Brain-derived neurotrophic factor (BDNF) in children with ASD and their parents: a 3-year follow-up.

OBJECTIVE: Several lines of evidence point to a probable relationship between brain-derived neurotrophic factor (BDNF) and autism spectrum disorder (ASD), but studies have yielded inconsistent findings on the BDNF serum level in ASD. The study aimed to assess those levels in children with ASD and their families. METHOD: BDNF serum levels were measured in 45 ASD children without intellectual disability (ID) and allergies, age 30-42 months and age-matched normal controls. BDNF serum levels in the parents of the ASD subjects were compared to normal controls. BDNF serum levels in the ASD subjects were followed up for 3 years and correlated with adaptive functioning changes. RESULTS: BDNF serum levels were measured to be lower in children with ASD and independent of all the major baseline characteristics of the subjects. Having a child with ASD raises the BDNF levels in parents comparing to controls. Prospectively, no correlation between the change of BDNF variables in time and the change of the Vineland scores was found. CONCLUSIONS: Our results contradict those from recent published meta-analyses with the age, the presence of ID and allergies being possible contributing factors. The parents' data indeed point to a role of BDNF in the pathophysiology of ASD.

Evaluation of the impact of tumor HPV status on outcome in patients with locally advanced unresectable head and neck squamous cell carcinoma (HNSCC) receiving cisplatin, 5-fluorouracil with or without docetaxel: a subset analysis of EORTC 24971 study.

BACKGROUND: EORTC 24971 was a phase III trial demonstrating superiority of induction regimen TPF (docetaxel, cisplatin, 5-fluorouracil) over PF (cisplatin/5-fluorouracil), in terms of progression-free (PFS) and overall survival (OS) in locoregionally advanced unresectable head and neck squamous cell carcinomas. We conducted a retrospective analysis of prospectively collected data aiming to evaluate whether only HPV(-) patients (pts) benefit from adding docetaxel to PF, in which case deintensifying induction treatment in HPV(+) pts could be considered. PATIENTS AND METHODS: Pretherapy tumor biopsies (blocks or slides) were assessed for high-risk HPV by p16 immunohistochemistry, PCR and quantitative PCR. HPV-DNA+ and/or p16+ tumors were subjected to in situ hybridization (ISH) and HPV E6 oncogene expression qRT-PCR analysis. Primary and secondary objectives were to evaluate the value of HPV/p16 status as predictive factor of treatment benefit in terms of PFS and OS. The predictive effect was analyzed based on the model used in the primary analysis of the study with the addition of a treatment by marker interaction term and tested at two-sided 5% significance level. RESULTS: Of 358, 119 pts had available tumor samples and 58 of them had oropharyngeal cancer. Median follow-up was 8.7 years. Sixteen of 119 (14%) evaluable samples were p16+ and 20 of 79 (25%) evaluable tumors were HPV-DNA+. 13 of 40 pts (33%) assessed with HPV-DNA ISH and 12 of 28 pts (43%) assessed for HPV E6 mRNA were positive. The preplanned analysis showed no statistical evidence of predictive value of HPV/p16 status for PFS (P = 0.287) or OS (P = 0.118). CONCLUSIONS: The incidence of HPV positivity was low in the subset of EORTC 24971 pts analyzed. In this analysis only powered to detect a large treatment by marker interaction, there was no statistical evidence that treatment effect found overall was different in magnitude in HPV(+) or HPV(-) pts. These results do not justify selection of TPF versus PF according to HPV status.

Population-based study of human papillomavirus infection and cervical neoplasia in Athens, Greece.

The aim of our study is to describe the prevalence of the different HPV types in women with pre-neoplastic lesions of the cervix in Greece. Cervical scrapes from 841 women were obtained for both cytological evaluation and analysis for the presence of HPV DNA. PCR was performed on specimens from these 841 women. The Pap test results were normal or showed benign cellular changes in 45.8% of the women, atypical squamous cells of undetermined significance (ASCUS) in 23.2%, low-grade squamous intra-epithelial lesion (LSIL) in 27.9% and high-grade squamous intra-epithelial lesion (HSIL) in 3.1%. HPV DNA was demonstrated in 23.6% of cytologically normal women. We detected HPV in 60% of the total samples. Of these, HPV-16 was the most common HPV DNA detected. Interestingly, HPV-58 was inversely correlated with positive cytological findings. A clear pattern of decreasing prevalence of HPV with age was also observed. Our results indicate that HPV infections, especially those with HPV-16, represent a significant public health concern in Greece.

