzyme ST3Gal6. We provided strong evidence that an increased local synthesis of sialic acids is a trait of the Nurse cell of T. spiralis, and at least in part due to an overexpression of α-dystroglycan. In addition, circumstantially we suggest that the enzyme ST3Gal6 is engaged in the process of sialylation of the major oligosaccharide component of α-dystroglycan.

Identification of the novel HLA-A*11:192 allele by HLA typing of a Caucasian individual.

We report the new HLA-A*11:192 allele differing from A*11:01 by one nucleotide in exon 2.

Increased number of multi-oocyte follicles (MOFs) in juvenile p27Kip1 mutant mice: potential role of granulosa cells.

STUDY QUESTION: Why are female mice that lack a functional p27 protein infertile? SUMMARY ANSWER: The absence of a functional p27 leads to a dramatic increase in the number of multi-oocyte follicles (MOFs) in juvenile female mice; p27 would promote the individualization of follicles favoring the development of fertile eggs. WHAT IS KNOWN ALREADY: p27-/- female mice are infertile. p27 suppresses excessive follicular endowment and activation and promotes follicular atresia in mice. MATERIALS AND METHODS: Ovaries from wild type (WT) and p27Kip1 mutant mice aged 2, 4 and 12 weeks were subjected to immunohistochemistry/immunofluorescence. The slides with whole organs serially sectioned were scanned and examined by image analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Compared with WT, p27Kip1 mutant pre-pubertal mice had a greater number of oocytes, a greater number of growing follicles and a greater number of MOFs. These differences were statistically significant (P < 0.05), particularly in the case of MOFs (P > 0.001). The unusually large number of MOFs in juvenile p27-deficient mice is a novel observation. In WT mice p27 protein remains present in the oocyte nucleus but gradually decreases in the ooplasm during follicular growth, while granulosa cells show dynamic, follicle stage-related changes. LIMITATIONS, REASONS FOR CAUTION: These results have been obtained in mice and they cannot be directly extrapolated to humans. WIDER IMPLICATIONS OF THE FINDINGS: The dramatic increase in the numbers of MOFs in juvenile p27 mutants has not been previously reported. The number of MOFs declines sharply as the mice become sexually mature, pointing to their negative selection. These findings open a new approach to the study of sterility. STUDY FUNDING/COMPETING INTERESTS: This study has been funded by the Basque Government, Dept. of Health grant 2007111063 and Dept. of Industry (Saiotek) grant S-PC11UN008. Jairo Perez-Sanz was the recipient of a grant from Fundación Jesús de Gangoiti Barrera. The authors have no conflicts of interest to declare.

Detection of a novel HLA-A allele, designated A*02:334.

We report a novel allele HLA-A*02:334 with the transition G→A at nucleotide position 282.

Detection of a novel HLA-DQB1 allele, designated DQB1*06:49.

We report a novel allele HLA-DQB1*06:49 with a G→T transversion, most closely resembling HLA-DQB1*06:02:01.

A new HLA-B*52 allele, B*52:23, detected in a patient before bone marrow transplantation.

The new allele HLA-B*52:23 differs from B*52:01:01 at position 208 in exon 2.

Identification of a novel HLA-C*02 allele, HLA-C*02:02:09.

We report HLA-C*02:02:09 as a novel allele with a transition C->T at position 354.

A new HLA-C*07 variant allele, C*07:108, identified by sequence-based typing.

The new HLA-C*07:108 differs from C*07:01:01 by two transversions in exon 3.

A novel HLA-DQB1*03:02 variant designated DQB1*03:02:05.

The new DQB1*03:02:05 differs from DQB1*03:02:01 by two nucleotide exchanges in exon 2.

Identification of a new HLA-DRB1* 14 allele, DRB1*14:99, by HLA typing of an Asian individual.

We report the new HLA-DRB1*14:99 allele differing from DRB1*14:01:01 by one nucleotide in exon 2.

[Screening for gestational diabetes in Bulgaria--preliminary results].

