Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth.

OBJECTIVES: The aim of this study was to determine whether normal human embryonic stem cells (hESC) would secrete factors that arrest growth of human epithelial cancer cell lines. MATERIALS AND METHODS: Cell proliferation was examined using the MTT assay then haemocytometer cell counts. Staining with propidium iodide followed by flow cytometry was used to detect cell cycle stages. Heat denaturation and molecular fractionation experiments were also performed. RESULTS: We found that hESC conditioned medium (hESC CM) inhibited SKOV-3 and HEY cell proliferation. Similar results were also obtained when we used breast and prostate cancer cell lines, whereas little or no inhibitory effect was observed when human fibroblasts were tested. Moreover, a co-culture model confirmed that inhibition of cancer cell proliferation is mediated by soluble factors produced by hESCs. We also determined that the proportion of cancer cells in G(1) phase was increased by hESC CM treatment, accompanied by decrease in cells in S and G(2)/M phases, suggesting that the factors slow progression of cancer cells by cell cycle inhibition. Heat denaturation and molecular fractionation experiments indicated a low molecular weight thermostable factor was responsible for these properties. CONCLUSIONS: Our findings provide evidence that the human embryonic microenvironment contains soluble factor(s) that are capable of inhibiting growth of cancer cells, and that exposure to such factors may represent a new cancer treatment strategy.

Trends in colorectal day case surgery in NHS Trusts between 1998 and 2005.

OBJECTIVE: Day case surgery is safe and offers potential benefits to both patients and healthcare providers. This study aimed to describe national changes in colorectal day case workload between 1998 and 2005. METHODS: Admission data relating to Office of Population Censuses and Surveys Classification of Surgical Operations and Procedures (4th revision) (OPCS-4) coloproctology operation codes were analysed using the Hospital Episode Statistics (HES) database. Day case rates (DCRs) were calculated as the proportion of elective cases performed on an ambulatory basis. RESULTS: In total, 3 119 058 colorectal admissions were recorded on the HES database between 1998 and 2005; 1 891 474 (61%) of these were for lower gastrointestinal endoscopies. Emergency cases accounted for 527 665 (17%), elective inpatient cases for 406 368 (13%) and elective day cases for 293 551 (9%) admissions. Throughout the study period the DCRs for five commonly performed elective colorectal procedures were: 0.70 for anal lesion excisions (OPCS-4 codes: H48.1, H48.2 and H48.3); 0.16 for haemorrhoidectomy (OPCS-4 code: H51.1); 0.63 for anal fissure procedures (OPCS-4 codes: H56.2 and H56.4); 0.39 for elective procedures for anal fistula (OPCS-4 codes: H55.1, H55.2, H55.3 and H55.4); 0.37 for elective pilonidal surgery (OPCS-4 codes: H59 and H60.2). Two emergency operations, drainage of perianal and pilonidal abscesses (OPCS-4 codes: H58.2 and H60.3 respectively), were identified as operations potentially amenable to day surgery. Over the seven study years, an annual average of 8559 (+/-SD 307) admissions were coded to drainage of a perianal abscess and 4676 (+/-SD 478) admissions to drainage of pilonidal abscess. The average annual bed usage associated with these procedures was 18 831 (+/-SD 718) and 7623 (+/-SD 436) bed days respectively. CONCLUSIONS: Colorectal day case surgery is currently under-exploited in the NHS. By lifting some of the barriers to day case surgery significant resource savings may be possible.

Phagocytosis of collagen by fibroblasts and invasive cancer cells is mediated by MT1-MMP.

Degradation of collagen is required for the physiological remodelling of connective tissues during growth and development, as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodelling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. While various steps in this pathway have been characterized, the enzyme required to fragment collagen fibrils for phagocytosis has not been identified. Laser confocal microscopy, transmission electron microscopy and biochemical assays were used to show that degradation of collagen substrates by fibroblasts correlated with the expression of the membrane-bound metalloproteinase MT1-MMP (membrane-type 1 matrix metalloproteinase). The MT1-MMP was localized to sites of collagen cleavage on the cell surface and also within the cells. In contrast with MT1-MMP, the gelatinase MMP-2 was not required for collagen phagocytosis. Similar analyses of several ovarian cancer, breast cancer and fibrosarcoma cells indicated that highly metastatic cells also degrade collagen through a phagocytic pathway that is mediated by MT1-MMP. Collectively, these studies demonstrate a pivotal role for catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation by normal and transformed cells.