Prognostic significance of soluble interleukin-2 receptor levels in patients with dilated cardiomyopathy.

BACKGROUND: Activation of T lymphocytes is thought to mediate myocardial dysfunction in dilated cardiomyopathy (CMP), probably through cytotoxic cytokines, but its value as a prognostic factor has not been evaluated. METHODS: For 2 years we prospectively followed 76 patients (65 males, 11 females, age 49 +/- 7 years) with CMP and New York Heart Association(NYHA) Class II-III heart failure; left ventricular (LV) function was assessed echocardiographically. Thirty-three patients (28 males, five females, age 52 +/- 6 years) with ischaemic heart disease (IHD) and similar NYHA and LV function characteristics were used as controls. Serum sIL-2R levels, peripheral blood lymphocyte proliferation (basal, + concanavalin A) and HLA-DQB1 genotyping was carried out in all patients. RESULTS: The CMP patients had increased sIL-2R levels (1259 +/- 130 pg mL-1) compared with the IHD patients (703 +/- 80 pg mL-1, P < 0.01, only 3 > 800 pg mL-1). In the CMP patients, there was a significant (r = +0.45, P= 0.04) correlation between sIL-2R and the LV end-diastolic diameter but not with the LV ejection fraction or NYHA Class. During the 24-month follow up, 17 of the CMP patients had an adverse clinical course (death, need for cardiac transplantation, or worsening heart failure). Of these, 14 (75%) had elevated (>or= 800 pg mL-1) sIL-2R levels (Group I) compared with only five (6%) with a stable clinical course (Group II). Neither [3H] thymidine incorporation into the peripheral blood lymphocytes nor the excess of HLA-DQB1-30 histidine homozygotes in the Group I patients (38% vs. 17%, P < 0.05) could predict the clinical outcome. CONCLUSION: Increased sIL-2R levels in CMP patients are an independent predictor of a more aggressive clinical course.

Germ line BRCA1 & BRCA2 mutations in Greek breast/ovarian cancer families: 5382insC is the most frequent mutation observed.

BRCA1 and BRCA2 genes were screened for loss-of-function mutations in a series of 85 patients having at least one first- or second-degree relative affected by breast and/or ovarian cancer. All BRCA1 exons and BRCA2 exons 10 and 11 were screened with a combination of methods including SSCP, PTT and direct sequencing. We have found disease-associated mutations in 14 families (16.5%), eleven in BRCA1 and three in BRCA2. The known founder mutation 5382insC of BRCA1 was identified in seven unrelated families. The other mutations identified include the non-sense R1751X, the splice junction variant 5586G>A of BRCA1 and three frameshifts, 2024del5, 3034del4, and 6631del5, of BRCA2. Nine out of these 14 families had a family history of three or more breast/ovarian cancer cases. A large number of polymorphic or unclassified variants is also reported. Combined with our previously published data 5382insC was found in nine out of 20 families (45%), suggesting that this mutation may represent a common founder mutation in the Greek population.

Hyperprolactinaemia in patients with heart failure: clinical and immunogenetic correlations.