Gestational diabetes mellitus (GDM) is defined as carbohydrate intolerance with onset or first recognition during pregnancy. There is no data regarding the prevalence of GDM in Bulgaria. The aim of this study is to present the preliminary results from a pilot study for GDM in Bulgaria, including the prevalence and the epidemiological differences between women with or without this disorder. A 2 h oral glucose tolerance test (OGTT) with 75g glucose was performed on 330 pregnant women between 24 and 28 weeks of gestation, using ADA criteria to define pregnancies complicated by GDM. Blood glucose levels, insulin levels, HbA1c, hsCRP and lipid parameters were evaluated. GDM was found in 11.3% of the patients. Women with GDM were older than the ones without the disorder (p < 0.006), had higher fasting glucose levels (p < 0.0001), higher fasting insulin levels (p < 0.0001), higher HOMA index (p < 0.0001) and higher HbA1c (p < 0.001). There was a significant difference in body mass index (BMI) before pregnancy (p < 0.003) and at the time of screening (p < 0.001) between GDM and control groups (p < 0.001). GDM women more frequently had thyroid disease, developed early preeclampsia, had family history of type 2 diabetes mellitus and history of PCOS. In conclusion the prevalence of GDM in high risk Bulgarian population is relatively high--11.3%. This confirms the necessity of establishing of a national screening program. If financial recourses are limited selective screening in high risk patients should be considered.

[Our and foreign experience in surgical treatment of CIN III-cold knife conisation versus LLETZ].

AIM: Our aim was to assess the treatment results of patients with CIN III- surgically treated with cold knife cone biopsy compared with the results of treatment with LLETZ and laser conisation. MATERIALS AND METHODS: 600 patients were assessed and researched for 10 years period. Some of them were from the Specialized Hospital for Active Treatment in Oncology-Sofia, some from RHW and some from the Medical University of Varna-Cathedra of Obstetrics and Gynaecology. 350 patients were operated by the classical method- cold knife conisation, 200 by LLETZ and 50 by laser cone biopsy. RESULTS: We didn't find any difference in patients age, their cytological results and the results of their treatment. The LLETZ showed good results according to resected margins. The same good results were observed with laser conisation. Invasive disease and recurrence were observed in 150 patients--50 of them having adenocarcinoma in situ. CONCLUSIONS: Our research showed that there is no difference in the results of both groups- treated by the classical way--cold knife conisation or by LLETZ and laser conisation.

[Frequency of miscarriages among pregnant women with autoimmune thyroid disorders].

AIM OF THE STUDY: To analyse the influence of antithyroid antibodies (ATA) on the frequency of spontaneous abortions (SA) by pregnant women with a normal thyroid gland function. DESIGN OF THE STUDY: Prospective clinical study on 42 selected pregnant women with a normal thyroid gland function divided into two groups: I-st group--ATA positive pregnant [n = 28] and II-end group ATA negative pregnant [n = 14]. RESULTS: Increased ATA have been found by 30 (71.4%) out of the studied 42 pregnant women. There has been no significant difference found among the values of FT3, FT4 and TSH for women with positive and negative ATA. SA have been observed at 63.3% (19/30 women) from the ATA positive ones and at 25% (4/12 women) from the ATA negative ones (P = 0.001). By the ATA positive women with SA the average values of antithyroglobulin autoantibody (Tg-Ab) (Tg-Ab positive.-189.6 +/- 49.8 IU/ml vs. Tg-Ab -negative 118.2 +/- 58.3 IU/ml, P = 0.02) and antithyroid peroxidase autoantibody (TPO-Ab) (TPO-Ab positive-176.9 +/- 57.4 IU/ml vs. TPO - negative 118.2 +/- 81.3 IU/ml, P = 0.004) are both found to be significantly higher. CONCLUSION: There is a correlation found between ATA and the increased risk of SA, where the increased concentration of ATA is combined with an increased frequency of SA.

Quantitative immunohistochemical detection of the molecular expression patterns in proliferative inflammatory atrophy.

The proliferative inflammatory atrophy (PIA) is considered as a possible precursor of prostate intraepithelial neoplasia (PIN) or prostate cancer (PCa). In this study we assessed quantitatively the expression of AMACR, p63, COX-2, GST and iNOS in serial paraffin-embedded tissue sections obtained after radical prostatectomy of PCa patients (n = 30). The applicability of these markers to distinguish PIA, PIN and PCa was evaluated. We also compared the immunohistochemical expression profiles of AMACR, COX-2 and GST in the luminal and basal cells in lesions of PIN arisen in PIA or PIA alone. Two different patterns of COX-2 expression according to the p63 status of the basal cells were found. This observation gives us grounds to hypothesize that the diverse COX-2 patterns resulted from an initial basal cell damage which subsequently propagated to its luminal secretory cells progeny.

