Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.

Diagnostic Utility of Immunohistochemical Detection of MEOX2, SOX11, INSM1 and EGFR in Gliomas.

Histological identification of dispersed glioma cells in small biopsies can be challenging, especially in tumours lacking the IDH1 R132H mutation or alterations in TP53. We postulated that immunohistochemical detection of proteins expressed preferentially in gliomas (EGFR, MEOX2, CD34) or during embryonal development (SOX11, INSM1) can be used to distinguish reactive gliosis from glioma. Tissue microarrays of 46 reactive glioses, 81 glioblastomas, 34 IDH1-mutant diffuse gliomas, and 23 gliomas of other types were analysed. Glial neoplasms were significantly more often (p < 0.001, χ2) positive for EGFR (34.1% vs. 0%), MEOX2 (49.3% vs. 2.3%), SOX11 (70.5% vs. 20.4%), and INSM1 (65.4% vs. 2.3%). In 94.3% (66/70) of the glioblastomas, the expression of at least two markers was observed, while no reactive gliosis showed coexpression of any of the proteins. Compared to IDH1-mutant tumours, glioblastomas showed significantly higher expression of EGFR, MEOX2, and CD34 and significantly lower positivity for SOX11. Non-diffuse gliomas were only rarely positive for any of the five markers tested. Our results indicate that immunohistochemical detection of EGFR, MEOX2, SOX11, and INSM1 can be useful for detection of glioblastoma cells in limited histological samples, especially when used in combination.

Insulin Sensitivity and Secretion in Obese Type 2 Diabetic Women after Various Bariatric Operations.

OBJECTIVE: To compare the effects of biliopancreatic diversion (BPD) and laparoscopic gastric banding (LAGB) on insulin sensitivity and secretion with the effects of laparoscopic gastric plication (P). METHODS: A total of 52 obese women (age 30-66 years) suffering from type 2 diabetes mellitus (T2DM) were prospectively recruited into three study groups: 16 BPD; 16 LAGB, and 20 P. Euglycemic clamps and mixed meal tolerance tests were performed before, at 1 month and at 6 months after bariatric surgery. Beta cell function derived from the meal test parameters was evaluated using mathematical modeling. RESULTS: Glucose disposal per kilogram of fat free mass (a marker of peripheral insulin sensitivity) increased significantly in all groups, especially after 1 month. Basal insulin secretion decreased significantly after all three types of operations, with the most marked decrease after BPD compared with P and LAGB. Total insulin secretion decreased significantly only following the BPD. Beta cell glucose sensitivity did not change significantly post-surgery in any of the study groups. CONCLUSION: We documented similar improvement in insulin sensitivity in obese T2DM women after all three study operations during the 6-month postoperative follow-up. Notably, only BPD led to decreased demand on beta cells (decreased integrated insulin secretion), but without increasing the beta cell glucose sensitivity.

Preliminary evidence of altered steroidogenesis in women with Alzheimer's disease: Have the patients "OLDER" adrenal zona reticularis?

Alzheimer's disease (AD) represents more than half of total dementias. Various factors including altered steroid biosynthesis may participate in its pathophysiology. We investigated how the circulating steroids (measured by GC-MS and RIA) may be altered in the presence of AD. Sixteen women with AD and 22 age- and BMI-corresponding controls aged over 65 years were enrolled in the study. The steroid levels (47 steroids and steroid polar conjugates) and their ratios in AD female patients indicated increased CYP11A1 activity, weakened activity of the CYP17A1C17,20 lyase metabolic step and attenuated sulfotransferase SULT2A1 activity at higher activity of the CYP17A1 17-hydroxylase step. The patients showed diminished HSD3B2 activity for C21 steroids, abated conversion of 17-hydroxyprogesterone to cortisol, and significantly elevated cortisol. The women with AD had also attenuated steroid 7α-hydroxylation forming immunoprotective Δ(5)-C19 steroids, attenuated aromatase activity forming estradiol that induces autoimmunity and a shift from the 3ß-hydroxy-5α/ß-reduced C19 steroids to their neuroinhibitory and antiinflammatory GABAergic 3α-hydroxy- counterparts and showed higher levels of the 3α-hydroxy-5α/ß-reduced C21 steroids and pregnenolone sulfate (improves cognitive abilities but may be both protective and excitotoxic). Our preliminary data indicated functioning of alternative "backdoor" pathway in women with AD showing higher levels of both 5α/ß-reduced C21 steroids but reduced levels of both 5α/ß-reduced C21 steroids, which implied that the alternative "backdoor" pathway might include both 5α- and 5ß-reduced steroids. Our study suggested relationships between AD status in women based on the age of subjects and levels of 10 steroids measured by GC-MS.