MT1-MMP is the critical determinant of matrix degradation and invasion by ovarian cancer cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane metalloprotease that plays an important role in the invasion of many solid tumour types, promotes pericellular matrix degradation and may also stimulate tumour cell motility. As both these processes are key contributors to intraperitoneal ovarian tumour metastasis, we examined six ovarian cancer cell lines to determine whether MT1 is a critical mediator of invasive behaviour for this tumour type. Our results indicated that only those cell lines that expressed MT1 were capable of penetrating a type I collagen barrier, with the capacity for both matrix degradation and invasion reflecting endogenous MT1 expression level. Ectopic MT1 expression endowed an invasive phenotype upon cell lines lacking MT1 that were previously non-invasive, indicating the crucial role of this protease. Conversely, invasion was abolished by tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of MT1, yet was minimally affected when other (secreted) MMPs were inhibited using TIMP-1 and the gelatinase inhibitor SB-3CT. Whereas collagen I degradation was strikingly accelerated by ectopic MT1 expression, cell motility remained unchanged. We conclude that MT1 is necessary for collagen I invasion by ovarian cancer cells, and that its requisite activity is the promotion of matrix degradation, with no impact on cell motility.

Differentially androgen-modulated genes in ovarian epithelial cells from BRCA mutation carriers and control patients predict ovarian cancer survival and disease progression.

Epidemiological studies have implicated androgens in the etiology and progression of epithelial ovarian cancer. We previously reported that some androgen responses were dysregulated in malignant ovarian epithelial cells relative to control, non-malignant ovarian surface epithelial (OSE) cells. Moreover, dysregulated androgen responses were observed in OSE cells derived from patients with germline BRCA-1 or -2 mutations (OSEb), which account for the majority of familial ovarian cancer predisposition, and such altered responses may be involved in ovarian carcinogenesis or progression. In the present study, gene expression profiling using cDNA microarrays identified 17 genes differentially expressed in response to continuous androgen exposure in OSEb cells and ovarian cancer cells as compared to OSE cells derived from control patients. A subset of these differentially affected genes was selected and verified by quantitative real-time reverse transcription-polymerase chain reaction. Six of the gene products mapped to the OPHID protein-protein interaction database, and five were networked within two interacting partners. Basic leucine zipper transcription factor 2 (BACH2) and acetylcholinesterase (ACHE), which were upregulated by androgen in OSEb cells relative to OSE cells, were further investigated using an ovarian cancer tissue microarray from a separate set of 149 clinical samples. Both cytoplasmic ACHE and BACH2 immunostaining were significantly increased in ovarian cancer relative to benign cases. High levels of cytoplasmic ACHE staining correlated with decreased survival, whereas nuclear BACH2 staining correlated with decreased time to disease recurrence. The finding that products of genes differentially responsive to androgen in OSEb cells may predict survival and disease progression supports a role for altered androgen effects in ovarian cancer. In addition to BACH2 and ACHE, this study highlights a set of potentially functionally related genes for further investigation in ovarian cancer.

Cost-effectiveness of cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion: a systematic review and economic model.

