Association of maternal angiotensin II type 1 and type 2 receptor combination genotypes with susceptibility to early-onset preeclampsia.

Allelic variations affecting the activity of the maternal renin-angiotensin system may play a role in the development of hypertensive disorders of pregnancy like preeclampsia, its more severe early-onset form, and intrauterine growth restriction. We examined the association of common allelic variants of angiotensin II type 1 and type 2 receptor genes (AT1R and AT2R) sorted in five AT1R/AT2R receptor combination genotype groups with susceptibility to early-onset preeclampsia (EOP). The occurrence of AT1R (A1166C) and A2TR (C3123A) alleles in wild type (AA, CC), heterozygous (A/C, C/A), and homozygous (C/C, A/A) states was recorded in 84 women with a history of EOP and 84 age-matched controls sorted in five AT1R/AT2R receptor combination genotype (wild type: AA/CC, one mutant: AA/CA, AC/CC, two mutant: AC/CA, AA/AA, CC/CC, three mutants: AC/AA, CC/CA and four mutant: CC/AA) groups, by polymerase chain reaction-RFLP analysis. Three mutant receptor combination genotype carriers were more common in women with a history of EOP than in controls (26.18% vs. 4.76%, p = 0.003, OR = 8.25). Receptor combination genotyping may be of clinical value in: (a) maternal prediction of susceptibility to EOP, (b) disease subtyping for directed studies with receptor signaling antagonists, (c) the broader study of hypertension.

Follicular fluid oocyte/cumulus-free DNA concentrations as a potential biomolecular marker of embryo quality and IVF outcome.

The present prospective study examined the follicular fluid oocyte/cumulus-free DNA concentrations (ff o/c-free DNA) during ovarian stimulation and the possible association between ff o/c-free DNA and embryological results such as embryo quality and pregnancy rate. Eighty-three women undergoing IV/ICSI-ET treatments were prospectively included in this study. ff o/c-free DNA was determined by conventional quantitative real time PCR-Sybr green detection approach. The 83 ff samples were categorized in two groups: group 1 (n = 62) with cumulus oocytes complexes (CoCs) ≥2 and group 2 (n = 21) with CoCs = 1. Group 1 revealed significant higher embryo quality in terms of mean score of embryo transfer (MSET), but lower ff o/c-free DNA concentrations compared to group 2. The two groups showed comparable pregnancy rates (positive hCG and clinical pregnancy). The higher the ff o/c-free DNA concentration, the lower the number of produced oocytes. ff o/c-free DNA did not seem to have any direct role in the IVF outcome. Further research is required to clarify whether ff o/c-free DNA is a biomolecular marker of embryo quality and IVF outcome.

TLR4 gene polymorphisms: evidence for protection against type 2 diabetes but not for diabetes-associated ischaemic heart disease.

OBJECTIVE: Several factors either predisposing or protecting from the onset of diabetes mellitus type 2 (DM2) have been proposed. Two specific polymorphisms of toll-like receptor 4 (TLR4; Asp299Gly and Thr399Ile) have recently been identified either as candidate protector genes against DM2 and associated neuropathy or risk alleles for the manifestation of diabetic retinopathy. The impact of these alleles on the risk for ischaemic heart disease (IHD) is controversial while their role in diabetes-associated IHD has never been studied. DESIGN AND METHODS: In order to clarify the potential impact of TLR4 polymorphisms on the predisposition for DM2 as well as on diabetes-related IHD vulnerability, the distribution of the mutant TLR4 Asp299Gly and Thr399Ile alleles in 286 DM2 patients and 413 non-DM2 controls with or without IHD, was examined. RESULTS: Mutant alleles were predominantly detected in 79/413 non-diabetic individuals versus 15/286 DM2 patients (P<0.0001). The rates of positivity for mutant alleles were similar among diabetic patients with or without IHD (7/142 vs 8/144, P>0.1), whereas they proved different among non-diabetic individuals with or without IHD (39/145 vs 40/268, P=0.004). Following multivariate analysis, the difference between diabetic and non-diabetic subjects, with regard to TLR4 mutations alone, remained significant (P=0.04). CONCLUSIONS: Mutant TLR4 alleles confer protection against DM2. However, their presence does not seem to play any role, protective or aggravating, in the manifestation of IHD either in diabetic or in non-diabetic individuals.