[The prevalence of metabolic syndrome in women with polycystic ovary syndrome]. / Výskyt metabolického syndromu u zen se syndromem polycystických ovarií.

BACKGROUND: Polycystic ovary syndrome (PCOS) affects between 4-10% women of fertile age and is often connected with insulin resistance. We aimed to ascertain the prevalence of metabolic syndrome in Czech women with PCOS and to describe relations of different features of metabolic syndrome with insulin sensitivity. METHODS AND RESULTS: 179 women with PCOS. Clinical examination was done and blood lipid spectrum was measured. Euglycaemic hyperinsulinaemic clamp was done in 114 subjects. Metabolic syndrome (according to ATP III criteria) was detected in 28.7% women. The most frequent features were an increased waist circumference, decreased concentration of HDL - cholesterol (both in 96%), and increased blood pressure (88%). Increased triglycerides (49%) and impaired fasting blood glucose or diabetes mellitus type 2 (37.3%) were less common. The average insulin sensitivity described as corrected glucose disposal (Mk) was 34.9 +/- 12.70 micromol/kg/min. The most tight correlations was that of Mk and waist circumference (r = -0.896), weight (r = -0.875) and BMI (r = -0.844). CONCLUSION: The increased risk of metabolic syndrome and the decreased insulin sensitivity in polycystic ovary syndrome is tightly connected with obesity, especially with its abdominal type.

[(TTTTA), polymorphism in the promoter of the CYP11A1 gene in the pathogenesis of polycystic ovary syndrome]. / Promotorový polymorfismus (TTTTA)n genu CYP11A1 v patogenezi syndromu polycystických ovarií.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a common endocrinopathy which is characterized by ovarian androgen excess. PCOS has a strong genetic component but the pathogenetic mechanisms responsible for hyperandrogenemia are still unknown. The CYP11A1 encodes the cholesterol side-chain cleavage enzyme that catalyzes the first and rate-limiting step of steroidogenesis. A promoter polymorphism (TTTTA)n CYP11A1 has been reported to be related to the risk of PCOS but the results were controversial. METHODS AND RESULTS: We determined this polymorphism in a cohort of 256 PCOS and 109 healthy control women. Using two models (dominant model for allele with 4 repeats and dominant model for long alleles, i.e. 7 and more repeats) we did not find either the difference in allele and genotype distribution between PCOS and controls or the influence of polymorphism on serum testosterone and androstendione levels. However, the PCOS carriers of long alleles had lower FSH, total- and LDL-cholesterol compared to the carriers of short alleles (p = 0.007; p = 0.02; p = 0.02, ANOVA). In controls, the non-carriers of allele with 4 repeats had significantly higher DHEA-S (p = 0.02, ANOVA) levels than the carriers of allele with 4 repeats. CONCLUSIONS: Despite of some associations found, it seems that the promoter variability of CYP11A1 does not play a key role in the pathogenesis of PCOS.

[Relation between insulin secretion and action--study of genetic determination]. / Vztah sekrece a pusobení inzulínu--studium genetické determinace.

