Human adrenal corticocarcinoma NCI-H295R cells produce more androgens than NCI-H295A cells and differ in 3beta-hydroxysteroid dehydrogenase type 2 and 17,20 lyase activities

The human adrenal cortex produces mineralocorticoids, glucocorticoids, and androgens in a species-specific, hormonally regulated, zone-specific, and developmentally characteristic fashion. Most molecular studies of adrenal steroidogenesis use human adrenocortical NCI-H295A and NCI-H295R cells as a model because appropriate animal models do not exist. NCI-H295A and NCI-H295R cells originate from the same adrenocortical carcinoma which produced predominantly androgens but also smaller amounts of mineralocorticoids and glucocorticoids. Research data obtained from either NCI-H295A or NCI-H295R cells are generally compared, although for the same experiments no direct comparison between the two cell lines has been performed. Therefore, we compared the steroid profile and the expression pattern of important genes involved in steroidogenesis in both cell lines. We found that steroidogenesis differs profoundly. NCI-H295A cells produce more mineralocorticoids, whereas NCI-H295R cells produce more androgens. Expression of the 3beta-hydroxysteroid dehydrogenase (HSD3B2), cytochrome b5, and sulfonyltransferase genes is higher in NCI-H295A cells, whereas expression of the cytochrome P450c17 (CYP17), 21-hydroxylase (CYP21), and P450 oxidoreductase genes does not differ between the cell lines. We found lower 3beta-hydroxysteroid dehydrogenase type 2 but higher 17,20-lyase activity in NCI-H295R cells explaining the 'androgenic' steroid profile for these cells and resembling the zona reticularis of the human adrenal cortex. Both cell lines were found to express the ACTH receptor at low levels consistent with low stimulation by ACTH. By contrast, both cell lines were readily stimulated by 8Br-cAMP. The angiotensin type 1 receptor was highly expressed in NCI-H295R than NCI-H295A cells and angiotensin II stimulated steroidogenesis in NCI-H295R but not NCI-H295A cells. Our data suggest that comparative studies between NCI-H295A and NCI-H295R cells may help find important regulators of mineralocorticoid or androgen biosynthesis.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection

BACKGROUND: CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays. METHODOLOGY/PRINCIPAL FINDINGS: Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C. CONCLUSIONS/SIGNIFICANCE: During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Autonomic cardiovascular regulation in obesity

Obese persons suffer from an increased mortality risk supposedly due to cardiovascular disorders related to either continuously lowered parasympathetic or altered sympathetic activation. Our cross-sectional correlation study establishes the relationship between obesity and autonomic regulation as well as salivary cortisol levels. Three patient cohorts were sampled, covering ranges of body mass index (BMI) of 27-32 (n=17), 33-39 (n=13) and above 40 kg/m(2)(n=12), and stratified for age, sex and menopausal status. Autonomic cardiovascular regulation was assessed by use of heart rate variability and continuous blood pressure recordings. Spectral analytical calculation (discrete Fourier transformation) yields indices of sympathetic and parasympathetic activation and baroreflex sensitivity. Morning salivary cortisol was concurrently collected. Contrary to expectation, BMI and waist/hip ratio (WHR) were inversely correlated with sympathetic activity. This was true for resting conditions (r=-0.48, P<0.001; r=-0.33, P<0.05 for BMI and WHR respectively) and for mental challenge (r=-0.42, P<0.01 for BMI). Resting baroreflex sensitivity was strongly related to the degree of obesity at rest (BMI: r=-0.35, P<0.05) and for mental challenge (r=-0.53, P<0.001). Salivary cortisol correlated significantly with waist circumference (r=-0.34, P=0.05). With increasing weight, no overstimulation was found but a depression in sympathetic and parasympathetic activity together with a significant reduction in baroreflex functioning and in salivary cortisol levels.

Practice and perception--a nationwide survey of therapy habits in sepsis

OBJECTIVE: To simultaneously determine perceived vs. practiced adherence to recommended interventions for the treatment of severe sepsis or septic shock. DESIGN: One-day cross-sectional survey. SETTING: Representative sample of German intensive care units stratified by hospital size. PATIENTS: Adult patients with severe sepsis or septic shock. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Practice recommendations were selected by German Sepsis Competence Network (SepNet) investigators. External intensivists visited intensive care units randomly chosen and asked the responsible intensive care unit director how often these recommendations were used. Responses "always" and "frequently" were combined to depict perceived adherence. Thereafter patient files were audited. Three hundred sixty-six patients on 214 intensive care units fulfilled the criteria and received full support. One hundred fifty-two patients had acute lung injury or acute respiratory distress syndrome. Low-tidal volume ventilation < or = 6 mL/kg/predicted body weight was documented in 2.6% of these patients. A total of 17.1% patients had tidal volume between 6 and 8 mL/kg predicted body weight and 80.3% > 8 mL/kg predicted body weight. Mean tidal volume was 10.0 +/- 2.4 mL/kg predicted body weight. Perceived adherence to low-tidal volume ventilation was 79.9%. Euglycemia (4.4-6.1 mmol/L) was documented in 6.2% of 355 patients. A total of 33.8% of patients had blood glucose levels < or = 8.3 mmol/L and 66.2% were hyperglycemic (blood glucose > 8.3 mmol/L). Among 207 patients receiving insulin therapy, 1.9% were euglycemic, 20.8% had blood glucose levels < or = 8.3 mmol/L, and 1.0% were hypoglycemic. Overall, mean maximal glucose level was 10.0 +/- 3.6 mmol/L. Perceived adherence to strict glycemic control was 65.9%. Although perceived adherence to recommendations was higher in academic and larger hospitals, actual practice was not significantly influenced by hospital size or university affiliation. CONCLUSIONS: This representative survey shows that current therapy of severe sepsis in German intensive care units complies poorly with practice recommendations. Intensive care unit directors perceive adherence to be higher than it actually is. Implementation strategies involving all intensive care unit staff are needed to overcome this gap between current evidence-based knowledge, practice, and perception.