BACKGROUND: Prolactin represents a stimulatory link between the neuroendocrine and immune systems, but its involvement in the neurohumoral adaptations to heart failure (HF) has not been explored. METHODS: We prospectively studied 55 patients (45 males, 10 females, age 48 +/- 7 years) with NYHA Class II/III HF due either to dilated cardiomyopathy (CMP) (n = 33) or ischemic heart disease (IHD) (n = 22). Serum prolactin levels were determined by radioimmunoassay, soluble interleukin-2 receptor (sIL-2R) levels by enzyme-linked immunoassay and HLA-DQ genotyping with PCR. Left ventricular ejection fraction (LVEF) and end-diastolic diameter (LVEDd) were assessed echocardiographically. RESULTS: Hyperprolactinaemia (17.3 +/- 4 ng mL-1 [Group I] vs. 4.64 +/- 2 ng mL-1 [Group II], P < 0.01) was found in 14 patients (8 with IHD, 6 with CMP). The distribution of HLA-DQB1 alleles was compared in the two groups and showed a significant increase in the frequency of *0301 (86% in Group I vs. 45% in Group II, P < 0.05). Histidine at position 30 of the HLA-DQB1 gene was found in 22% of Group II but in none of Group I patients. Furthermore, there was an inverse correlation between the presence of histidine at position 30 and the levels of serum prolactin. Both sIL-2R levels, a marker of T-cell activation, and concanavalin A-stimulated lymphocyte proliferation were lower in Group I patients (561 +/- 106 vs. 804 +/- 109 pg mL-1 and 20.8 +/- 4 vs. 37.3 +/- 5 cpmX103 [3H] thymidine, respectively). LVEF was significantly higher (32 +/- 5%) and LVEDd smaller (62.0 +/- 6 mm) in Group I compared to Group II (25 +/- 4% and 68.0 +/- 5 mm, respectively, P < 0.01) patients. CONCLUSION: Hyperprolactinaemia presents in 25% of patients with HF and may reflect decreased activation of T-lymphocytes associated with relatively preserved LV systolic function which is under immune-genetic control at the HLA-DQ locus.

Physical training reduces peripheral markers of inflammation in patients with chronic heart failure.

AIMS: Previous studies have shown an abnormal expression of cellular adhesion molecules and cytokines in chronic heart failure, which may be related to endothelial dysfunction characterizing this syndrome. Our study investigates the effects of physical training on serum activity of some peripheral inflammatory markers associated with endothelial dysfunction, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage chemoattractant protein-1 (MCP-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with chronic heart failure. METHODS AND RESULTS: Serum levels of GM-CSF, MCP-1, sICAM-1 and sVCAM-1 were determined in 12 patients with stable chronic heart failure (ischaemic heart failure: 6/12, dilated cardiomyopathy: 6/12, New York Heart Association: II-III, ejection fraction: 24+/-2%) before and after a 12-week programme of physical training in a randomized crossover design. In addition, the functional status of chronic heart failure patients was evaluated by using a cardiorespiratory exercise stress test to measure peak oxygen consumption. Physical training produced a significant reduction in serum GM-CSF (28+/-2 vs 21+/-2 pg. ml(-1), P<0.001), MCP-1 (192+/-5 vs 174+/-6 pg. ml(-1), P<0.001), sICAM-1 (367+/-31 vs 314+/-29 ng. ml(-1), P<0.01) and sVCAM-1 (1247+/-103 vs 1095+/-100 ng. ml(-1), P<0.01) as well as a significant increase in peak oxygen consumption (14.6+/-0.5 vs 16.5+/-0.5 ml. kg(-1)min(-1), P<0.005). A significant correlation was found between the training-induced improvement in peak oxygen consumption and percentage reduction in soluble adhesion molecules sICAM-1 (r=-0.72, P<0.01) and sVCAM-1 (r=-0.67, P<0.02). CONCLUSION: Physical training affects beneficially peripheral inflammatory markers reflecting monocyte/macrophage-endothelial cell interaction. Training-induced improvement in exercise tolerance is correlated with the attenuation of the inflammatory process, indicating that inflammation may contribute significantly to the impaired exercise capacity seen in chronic heart failure.

BRCA1 mutation analysis in breast/ovarian cancer families from Greece.

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece.

Cell adhesion molecules in relation to simvastatin and hormone replacement therapy in coronary artery disease.