BACKGROUND: The disposition index represents insulin secretion related to the degree of insulin sensitivity, being constant for given degree of glucose tolerance. The aim of this study is to discern genetic determinants influencing the value of disposition index, e.g. predisposition to glucose intolerance. METHODS AND RESULTS: Two hundred and four non-diabetic subjects with varied glucose tolerance were divided into groups according to the values of disposition index. Glucose and lipid metabolism, anthropometric parameters and family history of type 2 diabetes mellitus (DM2) were examined. The genotype frequency of candidate genes was compared between the groups of individuals within the lowest (Q1) and the highest (Q4) quartiles of the disposition index values. Those groups were not different concerning age and female to male ratio. Fasting and stimulated parameters of glucose metabolism and lipid profile were worse in group Q1 compared to group Q4. Group Q1 is characterized with higher number of individuals with metabolic syndrome and family history of DM2. The examination of candidate genes revealed the differences in genotype frequency of B2AR (rs1042714), PPARA (rs1800206), KCNJ11 (rs5219), and SLC30A8 (rs13266634) between groups Q1 and Q4. CONCLUSIONS: Low value of disposition index is related to the deterioration of glucose tolerance and other signs of metabolic syndrome. It is associated with genes affecting insulin secretion and genes related to energy metabolism and obesity.

RET mutation Tyr791Phe: the genetic cause of different diseases derived from neural crest.

Activating germline RET mutations are presented in patients with familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasia (MEN) types 2A and 2B, whereas inactivating germline mutations in patients with Hirschsprung's disease (HSCR). The aim of this study was to evaluate genotype-phenotype correlations of the frequently discussed Tyr791Phe mutation in exon 13 of the RET proto-oncogene. Screening of three groups of patients was performed (276 families with medullary thyroid carcinoma (MTC), 122 families with HSCR, and 29 patients with pheochromocytoma). We found this mutation in 3 families with apparently sporadic MTC, 3 families with FMTC/MEN2, 1 patient with pheochromocytoma, and 3 families with HSCR. All gene mutation carriers have a silent polymorphism Leu769Leu in exon 13. In three families second germline mutations were detected: Cys620Phe (exon 10) in MEN2A family, Met918Thr (exon 16) in MEN2B family, and Ser649Leu (exon 11) in HSCR patient. Detection of the Tyr791Phe mutation in MEN2/MTC and also in HSCR families leads to the question whether this mutation has a dual character (gain-of-function as well as loss-of-function). A rare case of malignant pheochromocytoma in a patient with the Tyr791Phe mutation is presented. This study shows various clinical characteristics of the frequently discussed Tyr791Phe mutation.

Beta cell function and insulin sensitivity in women with polycystic ovary syndrome: influence of the family history of type 2 diabetes mellitus.

AIM: To study the impact of family history (FH) of type 2 diabetes mellitus on beta-cell compensatory mechanism in women with polycystic ovary syndrome (PCOS). SUBJECTS AND METHODS: A total of 70 women with PCOS, 14 with first-degree relative with type 2 diabetes mellitus (T2DM) (FH+), 56 with negative FH of T2DM (FH-) and 72 age and BMI matched control healthy women (CNT) underwent oral glucose tolerance test (OGTT). Insulin resistance was evaluated as oral glucose index (OGIS); insulin and C-peptide secretion as the insulinogenic index in 30th min of OGTT. RESULTS: Fasting blood glucose levels were significantly higher in FH+ than in FH- (p < 0.05). Fasting insulin was higher in FH+ than in CNT (p < 0.05). Fasting C-peptide was significantly higher in both FH- and FH+ than in CNT (p < 0.05 and p < 0.01, respectively). OGIS was lower in FH+ than in FH- or in CNT (p < 0.05). Insulinogenic index calculated from C-peptide values (II-Cp) was lower in FH+ than in CNT (p < 0.05). Adaptation index calculated from the values of OGIS and insulinogenic index was significantly lower in FH+ than in CNT or in FH- (p < 0.0001 and p < 0.01, respectively). CONCLUSIONS: Insulin resistance and defective early-phase insulin secretion is present only in those PCOS-affected subjects who had positive FH of T2DM.

Incretin levels in polycystic ovary syndrome.