Modulating effect of leptin on basal and follicle stimulating hormone stimulated steroidogenesis in cultured human lutein granulosa cells.

BACKGROUND: In vitro data have shown conflicting results in terms of the effect of leptin on granulosa cells steroidogenesis. AIM: The aim of the present study was to investigate the effect of low and high doses of leptin on basal and FSH-induced steroids secretion by human luteinized granulosa cells in culture. MATERIALS AND METHODS: Granulosa cells were obtained from normal women undergoing in vitro fertilization (IVF) treatment and were cultured in serum-free conditions for 72 h. A one-way analysis of variance design was set to study the effect of leptin on basal and FSH-induced steroidogenesis. RESULTS: Leptin affected basal estradiol and progesterone secretion in a dose-related manner. In particular, leptin at low concentrations stimulated the secretion of estradiol (1 and 10 ng/ml) and progesterone (10 ng/ml), while at a high concentration (100 ng/ml) it suppressed the secretion of both steroids. A dose-related effect of leptin on FSH-induced steroidogenesis was not evident, since only the suppressive effect of the high concentration of leptin (100 ng/ml) reached statistical significance for both steroids. CONCLUSIONS: These results demonstrate that leptin affects the secretion of steroids in luteinized granulosa cells in a dose-dependent manner. Although a physiological role for leptin is possible, it is suggested that this protein is a mediator of negative rather than positive influential interactions on ovarian function that may compromise fertility.

Surgical complications of hyperglycaemia.

We review the mechanisms leading to hyperglycaemic damage and draw functional extrapolations aiming to an improved management of surgical complications, which are common among diabetic patients.

The fat content of small primary breast cancer interferes with radiofrequency-induced thermal ablation.

BACKGROUND: Radiofrequency (RF) thermal ablation is a minimally invasive technique of local mass elimination with variable efficiency. METHODS: Ten patients with small primary breast cancer diagnosed preoperatively by core needle biopsy were ablated percutaneously by an RF (Radionics Cool-tip) device operating on impedance control mode. The percent fat-containing area was calculated in each slide of a total of 47 slides introduced to IQ materials software image analysis. RESULTS: Seven of 10 tumors with tumor diameter less than 2.8 cm and fat content less than 12.47% were totally ablated (score 3). One of 10 with 3 cm tumor diameter and 5.45% fat content showed an intermediate degree of ablated tissue (score 2), and the last 2 with 2 cm and 2.2 cm tumor diameter and more than 19.74% tumor fat content were minimally ablated (score 1). Our present exploratory study on 10 patients suggests dependence of the degree of thermal damage on tumor fat content. CONCLUSIONS: We conclude that the fat content of small primary breast cancer could serve as a 'heat sink' and should be considered as a preventing factor of complete local tumor destruction by RF thermal ablation.

Association of an oversized adrenal cortical adenoma with expression of pheochromocytoma-like neurosecretory features.

Abnormal stimulation of adrenal function may be either direct, affecting similarly cortical and medullary secretion, or indirect affecting primarily the medulla. Indirect activation of clinically detectable adrenomedullary function may develop as a physical consequence of a non-functional adrenal tumor exerting pressure on the medulla by its size, location and direction of growth. Our case of an oversized and overweight adrenal tumor associated with expression of late-onset pheochromocytoma-like clinical symptoms may be explained by the physical indirect rather than the biological direct activation of adrenomedullary function like hyperplasia or cancer.

Role of hyperglycemia in isogeneic islet transplantation: an experimental animal study.