OBJECTIVES: To compare patient outcomes, resource use and costs to the NHS and NHS Blood Transfusion Authority (BTA) associated with cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion. DATA SOURCES: Electronic databases covering the period 1996-2004 for systematic reviews and 1994-2004 for economic evidence. REVIEW METHODS: Existing systematic reviews were updated with data from selected randomised controlled trials (RCTs) that involved adults scheduled for elective non-urgent surgery. Any resource use or cost data were extracted for potential use in populating an economic model. Relative risks or weighted mean difference of each outcome for each intervention were assessed, taking into account the number of RCTs included in each outcome and intervention and the presence of any heterogeneity. This allowed indirect comparison of the relative effectiveness of each intervention when the intervention is compared with allogeneic blood transfusion. A decision analytic model synthesised clinical and economic data from several sources, to estimate the relative cost-effectiveness of cell salvage for people undergoing elective surgery with moderate to major expected blood loss. The perspective of the NHS and patients and a time horizon of 1 month were used. The economic model was developed from reviews of effectiveness and cost-effectiveness and clinical experts. Secondary analysis explored the robustness of the results to changes in the timing and costs of cell salvage equipment, surgical procedure, use of transfusion protocols and time horizon of analysis. RESULTS: Overall, 668 studies were identified electronically for the update of the two systematic reviews. This included five RCTs, of which two were cell salvage and three preoperative autologous donation (PAD). Five published systematic reviews were identified for antifibrinolytics, fibrin sealants and restrictive transfusion triggers, PAD plus erythropoietin, erythropoietin alone and acute normovolaemic haemodilution (ANH). Twelve published studies reported full economic evaluations. All but two of the transfusion strategies significantly reduced exposure to allogeneic blood. The relative risk of exposure to allogeneic blood was 0.59 for the pooled trials of cell salvage (95% confidence interval: 0.48 to 0.73). This varied by the type and timing of cell salvage and type of surgical procedure. For cell salvage, the relative risk of allogeneic blood transfusion was higher in cardiac surgery than in orthopaedic surgery. Cell salvage had lower costs and slightly higher quality-adjusted life years compared with all of the alternative transfusion strategies except ANH. The likelihood that cell salvage is cost-effective compared with strategies other than ANH is over 50%. Most of the secondary analyses indicated similar results to the primary analysis. However, the primary and secondary analyses indicated that ANH may be more cost-effective than cell salvage. CONCLUSIONS: The available evidence indicates that cell salvage may be a cost-effective method to reduce exposure to allogeneic blood transfusion. However, ANH may be more cost-effective than cell salvage. The results of this analysis are subject to the low quality and reliability of the data used and the use of indirect comparisons. This may affect the reliability and robustness of the clinical and economic results. There is a need for further research that includes adequately powered high-quality RCTs to compare directly various blood transfusion strategies. These should include measures of health status, health-related quality of life and patient preferences for alternative transfusion strategies. Observational and tracking studies are needed to estimate reliably the incidence of adverse events and infections transmitted during blood transfusion and to identify the lifetime consequences of the serious hazards of transfusion on mortality, health status and health-related quality of life.

Systematic review of the long-term effects and economic consequences of treatments for obesity and implications for health improvement.

OBJECTIVES: To undertake a systematic review of the long-term effects of obesity treatments on body weight, risk factors for disease, and disease. METHODS: The study encompassed three systematic reviews that examined different aspects of obesity treatments. (1) A systematic review of obesity treatments in adults where the methods of the Cochrane Collaboration were applied and randomised controlled trials (RCTs) with a follow-up of at least 1 year were evaluated. (2) A systematic epidemiological review, where studies were sought on long-term effects of weight loss on morbidity and/or mortality, and examined through epidemiological modelling. (3) A systematic economic review that sought reports with both costs and outcomes of treatment, including recent reports that assessed the cost-effectiveness of pharmaceutical and surgical interventions. A Markov model was also adopted to examine the cost-effectiveness of a low-fat diet and exercise intervention in adults with obesity and impaired glucose tolerance. RESULTS: The addition of the drugs orlistat or sibutramine was associated with weight loss and generally improved risk factors, apart from diastolic blood pressure for sibutramine. Metformin was associated with decreased mortality after 10 years in obese people with type 2 diabetes. Low-fat diets were associated with continuing weight loss for 3 years and improvements in risk factors, as well as prevention of type 2 diabetes and improved control of hypertension. Insufficient evidence was available to demonstrate the benefits of low calorie or very low calorie diets. The addition of an exercise or behaviour programme to diet was associated with improved weight loss and risk factors for at least 1 year. Studies combining low-fat diets, exercise and behaviour therapy suggested improved hypertension and cardiovascular disease. Family therapy was associated with improved weight loss for 2 years compared to individual therapy. There was insufficient evidence to conclude that individual therapy was more beneficial than group therapy. Weight lost more quickly (within 1 year), from the epidemiology review, may be more beneficial with respect to the risk of mortality. The effects of intentional weight loss need further investigation. Weight loss from surgical and non-surgical interventions for people suffering from obesity was associated with decreased risk of development of diabetes, and a reduction in low-density lipoprotein cholesterol, total cholesterol and blood pressure, in the long term. Targeting high-risk individuals with drugs or surgery was likely to result in a cost per additional life-year or quality-adjusted life-year (QALY) of no more than 13,000 British pounds. There was also suggestive evidence of cost saving from treatment of people with type 2 diabetes with metformin. Targeting surgery on people with severe obesity and impaired glucose tolerance was likely to be more cost-effective at 2329 British pounds per additional life-year. Economic modelling over 6 years for diet and exercise for people with impaired glucose tolerance was associated with a high initial cost per additional QALY, but by the sixth year the cost per QALY was 13,389 British pounds. Results did not include cost savings from diseases other than diabetes, and therefore may be conservative. CONCLUSIONS: The drugs orlistat and sibutramine appear beneficial for the treatment of adults with obesity, and metformin for obese patients with type 2 diabetes. Exercise and/or behaviour therapy appear to improve weight loss when added to diet. Low-fat diets with exercise, or with exercise and behaviour therapy are associated with the prevention of type 2 diabetes and hypertension. Long-term weight loss in epidemiological studies was associated with reduced risk of type 2 diabetes, and may be beneficial for cardiovascular disease. Low-fat diets and exercise interventions in individuals at risk of obesity-related illness are of comparable cost to drug treatments. Long-term pragmatic RCTs of obesity treatments in populations with obesity-related illness or at high risk of developing such illness are needed (to include an evaluation of risk factors, morbidity, quality of life and economic evaluations). Drug trials that include dietary advice, plus exercise and/or behaviour therapy are also needed. Research exploring effective types of exercise, diet or behaviour and also interventions to prevent obesity in adults is required.