OBJECTIVE: Polycystic ovary syndrome (PCOS) has been linked to a high risk of type 2 diabetes mellitus. Disturbances in the secretion of the incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) have been observed in states with impaired glucose regulation. This paper considers the secretion of GIP and GLP-1 after oral glucose load in a group of lean, glucose-tolerant PCOS women in comparison with age- and body mass index (BMI)-matched healthy women. DESIGN: Case control. METHODS: PCOS (n=21, 25.8+/-4.1 years, BMI 21.6+/-1.7 kg/m(2)) and control healthy women (CT, n=13, 28.5+/-7.2 years, BMI 20.3+/-2.5 kg/m(2)) underwent oral glucose tolerance test (OGTT) with blood sampling for glucose, insulin, C-peptide, total GIP, and active GLP-1. Insulin sensitivity was determined both at fasting and during the test. STATISTICS: Repeated measures ANOVA. RESULTS: Glucose levels and insulin sensitivity did not differ between PCOS and CT. PCOS had significantly higher levels of C-peptide (P<0.05) and tended to have higher insulin levels. The levels of total GIP were significantly higher in PCOS than in CT (P<0.001). Active GLP-1 levels exhibited a significantly different time-dependent pattern in PCOS (P<0.002 for PCOS versus time interaction). GLP-1 concentrations were similar in PCOS and CT in the early phase of OGTT and then reached significantly lower levels in PCOS than in CT at 180 min (P<0.05). CONCLUSIONS: Increased total GIP and lower late phase active GLP-1 concentrations during OGTT characterize PCOS women with higher C-peptide secretion in comparison with healthy controls, and may be the early markers of a pre-diabetic state.

Prevalence of insulin resistance and prediction of glucose intolerance and type 2 diabetes mellitus in women with polycystic ovary syndrome.

BACKGROUND: Diabetes mellitus type 2 (DM2) affects 10% of women with polycystic ovary syndrome (PCOS). We evaluated the sensitivity and specificity of clinical and fasting biochemical parameters in screening for impaired glucose tolerance (IGT) and DM2. METHODS: Women with PCOS [n=244, age 27.4+/-7.5 years, body mass index (BMI) 27.5+/-6.9 kg/m(2)] and healthy women (n=57, age 26.8+/-5.8 years, BMI 21.3+/-2.1 kg/m(2)) underwent basal blood sampling and an oral glucose tolerance test (oGTT). RESULTS: Insulin resistance was identified in 40.2% of PCOS women. Impaired fasting glucose (5.6-6.9 mmol/L) was found in 30 subjects (12.3%), but the oGTT revealed IGT in only six of these cases and DM2 in one subject. IGT was found in 23 (9.4%) and DM2 in four (1.6%) of the women with PCOS. The conventional upper limits for total cholesterol, triglycerides, systolic and diastolic blood pressure and fasting glucose revealed low sensitivity for the identification of impaired glucose metabolism. CONCLUSIONS: No single parameter nor any combination of them showed an accuracy sufficient for screening of IGT or DM2 in PCOS patients. All PCOS patients should be screened using an oGTT to identify disturbances in glucose metabolism.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient antibiotic use in Europe.

BACKGROUND: The ESAC project, granted by DG SANCO of the European Commission, is an international network of surveillance systems, aiming to collect comparable and reliable data on antibiotic use in Europe. Data on outpatient antibiotic use were collected from 34 countries using the ATC/DDD methodology. METHODS: For the period 1997-2003, data on outpatient use of systemic antibiotics aggregated at the level of the active substance were collected and expressed in DDD (WHO, version 2004) per 1000 inhabitants per day (DID). Outpatient antibiotic (ATC J01) use in 25 European countries, able to deliver valid data, was analysed. RESULTS: Total outpatient antibiotic use in 2003 varied by a factor of 3 between the country with the highest (31.4 DID in Greece) and the country with the lowest (9.8 DID in the Netherlands) use. General use patterns in individual countries as well as trends during the period 1997-2003 are described in this paper, while major antibiotic classes (penicillins, cephalosporins, macrolides/lincosamides/streptogramins and quinolones) will be analysed in detail in separate papers. CONCLUSION: The ESAC project established for the first time a credible alternative to industry sources for the collection of internationally comparable data on antibiotic use in Europe, based on cooperation between regulatory authorities, scientific societies, health insurers and professional organizations. These data provide a tool for assessing public health strategies aiming to optimize antibiotic prescribing.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient penicillin use in Europe.