OBJECTIVE: Study the role of hyperglycemia-induced beta cell loss on grafted islet destruction. DESIGN: Male inbred rats were made diabetic by streptozotocin administration and used as islet donors and/or isograft recipients to probe directly the role of hyperglycemia as an important determinant of transplanted islet fate, following exclusion of immune-related causes of islet graft destruction like allograft immunity and disease recurrence. RESULTS: Our studies showed that: a) Hyperglycemia destroyed islet but not pituitary isografts and b) Tight control of normoglycemia by sufficient islet mass engraftment prevented graft damage. CONCLUSION: While sustained hyperglycemia caused destruction of transplanted islet isografts, induction of normoglycemia by transplantation of sufficient islet mass to diabetic recipients had a beneficial long term effect on their functional engraftment.

Reevaluating pancreatic duct ligation in Whipple procedure.

In an effort to avoid the morbidity and mortality related to pancreaticojejunal anastomosis after pancreaticoduodenectomy (PPD), we report on the treatment of the pancreatic stump by pancreatic duct ligation (PDL) following Whipple procedure. We studied a series of 9 consecutive unselected patients (8 with pancreatic cancer and 1 with chronic pancreatitis). Of those, pancreatic fistula occurred in 4 patients and persisted for 14 to 58 days (mean 35.4 days). Two patients died within 30 days after surgery from causes not related to PDL. None of our patients developed diabetes mellitus following PDL surgery, nor any of the other frequently mentioned postoperative complications such as acute pancreatitis or pancreatic insufficiency. In conclusion, PDL may occasionally lead to a controlled pancreaticocutaneous fistula with fading inactive contents over time not causing further metabolic complications but is a safe, simple and fast alternative to pancreaticojejunostomy.

Comparing normal primary endocervical adenoepithelial cells to uninfected and influenza B virus infected human cervical adenocarcinoma HeLa cells.

Human adenocarcinoma HeLa cells surviving infection with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers were compared to their uninfected precursors and to normal endocervical adenoepithelial and metaplastic cells using Papanikolaou-staining method and immunocytochemistry. Normal primary endocervical and infected HeLa cells surviving infection shared similar morphologic, phenotypic, and divisional patterns that differed drastically from those of uninfected HeLa cells. The number of infected hosts surviving 6-7 days of viral exposure did not change during 3-week follow-up period, and their cyclin E levels suggested that they had been arrested to the G1 phase of the cell cycle by viral stress. Our findings suggest that in addition to apoptosis, nononcogenic viral stress activated the expression of endocervical metaplastic-like motifs in surviving hosts. A mechanism of cell response to nononcogenic viral stress was proposed to explain these findings. We conclude that nononcogenic respiratory viruses specifically target and eliminate abnormal cells ectopically overexpressing appropriate receptors and may complement current treatments of cervical cancer.

Telomerase reverse transcriptase mRNA expression in peripheral lymphocytes of patients with chronic HBV and HCV infections.

SUMMARY: Telomerase activity is present at low levels in peripheral lymphocytes (PL) and is upregulated upon activation, possibly protecting PL from telomere shortening. As decreased telomere length is considered a sign of cellular senescence, telomerase may, therefore, play an important role on immune function, organ regeneration and carcinogenesis. So far, quantification of human telomerase reverse transcriptase levels (hTERT) in PL, has not been reported. We determined hTERT mRNA levels in PL of hepatitis B virus (HBV) and hepatitis C virus (HCV) patients, in an attempt to address whether hTERT transcripts in PL are altered in these viral diseases, which are characterized by immune dysfunction and increased incidence of hepatocarcinogenesis. hTERT mRNA levels in PL of HBV (n = 17), HCV (n = 24) patients and healthy controls (n = 22) were quantified by real-time polymerase chain reaction. We observed significantly lower hTERT mRNA levels in HBV and HCV patients compared with healthy individuals (P < 0.05). hTERT mRNA levels were not associated with the patients' clinical status (inactive, hepatitis and cirrhosis). Also no correlation was observed between hTERT mRNA expression, and HBV and HCV replicative activity. In the inactive group (n = 18) we observed a negative correlation between hTERT mRNA expression and disease duration (rs = -0.52, P < 0.03). We performed for the first time an accurate quantification of hTERT mRNA expression in PL of HBV and HCV patients. The observed low levels of hTERT mRNA expression in the above patients may suggest its involvement in the immunopathogenesis of chronic viral hepatitis.