Motion analysis.

BACKGROUND: The ability to make an objective evaluation of a surgeon's operative ability remains an elusive goal. In this study, we used motion analysis as a measure of dexterity in the performance of a simulated operation. METHODS: Fifteen surgeons performed a total of 45 laboratory-based laparoscopic cholecystectomies on a cadaveric porcine liver model. Subjects were assigned to one of three groups according to their level of experience in human laparoscopic cholecystectomy. Electromagnetic tracking devices were used to analyze the surgeon's hand movements as they performed the procedure. Movement data (time, distance, number of movements, and speed of movement) were then compared. RESULTS: Analysis of variance (ANOVA) movement scores across the three groups showed significantly better performance among the experienced laparoscopic surgeons than the novices. Learning curves across repetitions of procedures were plotted. Novices made more improvement than experts. CONCLUSIONS: Motion analysis provides useful data for the assessment of laparoscopic dexterity, and the porcine liver model is a valid simulation of the real procedure.

Loss of androgen receptor associated protein 70 (ARA70) expression in a subset of HER2-positive breast cancers.

Co-transfection studies indicate that HER2 (erbB-2) overexpression results in the phosphorylation and enhanced transcriptional activity of the androgen receptor (AR). This amplification of AR action is further enhanced by the expression of ARA70, a putative co-activator with a predilection for the AR. Because androgens inhibit the growth of breast cancer cells whereas HER2 overexpression stimulates the growth of these cells, it seems possible that loss of expression of AR or ARA70 in some HER2 overexpressing tumors might confer a growth advantage to these cells. We examined ARA70 and AR expression in 20 HER2-positive (overexpressing) and 21 HER2-negative cases of breast invasive ductal carcinoma (IDC) to determine the relationship between loss of ARA70 and/or AR with HER2 overexpression. Strong ARA70 immunostaining was observed in all normal and breast epithelial cells in fibrocystic change and in in situ carcinoma present in the patient samples. Of the 41 cases of IDC, focal or complete loss of ARA70 protein expression was observed in 46% of the cases, with 60% of HER2-positive versus 33% of HER2-negative cases showing loss. Loss of AR expression was observed in 60% of HER2-positive versus 43% of HER2-negative cases. Remarkably, only 20% of HER2-positive tumors expressed both AR and ARA70, while 43% of HER2-negative tumors expressed both of these elements of the AR signaling pathway. This trend is consistent with a possible clinical relevance of the potential crosstalk between the HER2 and AR signaling pathways. Western blot analysis for ARA70 expression performed on frozen breast biopsies of normal or malignant breast tissue from four patients revealed a 70 kDa immunoreactive band in all four normal tissue samples, with an additional 35 kDa band in two of the breast cancer samples and in human breast cancer MCF-7 cells. This may reflect aberrant splicing in some breast cancers, leading to the emergence of the 35 kDa isoform.