BACKGROUND: Data on outpatient penicillin use in Europe were collected from 25 countries within the ESAC project, funded by DG SANCO of the European Commission, using the WHO ATC/DDD methodology. METHODS: For the period 1997-2003, data on outpatient use of systemic penicillins aggregated at the level of the active substance were collected and expressed in DDD (WHO, version 2004) per 1000 inhabitants per day (DID). Of the 'Penicillins' (J01C), outpatient use of narrow-spectrum penicillins (J01CE), broad-spectrum penicillins (J01CA), penicillinase-resistant penicillins (J01CF) and combinations with beta-lactamase inhibitors (J01CR) in 25 European countries was analysed in detail. RESULTS: Total outpatient penicillin use in 2003 varied by a factor of 4 between the country with the highest (15.27 DID in Slovakia) and lowest use (3.86 DID in the Netherlands). Narrow-spectrum penicillins, broad-spectrum penicillins and combinations with beta-lactamase inhibitors were used most in 4, 12 and 9 countries, respectively. Penicillin use increased by more than 1 DID in nine countries, whereas it decreased by more than 1 DID in two countries (Czech Republic, France). An increase of the use of combinations with beta-lactamase inhibitors by more than 10% in 10 countries coincided with an equal decrease of broad-spectrum penicillins in seven countries and narrow-spectrum penicillins in three countries. CONCLUSION: Penicillins represent the most widely used antibiotic class in all 25 participating countries; albeit with considerable variation of their use patterns. A distinct shift from narrow-spectrum penicillins to broad-spectrum penicillins, and specifically their combinations with beta-lactamase inhibitors, was observed during the period 1997-2003.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient cephalosporin use in Europe.

BACKGROUND: Data on outpatient cephalosporin use in Europe were collected from 25 countries within the ESAC project, funded by DG SANCO of the European Commission, using the WHO ATC/DDD methodology. METHODS: For the period 1997-2003, data on outpatient use of systemic cephalosporins aggregated at the level of the active substance were collected and expressed in DDD (WHO, version 2004) per 1000 inhabitants per day (DID). Use was analysed in detail, using the new ATC codes J01DB, J01DC, J01DD and J01DE, introduced in the 2005 issue of the WHO ATC index and assigned to the four cephalosporin generations. RESULTS: Total outpatient cephalosporin use in 2003 varied by a factor of 270 between the country with the highest (6.18 DID in Greece) and lowest (0.02 DID in Denmark) use. First-, second- and third-generation cephalosporins were used most in 6, 16 and 3 countries, respectively. We observed fourth-generation use (mainly cefepime) in ambulatory care in 11 countries. From 1997 to 2003 cephalosporin use decreased in 13 countries, in France by more than 1 DID. A relative increase of second-generation (mainly cefuroxime) or third-generation use (mainly cefpodoxime or cefixime) by more than 10% in 12 countries coincided with an equally large decrease of first-generation use in eight countries (mainly cefadroxil, cefalexin or cefatrizine). In six countries, first-generation use increased, second-generation use decreased or both occurred. CONCLUSION: The new ATC codes allow a more detailed description of outpatient cephalosporin use. The variation in antibiotic use in Europe is most extreme for this class of antibiotics, suggesting that in many countries in Europe these antibiotics are prescribed inappropriately.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient quinolone use in Europe.