The role of human telomerase catalytic subunit mRNA expression in cervical dysplasias.

Telomerase activity and human telomerase reverse transcriptase (hTERT) mRNA expression were investigated in cervical specimens and were correlated with cytologic findings and the presence of human papilloma virus (HPV) infection. Telomerase activity was evaluated by the telomeric repeat protocol assay and hTERT mRNA expression was evaluated by reverse transcriptase polymerase chain reaction (PCR). HPV DNA was detected by PCR, as well as restriction endonuclease digestion. HPV DNA was detected in all 82 specimens with abnormal cytologic findings and in 4 of 34 normal samples. Low-grade squamous intraepithelial lesions (LGSILs) were present in 74 of 82 specimens (90.2%) and high-grade squamous intraepithelial lesions (HGSILs) were present in 8 of 82 (9.75%) specimens. Seven of the eight HGSIL (87.5%) and 26 of 74 LGSIL (35.1%) specimens were hTERT positive, whereas all normal specimens were hTERT mRNA negative. Telomerase activity was detected in 21 of 74 (28.4%) LGSIL/atypical squamous epithelial cells of undetermined significance (ASCUS) and in five of eight (62.5%) HGSIL samples. A correlation was observed among telomerase activity, hTERT mRNA expression, and high-risk HPV infection in HGSIL samples (P < 0.001). High-risk HPV infection assessment showed 75% sensitivity and 72.2% specificity for HGSILs. Telomerase activity assessment in cervical smears showed sensitivity and negative predictive value (NPV) for HGSILs 62.5% and 96.7%, whereas specificity and positive predictive value (PPV) were 80.5% and 19.2%, respectively. hTERT mRNA expression assessment showed 87.5% sensitivity and 98.7% NPV for HGSILs, whereas specificity and PPV were 76% and 21.2%, respectively. Based on the above-described telomerase assessment values, it is suggested that the telomerase system might not be an appropriate diagnostic marker for cytology, given that the final evaluation must rely on a combination of all available test assessment data, clinical diagnosis, as well as the follow-up of all LGSIL samples that were positive for telomerase activation.

Estradiol and leptin as conditional prognostic IVF markers.

We studied the concentration of serum estradiol and serum and follicular fluid leptin in 200 women undergoing their first in vitro fertilization with embryo transfer (IVF-ET) program at the time of human chorionic gonadotrophin administration and oocyte retrieval, in an attempt to assess their concerted role on embryo quality and the prognosis of IVF outcome. Low serum (46.49 +/- 8.4 ng/ml) and follicular fluid (52 +/- 9.8 ng/ml) leptin levels were associated with a high number of 'good-quality' embryos (73.6%) and high implantation (11.2%) and pregnancy (35.8%) rates and were observed in women with normal peak estradiol levels of between 1000 and 2000 pg/ml. It appears that leptin and estradiol interact coordinately in a concentration-dependent manner to control IVF outcome. Further studies will be required to substantiate and clarify the mechanism of proposed conditional interaction between the two hormonal systems.

Absence of leptin expression and secretion by human luteinized granulosa cells.