CD8 T lymphocytes and macrophages infiltrate coronary artery aneurysms in acute Kawasaki disease.

The pathogenesis of coronary arterial inflammation in acute Kawasaki disease (KD) is unclear. To test the hypothesis that the KD vascular lesion is an activated T lymphocyte-dependent process, immunohistochemical studies were done on coronary artery aneurysms from 8 fatal acute KD cases by using antibodies to CD45RO (activated or memory T lymphocyte), CD8 (cytotoxic T lymphocyte), CD4 (helper T lymphocyte), HAM56 (macrophage), and CD20 (B lymphocyte). Acute KD coronary arteritis was characterized by transmural infiltration of CD45RO T lymphocytes with CD8 T lymphocytes predominating over CD4 T lymphocytes. Macrophages were present primarily in the adventitial layer; B lymphocytes were notably absent. These data lend support to the hypotheses that KD results from infection with an intracellular pathogen, such as a virus, whose antigens are presented by major histocompatibility complex class I molecules, and that CD8 T lymphocytes and macrophages are important in the pathogenesis of KD coronary aneurysms.

Perineural invasion of cutaneous malignancies.

BACKGROUND: Perineural invasion is an important mode of tumor spread and is associated with increased aggressiveness and a propensity for recurrence among cutaneous malignancies. OBJECTIVE: To review the pathogenesis, diagnosis, and treatment of cutaneous tumors exhibiting perineural invasion. METHODS: This article is based on a review of the medical literature concerning tumors with perineural involvement. RESULTS: This article describes the clinical signs and histologic features of cutaneous malignancies exhibiting perineural involvement. CONCLUSION: Appropriate patient care mandates consideration of perineural invasion in the evaluation of cutaneous tumors. As the majority of patients present without symptoms of neural involvement, physicians must be vigilant in the search for this type of tumor spread.

Glucagon-like peptide (GLP)-2 action in the murine central nervous system is enhanced by elimination of GLP-1 receptor signaling.

Glucagon-like peptide-2 (GLP-2) regulates energy homeostasis via effects on nutrient absorption and maintenance of gut mucosal epithelial integrity. The biological actions of GLP-2 in the central nervous system (CNS) remain poorly understood. We studied the sites of endogenous GLP-2 receptor (GLP-2R) expression, the localization of transgenic LacZ expression under the control of the mouse GLP-2R promoter, and the actions of GLP-2 in the murine CNS. GLP-2R expression was detected in multiple extrahypothalamic regions of the mouse and rat CNS, including cell groups in the cerebellum, medulla, amygdala, hippocampus, dentate gyrus, pons, cerebral cortex, and pituitary. A 1.5-kilobase fragment of the mouse GLP-2R promoter directed LacZ expression to the gastrointestinal tract and CNS regions in the mouse that exhibited endogenous GLP-2R expression, including the cerebellum, amygdala, hippocampus, and dentate gyrus. Intracerebroventricular injection of GLP-2 significantly inhibited food intake during dark-phase feeding in wild-type mice. Disruption of glucagon-like peptide-1 receptor (GLP-1R) signaling with the antagonist exendin-(9-39) in wild-type mice or genetically in GLP-1R(-)/- mice significantly potentiated the anorectic actions of GLP-2. These findings illustrate that CNS GLP-2R expression is not restricted to hypothalamic nuclei and demonstrate that the anorectic effects of GLP-2 are transient and modulated by the presence or absence of GLP-1R signaling in vivo.

Activation of androgen receptor-associated protein 70 (ARA70) mRNA expression in ovarian cancer.