BACKGROUND: Data on outpatient quinolone use in Europe were collected from 25 countries within the ESAC project, funded by DG SANCO of the European Commission, using the WHO ATC/DDD methodology. METHODS: For the period 1997-2003, data on outpatient use of systemic quinolones aggregated at the level of the active substance were collected and expressed in DDD (WHO, version 2004) per 1000 inhabitants per day (DID). Because a new DDD for levofloxacin was published in the ATC 2004 index (0.5 g instead of 0.25 g) all data were recalculated accordingly. Quinolone use was analysed in detail, using a classification into three generations based on their pharmacokinetic and in vitro potency profiles, which determines the area of clinical use. RESULTS: Total outpatient quinolone use in 2003 varied by a factor of 12 between the country with the highest (3.10 DID in Portugal) and lowest (0.25 DID in Denmark) quinolone use. The second-generation quinolones represented more than 50% of the quinolone use (mainly ciprofloxacin) except for Croatia, where the first-generation was used most (mainly norfloxacin). In 22 countries, the use of second and/or third-generation quinolones increased at the expense of the use of first-generation quinolones. The new so-called respiratory quinolones (levofloxacin and moxifloxacin) represented more than 10% of quinolone use in 12 countries, with extreme seasonal variation in all these countries except for one. CONCLUSION: There has been a substantial change in the use pattern of quinolones between 1997 and 2003, since the introduction of quinolones that are effective for the treatment of respiratory tract infections. These quinolones are not the first-line antibiotics for this indication and therefore quinolone use should in general still be limited and not show substantial seasonal variation.

Reduced Pms2 expression in non-neoplastic flat mucosa from patients with colon cancer correlates with reduced apoptosis competence.

Pms2 protein is a component of the DNA mismatch repair complex responsible both for post-replication correction of DNA nucleotide mispairs and for early steps in apoptosis. Germline mutations in DNA mismatch repair genes give rise to hereditary non-polyposis colon cancer, which accounts for about 4% of colon cancers. However, little is known about the expression of mismatch repair proteins in relation to sporadic colon cancer, which accounts for the great majority of colon cancers. Multiple samples were taken from the non-neoplastic flat mucosa of colon resections from patients with no colonic neoplasia, a tubulovillous adenoma, or an adenocarcinoma. Expression of Pms2 was assessed using semiquantitative immunohistochemistry. Apoptosis was assessed in polychrome-stained epoxy sections using morphologic criteria. Samples from patients without colonic neoplasia had moderate to strong staining for Pms2 in cell nuclei at the base of crypts, while samples from 2 of the 3 colons with a tubulovillous adenoma, and from 6 of the 10 colons with adenocarcinomas, showed reduced Pms2 expression. Samples from patients with an adenocarcinoma that had reduced Pms2 expression also exhibited reduced apoptosis capability in nearby tissue samples, evidenced when this paired tissue was stressed ex vivo with bile acid. Reduced Pms2 expression in the colonic mucosa may be an early step in progression to colon cancer. This reduction may cause decreased mismatch repair, increased genetic instability, and/or reduced apoptotic capability. Immunohistochemical determination of reduced Pms2 expression, upon further testing, may prove to be a promising early biomarker of risk of progression to malignancy.

A novel Czech kindred with familial medullary thyroid carcinoma and Hirschsprung's disease.

PURPOSE: The RET proto-oncogene is involved in neural crest disorders. Activating germline mutations in the RET proto-oncogene cause the development of familial medullary thyroid carcinoma (FMTC) or medullary thyroid carcinoma (MTC) as a part of multiple endocrine neoplasia type 2 (MEN2) syndrome. Inactivating germline mutations in the RET proto-oncogene are detected in Hirschsprung's disease (HSCR). Only in a very small number of families are these 2 diseases expressed together. METHODS: This study presents a novel Czech kindred with FMTC-HSCR phenotype. Two family members (mother and daughter) were tested for RET germline mutations in exons 10, 11, 13, 14, 15, and 16. RESULTS: Direct fluorescent sequencing of genomic DNA revealed a heterozygous mutation in the RET proto-oncogene in exon 10 at codon C609Y in both persons tested. This family was reclassified, thanks to genetic screening from the apparently sporadic MTC-HSCR to FMTC-HSCR. CONCLUSION: The germline mutation was detected because of the systematic genetic screening of the RET proto-oncogene, which is useful for genetic counseling of potential risk of HSCR and MTC in other family members. This family could be added to the small worldwide cohort of families with MEN2A/FMTC-HSCR.