Whether leptin is secreted by the human ovary is not known. The available data on leptin gene (ob gene) expression by human granulosa cells are conflicting. The aim of the present study was first to re-examine the expression of leptin messenger RNA (mRNA) by human granulosa cells and second to investigate if these cells have the ability to secrete leptin in cultures. Human luteinized granulosa cells were obtained from normal women undergoing in vitro fertilisation treatment after ovarian stimulation and follicle aspiration. The expression of ob gene was studied by Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) both in primary granulosa cells treated immediately after oocyte recovery and in cells cultured up to 24 h under baseline and hormonally stimulated conditions (FSH: 100 ng/ml, LH: 100 ng/ml). ob mRNA transcripts were not detected in luteinized granulosa cells, while they were present in adipose tIssue cDNA. Actin gene expression was detected in all studied samples. Using a sensitive radioimmunoassay (lower limit of detection 0.05 ng/ml), leptin was undetectable in the culture media at all points during the 72 h cultures, while at the same time significant amounts of oestradiol and progesterone were produced particularly after the addition of androstendione (1 microM) to the incubation media. These results demonstrate for the first time that leptin is not secreted by human luteinized granulosa cells in cultures. From a physiological point of view, this may contribute to the development of the optimal follicular environment for oocyte maturation during the preovulatory period.

Molecular and phylogenetic analysis of haemagglutinin and neuraminidase sequences from recent human influenza type A (H3N2) viral isolates in Southern Greece.

Eighteen haemagglutinin (HA1) gene segments and eleven neuraminidase (NA) genes of human influenza type A (H3N2) viruses isolated from non-vaccinated individuals presenting severe influenza-like illness at peak influenza activity in Southern Greece during the surveillance period 1996-1999, were subjected to sequence and phylogenetic analyses following propagation in embryonated hen's eggs. The HA1 gene segment of the clinical isolates differed from the recent reference influenza type A (H3N2) vaccine strains in an Ile at residue 186, a Val at residue 194 and a Val at residue 226 for one, two and thirteen isolates of the 1996-1997 and 1996-1999 periods, respectively. The analogous differences in the NA gene were confined in an Asp to Asn substitution at residue 198 in one A/Wuhan/359/95 (H3N2)-like isolate of the 1996-1997 period, primarily. In addition, phylogenetic analysis revealed that an isolate of the 1997-1998 period was a recombinant with its HA1 gene segment being closely related to that of A/Wuhan/359/95-like viruses and its NA to viruses of the A/Sydney/5/97 (H3N2) lineage. These findings confirmed the profound genetic instability of influenza type A (H3N2) viruses and underscored the importance for periodic molecular surveys of HA and NA in the effective prevention and management of viral outbreaks. Most importantly, however, they contributed the first complete epidemiological material for influenza in Southern Greece, the archival nature of which constitutes valuable reference for future surveys.

Correlating functional staging to effective treatment of acute surgical illness.

BACKGROUND: Proinflammatory and anti-inflammatory events may eventually trigger host response, which acting via a broad spectrum of complex biological processes and molecular interactions may either enhance or resolve the symptoms of acute surgical illness (ASI). Staging the sequence of biological events that take place at the cellular level during the development of ASI may provide leads to effective stage-specific treatments. In line with the hypothesis that proper timing of therapeutic intervention may be crucial to the management of the disease, we have attempted in this review to correlate functional staging to effective treatment of ASI. DATA SOURCE: The present report proposes a conceptual synthesis on the biogenesis and treatment of ASI that is based on known molecular and cellular aspects of human inflammatory sequence and patient data from clinical trials. It also introduces proper timing of therapeutic intervention as a potentially important determinant for the successful outcome of the disease process. CONCLUSIONS: Progress in understanding the biogenesis of ASI did not result in successful therapeutic developments as yet. The challenge ahead should be a better understanding of the dynamics of the various processes and regulators in appropriate animal and clinical models of ASI, in order to properly intervene and direct effective therapies for the benefit of critically ill patients.

Heroin-induced changes of catecholamine-containing particles in male rat cerebellar cortex.