A pharmacoeconomical model for choice of a treatment for pregnant women with gestational diabetes.

This study discusses two main questions: the direct medical costs and the clinical effectiveness of the hospital treatment with insulin of pregnant women with gestational diabetes (GD). A prospective study that includes 50 women with GD is performed. The pregnant women are divided into 2 groups: Group I (n=30) - pregnant women treated only with a diet; and Group II (n=20) - pregnant women treated with diet and insulin. We found that the metabolite compensation degree is improved after the applied treatment with insulin. The coefficient cost/effectiveness is 6954 lv./100 women. The analysis decision tree confirms in a very convenient way the fact that insulin treatment is a clinically more effective and financially more profitable strategy.

[Is it possible to reduce the incidence of preeclampsia in pregnant women with type I diabetes mellitus].

AIM OF STUDY: To follow the meaning of diabetic metabolic control in the early pregnancy springing up the preeclampsia (PR) in pregnant women with type I diabetes mellitus (T1DM). DESIGN OF STUDY: A prospective study has been done among 105 pregnant women with T1DM. The diabetic pregnant have been divided into two groups: 1st group--43 women with preplanning pregnancy and 2nd group--62 women with non-planning pregnancy. RESULTS: There was no difference in the frequency of PR among women with planning and non-planning pregnancy (25.6% vs. 30.6%; P=0.36). The mild and moderate cases of PR among the women with planning pregnancy are prevailing, while moderate and severe cases of PR were observed in non-planning pregnancy. In pregnant women with planning pregnancy there is no difference between the initial level HbA1c of women without PR and PR (7%+/-0.7 vs. 7.1%+/-0.9%; P=0.13). Pregnant women with non-planning pregnancy have significantly higher initial level of HbA1c (9.7%+/-1.7 vs. 8.3%+/-1.3%; P=0.01). Pregnant women with PR have significantly higher body weight, creatinin, diurnal urine albumin excretion and diurnal insulin dose. CONCLUSION: The chronicle hyperglycemia in the early pregnancy in combination with the established factors are increasing the risk of preeclampsia in women with non-planning pregnancy and poor glycaemic control.

[Incidence of preeclampsia in pregnant women with type 1 diabetes mellitus].

UNLABELLED: Preeclampsia (PE) is a complication unique to human pregnancy. Up to 39% of pregnant women suffering from type 1 diabetes (T1DM) are affected. It is supposed, that hyperglycemia, causing vascular endothelial dysfunction in placental vessels is the major mechanism, leading to PE. OBJECTIVE: To study the incidence of PE in women with T1D and to study the correlation between development of PE and metabolic control of diabetes. STUDY DESIGN: 105 pregnant women'with successful pregnancy outcome, suffering from TID have been included in a prospective study during the period 2002-2005. Starving, postprandial blood glucose and HbA1c have been measured at 12 wks and at 36 wks of gestation. The influence of risk factors has been assessed. RESULTS: The incidence of PE in the studied group is 28.6%. HbA1-c in women developing PE is significantly higher (8.2 +/- 1.6 and 7.6 +/- 1.2; P = 0.02 respectively). Hyperglycemia during pregnancy is a reliable predictor for development of PE. Elevation of HbA1-c with 1% is associated with 1.43 times increased risk for PE (OR = 1.43; 95% Cl = 1.11-12.7; P = 0.03). Combination of hyperglycemia and diabetic vascular disease increases significantly the risk for PE- OR 1.17 with 88.6% predictive value (P = 0.001). CONCLUSIONS: Normoglycaemic control of diabetes before and during pregnancy could reduce the incidence of PE.

[Risk of thrombophilia in carriers of thrombophilic genetic factors in unsuccessful assisted reproduction].