OBJECTIVE: Androgens have been implicated in ovarian cancer and androgen receptor expression has been reported in 70-95% of ovarian adenocarcinomas, implying a role in ovarian cancer cell biology. Androgen receptor-associated protein 70 (ARA70) is a reported androgen receptor coactivator that enhances the transactivational potential of the androgen receptor up to 10-fold. Because ARA70 expression could amplify androgen action in ovarian cancer cells, we examined patient samples of ovarian cancer for ARA70 expression. METHODS: Twenty invasive ovarian carcinomas and four nonmalignant ovaries were tested for ARA70 mRNA expression by in situ hybridization using a 35S-labeled riboprobe. RESULTS: The probe was first assessed using a sample of human benign prostatic hyperplasia. Expression was restricted to cells within the epithelial glands, which are known to express the highest levels of androgen receptor. In the nonmalignant ovary, ARA70 mRNA was expressed in moderate levels in thecal cells associated with antral follicles, with less labeling observed in granulosa cells and stroma. The surface epithelium was negative for ARA70 transcripts, with only low levels observed in occasional cells. In contrast, a high level of ARA70 expression was observed in 17 of the 20 ovarian carcinomas of various histological types. Labeling was associated with the tumor cells while little if any ARA70 mRNA was observed in stromal cells associated with the carcinoma. CONCLUSION: These observations indicate that ARA70 expression is activated in invasive ovarian cancer tumor cells, and suggest that amplification of androgen action by ARA70 may be involved in the etiology/progression of this disease.

Studies of isopenicillin N synthase enzymatic properties using a continuous spectrophotometric assay.

Isopenicillin N synthase (IPNS) from Aspergillus nidulans is a no-heme iron(II)-dependent oxygenase which catalyses, in a single reaction, the bicyclisation of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine into isopenicillin N, the precursor of all other penicillins, cephalosporins and cephamycins. The IPNS reaction can be followed directly and continuously by a new assay which monitors the absorbance increase at 235 nm characteristic of penicillin nucleus formation. Using this assay, the effects of influential factors affecting the in vitro IPNS enzymatic reaction were investigated. Even under optimal conditions, enzyme inactivation occurred during catalysis. Iron(II) depletion and product inhibition were not the cause of this phenomenon, the addition of antioxidants or reducing agents failed to slow down inactivation or reactivate the enzyme. Therefore, this phenomenon appears to be irreversible and is attributed to oxidative damage caused to the enzyme by reactive oxygen species generated in solution during catalysis. Nevertheless, the steady-state kinetic parameters for the IPNS reaction were determined.

Complete spontaneous regression of Merkel cell carcinoma: a review of the 10 reported cases.

BACKGROUND: Merkel cell (neuroendocrine) carcinoma (MCC) is a very aggressive primary cutaneous neoplasm occurring most often on the head and neck of the elderly. Complete spontaneous regression (CSR) of MCC was first described in 1986. Since then other cases have been reported bringing the total to 10. OBJECTIVE: To review these 10 cases and obtain long-term follow-up data, to compare them for similarities and differences. METHOD: Each original case report was extensively reviewed and authors contacted in most cases for confirmation and updated information. RESULTS: In no case did MCC recur after CSR was noted, although follow-up information in some cases was short. When CSR occurred, it was swift and dramatic with complete regression of skin and lymph node metastasis in 1-3 months. CONCLUSION: While only 10 cases of CSR is a small number, MCC is itself a rare malignancy with just over 600 reported cases. Today most cases of MCC receive aggressive combined therapy effectively precluding diagnosis of CSR. The nature of regression in these 10 cases may point toward future immunologic therapy just as similar cases of CRS in patients with melanoma have led to advances in the immunologic treatment for that malignancy.

Prevalence and strain differentiation of Giardia intestinalis in calves in the Manawatu and Waikato regions of North Island, New Zealand.