Cytoskeleton localization in the sperm head prior to fertilization.

Three major cytoskeletal proteins, actin, tubulin and spectrin, are present in the head of mammalian spermatozoa. Although cytoskeletal proteins are implicated in the regulation of capacitation and the acrosome reaction (AR), their exact role remains poorly understood. The aim of this study was to compare the distribution of the sperm head cytoskeleton before and after the AR in spermatozoa representing a range of acrosome size and shape. Spermatozoa from the human and three rodents (rat, hamster and grey squirrel) were fixed before and after the AR in appropriate medium in vitro. Indirect immunofluorescent localization of cytoskeletal proteins was undertaken with antibodies recognizing actin, spectrin and alpha-tubulin. Preparations were counterstained with propidium iodide and examined by epifluorescent and confocal microscopy. Our results clearly demonstrated changes in localization of cytoskeleton during the AR, mainly in the apical acrosome with further changes to the equatorial segment and post-acrosomal regions. The pattern of cytoskeletal proteins in the sperm head of all the species was similar in respect to various sub-compartments. These observations indicated that the sperm head cortical cytoskeleton exhibits significant changes during the AR and, therefore, support the image of cytoskeletal proteins as highly dynamic structures participating actively in processes prior to fertilization.

Determinants of circulating adiponectin in women with polycystic ovary syndrome.

BACKGROUND AND AIM: Adiponectin is regarded as a possible link between adiposity and insulin resistance. Ghrelin and leptin are considered as signals of energy status. We evaluated the relationships between these peptides, androgens and insulin sensitivity in women affected by polycystic ovary syndrome. METHODS: Thirty-six women with PCOS were examined with euglycemic hyperinsulinemic clamp (to determine M/I, index of insulin sensitivity). Leptin, ghrelin, adiponectin, androgens, and SHBG were determined. Statistics was done using correlation analysis and backward stepwise multiple regression. RESULTS: The positive correlation of adiponectin with testosterone remains significant even after adjustment for BMI (p = 0.01), M/I (p = 0.009) and for both M/I and BMI (p = 0.02). In multiple regression with testosterone, M/I, leptin and ghrelin as independent variables, the model including testosterone (p = 0.03) and ghrelin (p = 0.002) explained 49% of the variability (p < 0.0012) of adiponectin. CONCLUSIONS: Both adiponectin and ghrelin can be involved in the pathophysiology of PCOS but their relation must be delineated further.

Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa.

Apoptosis competence is central to the prevention of cancer. Frequency of apoptotic cells, after a sample of colonic tissue is stressed, can be used to gauge apoptosis competence and, thus, possible susceptibility to colon cancer. The gold standard for assessment of apoptosis is morphological evaluation, but this requires an experienced microscopist. Easier-to-use immunohistochemical markers of apoptosis, applicable in archived paraffin-embedded tissue, have been commercially developed. Potentially useful apoptosis markers include cleaved cytokeratin-18 (c-CK18), cleaved caspase-3 (c-cas-3), cleaved lamin A (c-lam-A), phosphorylated histone H2AX (gammaH2AX), cleaved poly(ADP ribose) polymerase (c-PARP), and translocation of apoptosis-inducing factor (AIF). When tissue samples from freshly resected colon segments were challenged ex vivo with the bile acid deoxycholate, approximately 50% of goblet cells became apoptotic by morphologic criteria. This high level of morphologic apoptosis allowed quantitative comparison with the usefulness and specificity of immunohistochemical markers of apoptosis. The antibody to c-CK18 was almost as useful and about as specific as morphology for identifying apoptotic colonic epithelial cells. Antibodies to c-cas-3, c-lam-A, and gammaH2AX, though specific for apoptotic cells, were less useful. The antibody to c-PARP, though specific for apoptotic cells, had low usefulness, and the antibody to AIF was relatively nonspecific, under our conditions.