The aim of this study was to evaluate an association of carrier status of common inherited thrombophilic genetic mutations and implantation failure after assisted reproduction (ART): IVF and ICSI. Sixty seven women with failure of embryo implantation and ninety six controls--women without obstetric complication were investigated for carriage of factor V Leiden (FVL), G20210A prothrombin gene mutation, genetic variant C677T in methylentetrahydrofolate reductase gene (MTHFR) and polymorphism A2 in platelet glycoprotein IIb/IIIa (GIPr IIb/IIIa). A significantly higher prevalence of polymorphism A2 in GIPr IIb/IIIa was found in women with implantation failure in ART compared to controls (respectively 26.1% and 12.5%; OR: 2.571, 95% CI: 1.066-6.258, p = 0.033). A higher but not significant prevalence of G20210A prothrombin gene mutation carriage was found inpatients compared to controls (respectively 5.8% and 3.13%, OR: 1.968, 95% CI 0.356-11.539). The carriage of FVL was a little but not significantly higher in controls. The carriage of genetic variant C677T in MTHFR was the same in both groups. These data suggest that polymorphism A2 in GIPr IIb/IIIa and G20210A prothrombin gene mutation could be play a role in the etiology of IVF failures and the carriers of GIPr IIb/IIIa A1/A2 and G20210A prothrombin gene mutation are at higher risk of implantation failure and not successful ART outcome. The carriage of these two genetic defects should be investigated in women undergoing IVF and the antithrombotic or anticoagulant prophylaxis should be indicated for carriers of these two factors.

[Selenium and glutathion peroxidase enzyme levels in diabetic patients with early spontaneous abortions].

UNLABELLED: The incidence of spontaneous abortions in women with type 1 diabetes mellitus varies between 10-30%. The etiology of this is still unclear despite numerous experimental studies. Pregnancy is a condition of increased oxidative stress due to impaired balance between pro- and antioxidants. Glutathion and related enzymes perform the best antioxidant protection. Some authors point to a possible correlation between spontaneous abortions and low plasma Se levels as well as low intracellular activity of glutathion peroxidase enzyme. Others report that Hb A1-c, values over 1SD above normal increase the risk of spontaneous abortions with 3% and Hb A1-C values between 10-12% are critically high for the occurrence of spontaneous abortions. The purpose of the study was to evaluate the levels of Se and glutathion peroxidase enzyme (Gl-Px) in pregnant women with type 1 diabetes mellitus in the first trimester of pregnancy and to find out is there a correlation between glycemic control of diabetes and the incidence of spontaneous abortions. DESCRIPTION OF PROJECT: 75 pregnant women enrolled in an- 1 year prospective study divided in 3 groups according to pregnancy outcome: gr. 1 - n = 30 with type diabetes mellitus, no abortions, gr.2 - n = 16 with type diabetes mellitus with first trimester spontaneous abortion and gr. 3 - n = 29 healthy pregnant women. Women with type 1 diabetes mellitus were divided into three subgr. according to glycemic control - subgr. 1 - n = 12 (Hb A1-c < 7%), subgr.2 - n = 18 (Hb A1-c > 7< 8%), subgr.3 - n = 16 (Hb A1-c > 8%). Gl-Px activity was determined in Er hemolisate with test reagents of Randox Ransel, with ref.values 27.5 - 73 U/g Hb. Selen concentration was determined in whole blood sample by atomic absorption spectrophotometry with ref. values 0.12-1.1 micromol/l. HbA 1-C was measured by affinity chromatography with ref. values 4.5-6.3%. Statistical methods used were: dispersion, correlation analysis - SPSS package version 11.01.01. RESULTS: Basic Se levels were low in all pregnant women in early pregnancy. The metabolic control level did not influence the levels of Se in pregnant women with diabetes mellitus type1. Gl -Px activity was within the normal limits in all women. There was no correlation between Se levels and Gl -Px activity in pregnant diabetics with and without abortions. There was a correlation between Se levels and Gl -Px activity only in healthy pregnant women. Pregnant women with poor glycemic control had higher incidence of spontaneous abortions. CONCLUSIONS: We could not support the hypothesis of reduced antioxidant protection (low Se and Gl-Px levels) as a causative factor in the pathogenesis of spontaneous abortions in diabetic patients. Our study results showed that poor metabolic control of diabetes (high Hb A1-c) in the first trimester of pregnancy had a primary role in the occurrence of early abortions. We could speculate that the early hyperglycemic maternal-fetal environment most probably plays a role of an additional stress to the developing embryo.