Giardia intestinalis has been reported in newborn calves world-wide; however, information on the extent of G. intestinalis in New Zealand calves has to date been very limited. The current study attempted to establish the prevalence rate of G. intestinalis in calves up to 8 weeks old in New Zealand. More than 700 calf fecal specimens were collected during the spring calving seasons of 1998 and 1999 from two regions in North Island, New Zealand (Manawatu and Waikato) and tested for the presence of G. intestinalis. In addition to determining the presence of G. intestinalis in newborn calves, sequence analysis was performed using specific amplification primers developed to target a section of the ribosomal DNA (rDNA). This locus is considered to be rapidly evolving, and therefore, suitable for use in the elucidation of phylogenetic relationships between G. intestinalis isolates. Sequencing was performed using G. intestinalis DNA extracted from cysts collected directly from the calf fecal matter. There was no culturing of the G. intestinalis isolates either in vivo or in vitro. Over 40% of all collected calf fecal specimens contained G. intestinalis cysts and rDNA sequence analysis revealed two different sequences among calf isolates. These sequence differences were not found to correspond to a particular season, geographical region or farming practice. Preliminary phylogenetic analysis suggests that these two rDNA sequence types are indicative of calf hosts.

Down-regulation of transforming growth factor beta receptors by androgen in ovarian cancer cells.

Steroid hormones have been implicated in the etiology and/or progression of epithelial ovarian cancer. As ovarian surface epithelial cells are growth inhibited by transforming growth factor beta (TGF-beta), we tested whether steroid hormones could regulate the expression of TGF-beta1 or its receptors in ovarian cancer cells, as assessed by quantitative reverse transcription-PCR. Treatment of ovarian cancer HEY cells with 500 nM 5alpha-dihydrotestosterone (DHT), but not estradiol-17beta or progesterone, for 60 h down-regulated the expression of mRNA for TGF-beta receptors I and II (TbetaR-I and TbetaR-II), betaglycan, and endoglin but had no effect on TGF-beta1 mRNA levels. Androgen receptor (AR) mRNA expression in HEY cells was compared to other ovarian cancer cell lines. OVCAR-3 cells expressed AR mRNA levels similar to that of androgen-responsive LNCaP prostate cancer cells, whereas SKOV-3 and HEY cells expressed only 3 and 0.01%, respectively. Western blot analysis and saturation binding assays confirmed the expression of AR protein in these three cell lines, but at the limit of detection in SKOV-3 and HEY cells. Treatment of SKOV-3 and HEY cells for 24 h with 1-50 nM DHT resulted in a dose-dependent down-regulation of TbetaR-II mRNA. The AR antagonist hydroxyflutamide did not reverse the effect of DHT on SKOV-3 cells but by itself down-regulated TbetaR-II mRNA. This apparent androgen-mimetic action of hydroxyflutamide and the ability of SKOV-3 and HEY cells to respond to DHT may be due to their expression of AR-associating protein 70, an AR co-activator reported to amplify AR transactivation and to result in agonist activity of AR antagonists. DHT was able to reverse TGF-beta1 growth-inhibitory action in SKOV-3 cells and in a primary culture of ovarian cancer cells derived from ascites. Thus, androgens may promote ovarian cancer progression in part by decreasing TGF-beta receptor levels, thereby allowing ovarian cancer cells to escape TGF-beta1 growth inhibition.

A new disorder of hyaluronan metabolism associated with generalized folding and thickening of the skin.

OBJECTIVE: To describe and characterize a new disorder of hyaluronan metabolism associated with marked abnormalities of cutaneous tissue and to determine whether a relationship with a phenotypically similar disorder in the shar-pei dog exists. METHODS: Biopsy specimens of the skin of a child with extreme cutaneous thickening and folding were examined by light and electron microscopy. The concentration of hyaluronan and the activity of hyaluronidase were measured in the patient's serum and plasma, respectively, and the activity of hyaluronan synthase was examined in cultured dermal fibroblasts. Hyaluronan concentration was also measured in the plasma of 23 shar-pei and 34 control dogs. RESULTS: The patient's skin displayed gross accumulation of hyaluronan, and the serum concentration of hyaluronan was markedly elevated (up to 3100 microg/L) during infancy. Hyaluronan synthase activity of cultured dermal fibroblasts was increased, whereas hyaluronidase activity in plasma was normal (5.5 +/- 0.08 IU/L). Plasma hyaluronan concentration was higher in the shar-pei dogs than in control dogs (median, 378 microg/L vs 73 microg/L, respectively). CONCLUSION: The child we describe has a novel disorder of hyaluronan metabolism, which appears to result from abnormal control of hyaluronan synthesis. An analogous disorder may be present in the shar-pei dog.