AIMS: To assess the effect of simvastatin, hormone replacement therapy and their combination on soluble cell adhesion molecules and plasma lipids, in hypercholesterolaemic post-menopausal women with coronary artery disease. METHODS: We studied 16 post-menopausal women with coronary artery disease and hypercholesterolaemia (total cholesterol >200mg x dl(-1) and LDL cholesterol >130 mg x dl(-1)). We compared simvastatin (20 mg daily) with hormone replacement therapy (0.625 mg conjugated oestrogen and 2.5 mg medroxyprogesterone acetate daily) and their combination, in a randomized, crossover, placebo controlled study. Each treatment period was 8 weeks long with a 4 week washout interval between treatments. Circulating cell adhesion molecules and plasma lipids were evaluated at the end of each treatment period. RESULTS: All three active treatments--simvastatin, hormone replacement therapy and the combination therapy--significantly reduced total and LDL cholesterol, compared to placebo (P<0.001). Only hormone replacement therapy, alone and in combination with simvastatin, significantly decreased lipoprotein(a) when compared to placebo (P<0.05), whereas simvastatin had no significant effect. Likewise, hormone replacement therapy and the combination therapy significantly reduced the intercellular adhesion molecule (ICAM-1) plasma levels (P=0.03 and P=0.02, respectively), while simvastatin, which was superior to hormone replacement therapy in lowering total and LDL cholesterol, did not modify ICAM-1 levels; the combination therapy was not more effective than hormone replacement therapy alone in ICAM-1 reduction. Neither the effect, on any treatment when compared to placebo, of VCAM-1 nor E-selectin levels differed significantly. CONCLUSIONS: Hormone replacement therapy may limit the inflammatory response to injury by modulating the expression of cell adhesion molecules from the endothelial cells, possibly in association with lipoprotein (a) reduction.

Prevalence of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations in a Greek population of blood donors.

The pathogenesis of venous thrombosis involves the interaction of genetic and environmental factors. In order to estimate the frequency of the factor V Leiden, the prothrombin G20210A, and the MTHFR C677T mutations in the Greek population, we analyzed 160 healthy Greek blood donors by PCR amplification and detected allele frequencies of 2.5%, 2.2%, and 35.3%, respectively. The allele frequencies were compared with reported frequencies of other populations of southern Europe. The identification of these common genetic risk factors for thrombosis should enable easy DNA diagnosis and carrier detection in a high proportion of cases and will contribute to a better understanding of the interaction of genetic and environmental risk factors.

ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger.

Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.

Dimerization of Thermomonospora fusca beta-1,4-endoglucanase E2.

Unboiled Thermomonospora fusca endoglucanase E2 electrophoresed on SDS-polyacrylamide gels migrated in the range of 80-90 kDa, but when boiled it migrated in the 40-42-kDa range. Sedimentation equilibrium centrifugation as well as chemical cross-linking experiments confirmed that E2 is a dimer. The dimer was reversibly dissociated at low pH. The E2 dimer was stable up to 70 degrees C, but began to dissociate at this temperature after a 30-60-min incubation. A nondimerizing mutant was obtained using region-specific chemical mutagenesis. DNA sequencing of this mutant revealed a single base change that substituted Gly for Glu-263. Chemical modification of carboxylic acid residues in E2 disrupted the dimer interaction.

Stable expression of the cardiac sodium-calcium exchanger in CHO cells.

A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchanger. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.

Cloning and expression of the bovine cardiac sodium-calcium exchanger.

Two clones (p17 and p13), each containing the complete coding sequence for the bovine cardiac Na+/Ca2+ exchanger, were obtained from a lambda gt10 cDNA library by screening with cDNA probes from the canine exchanger. The coding sequence of clone p17 was 92 and 98% identical to the canine cDNA at the nucleotide and amino acid levels, respectively. Nine of the 21 amino acid differences between the two exchangers were found within the 32-amino acid signal sequence. The sequenced portions of the 3' untranslated regions of the cow and dog clones were 88% identical. Na+/Ca2+ exchange activity was expressed in Xenopus laevis oocytes injected with cRNA from clone p17, and in COS cells transfected with expression vectors containing p17. Immunoprecipitation of 35S-labeled proteins from transfected cells with an antibody against the N-terminal portion of the bovine exchanger showed the presence of a 120-kDa protein corresponding to the intact cardiac exchanger. The second bovine clone (p13) did not express exchange activity in either of the above expression systems, presumably because it contained a 300-bp insert with multiple stop codons which interrupted the coding sequence. Comparison of the 5' untranslated regions of p13 and p17 revealed a 156-bp segment in p17 that was apparently spliced out of p13. This segment contained a short open reading frame. A chimera encoding the 5' untranslated region of p13 and the coding sequence of p17 exhibited only a modest (74%) increase in expressed exchange activity in transfected cells compared to p17, suggesting that the presence of the upstream open reading frame in p17 did not greatly reduce translation efficiency. The results suggest that alternate splicing mechanisms may be involved in processing mRNA for the bovine cardiac exchanger.