The content and distribution of catecholamine-containing formations in the cerebellum of untreated and heroin-treated male rats, was visualized by glyoxylic acid-induced histofluorescence, in an attempt to define the adaptive mechanisms leading to heroin dependent tolerance as well as identify a biological role for these formations. Repeated heroin administration increased the number of specifically organized intracellular catecholamine containing particles, including grain (diameter less than 0.8 microm) and aggregate (diameter greater than 1 microm) forms, in all cerebellar cortical layers examined one hour after the last injection of the drug, relative to controls. The number of grains in all cerebellar cortical layers examined and aggregates in the granular layer, returned to normal or near normal baseline levels within twenty four hours after the last injection of the drug. The analogous baseline of the aggregates in the Purkinje cell layer primarily and the Molecular layer secondarily remained significantly elevated by 86% and 50% respectively, relative to controls. Catecholamine-heroin interactions most likely mediated this elevation that was related directly to the heroin-dependent state of tolerance. These findings indicate that heroin administration to heroin-tolerant rats leads to the formation of unusually large intracellular aggregates with catecholamines in the Purkinje cells of the cerebellum primarily and support a direct role for these formations in the modulation of biogenic amine bioavailability. We conclude that adaptation to drug exposure involves multiple homeostatic interactions, with sympathetic activation at the level of catecholamine reorganization and redistribution playing a major role in rat cerebellar cortex.

High sequence divergence in the 5' non-coding region of reference Coxsackie B and ECHO viral strains and clinical isolates revealed by restriction fragment length polymorphism analysis.

We report the restriction fragment length polymorphism (RFLP) patterns of a 440-bp-long 5' non-coding region (5' NCR) amplification target of all 34 reference Coxsackie B and ECHO (enteric cytopathic human orphan) enterovirus strains and a total of 42 serotypically pre-assigned clinical isolates, in order to afford meaningful comparisons among these patterns and those of polioviruses. The RFLP patterns of reference Coxsackie B strains differed from one another and from those of polio and ECHO reference enteroviruses except from Coxsackie B1 and B2, which, although they differed from one another, had identical RFLP patterns with ECHO 17 and 13, respectively. The 28 ECHO reference strains formed a more variable viral group including strains with RFLP patterns distinct from one another and from those of polio and Coxsackie B enteroviruses, and others with RFLP pattern identities common to other ECHO viruses and Coxsackie B1 and B2 but not polioviruses. The RFLP patterns of the clinical isolates and their corresponding serotypically assigned reference Coxsackie B and ECHO strains presented the most notable variations. The observed differences between serotype and genotype-dependent assignments within the 440-bp long 5' NCR target sequence of Coxsackie B and ECHO enteroviruses were in sharp contrast to the analogous situation with polioviruses. These findings support the specificity of the described method for clinical diagnostic genotyping of polioviruses and demonstrate that the 440-bp-long target sequence follows a different evolutionary process in polio and non-polio enteroviruses that is particularly prominent between reference non-polio strains and their serotypically assigned clinical isolates.

Improved genotyping vaccine and wild-type poliovirus strains by restriction fragment length polymorphism analysis: clinical diagnostic implications.

The combination of preventive vaccination and diagnostic typing of viral isolates from patients with clinical poliomyelitis constitutes our main protective shield against polioviruses. The restriction fragment length polymorphism (RFLP) adaptation of the reverse transcriptase (RT)-PCR methodology has advanced diagnostic genotyping of polioviruses, although further improvements are definitely needed. We report here on an improved RFLP procedure for the genotyping of polioviruses. A highly conserved segment within the 5' noncoding region of polioviruses was selected for RT-PCR amplification by the UC(53)-UG(52) primer pair with the hope that it would be most resistant to the inescapable genetic alteration-drift experienced by the other segments of the viral genome. Complete inter- and intratypic genotyping of polioviruses by the present RFLP method was accomplished with a minimum set of four restriction endonucleases (HaeIII, DdeI, NcoI, and AvaI). To compensate for potential genetic drift within the recognition sites of HaeIII, DdeI, or NcoI in atypical clinical samples, the RFLP patterns generated with HpaII and StyI as replacements were analyzed. The specificity of the method was also successfully assessed by RFLP analysis of 55 reference nonpoliovirus enterovirus controls. The concerted implementation of these conditional protocols for diagnostic inter- and intratypic genotyping of polioviruses was evaluated with 21 clinical samples with absolute success.

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction.

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.