[Study protocol of the VISEP study. Response of the SepNet study group]

In the commentary by Zander et al. the authors appear concerned about the methods and results of our, at that time, unpublished sepsis trial evaluating hydroxyethyl starch (HES) and insulin therapy. Unfortunately, the authors' concerns are based on false assumptions about the design, conduct and modes of action of the compounds under investigation. For instance, in our study the HES solution was not used for maintenance of daily fluid requirements, so that the assumption of the authors that this colloid was used "exclusively" is wrong. Moreover, the manufacturer of Hemohes, the HES product we used, gives no cut-off value for creatinine, thus the assumption that this cut-off value was "doubled" in our study is also incorrect. Other claims by the authors such as that lactated solutions cause elevated lactate levels, iatrogenic hyperglycemia and increase O(2) consumption are unfounded. There is no randomized controlled trial supporting such a claim - this claim is neither consistent with our study data nor with any credible published sepsis guidelines or with routine practice worldwide. We fully support open scientific debate. Our study methods and results have now been published after a strict peer-reviewing process and this data is now open to critical and constructive reviewing. However, in our opinion this premature action based on wrong assumptions and containing comments by representatives of pharmaceutical companies does not contribute to a serious, unbiased scientific discourse.

Subarachnoid hemorrhage and intracerebral hematoma: incidence, prognostic factors, and outcome

OBJECTIVE: To analyze the incidence and impact of an intracerebral hematoma (ICH) on treatment and outcome in patients with aneurysmal subarachnoid hemorrhage. METHODS: Data of 585 consecutive patients with subarachnoid hemorrhage from June 1999 to December 2005 were prospectively entered in a database. ICH was diagnosed and size was measured by computed tomographic scan before aneurysm occlusion. Fifty patients (8.5%) presented with an ICH larger than 50 cm3. The treatment decision (coil, clip, or hematoma evacuation) was based on an interdisciplinary approach. Patients were stratified into good (Hunt and Hess Grades I-III) versus poor (Hunt and Hess Grades IV and V) grade, and outcome was assessed according to the modified Rankin Scale at 6 months. RESULTS: Overall, 358 patients presented in good grade, with 4 of them having ICH (1.1%); and 227 patients presented in poor grade, with 46 of them having ICH (20.3%, P < 0.01). In good-grade patients with an ICH (n = 4), a favorable outcome (modified Rankin Scale score of 0-2) was achieved in 1 patient (25%), and in 246 patients (75%) without an ICH (P = 0.053; odds ratio, 0.11). A favorable outcome was achieved in 5 poor-grade patients (12.8%) with an ICH and in 40 patients (23.7%) without an ICH (P = 0.19; odds ratio, 0.47). Time to treatment was significantly shorter in patients with an ICH than without an ICH (median, 7 versus 26 h; P < 0.001) and shortest in patients with favorable outcome (3.5 hours; P < 0.01). CONCLUSION: The current data confirm that the presence of an ICH is a predictor of unfavorable outcome. However, despite large ICHs, a significant number of patients have a good outcome. To achieve a favorable outcome, ultra-early treatment with hematoma evacuation and aneurysm obliteration seems to be mandatory.

RISK OF SHUNT-DEPENDENT HYDROCEPHALUS AFTER OCCLUSION OF RUPTURED INTRACRANIAL ANEURYSMS BY SURGICAL CLIPPING OR ENDOVASCULAR COILING: A SINGLE-INSTITUTION SERIES AND META-ANALYSIS

OBJECTIVE: To compare the risk of shunt-dependent hydrocephalus after treatment of ruptured intracranial aneurysms by clipping versus coiling. METHODS: We analyzed 596 patients prospectively added to our database from July of 1999 to November of 2005 concerning the risk of shunt dependency after clipping versus coiling. Factors analyzed included age; sex; Hunt and Hess grade; Fisher grade; acute hydrocephalus; intraventricular hemorrhage; angiographic vasospasm; and number, size, and location of aneurysms. In addition, a meta-analysis of available data from the literature was performed identifying four studies with quantitative data on the frequency of clip, coil, and shunt dependency. RESULTS: The institutional series revealed Hunt and Hess grade, Fisher grade, acute hydrocephalus, intraventricular hemorrhage, and angiographic vasospasm as significant (P < 0.05) risk factors for shunt dependency after a univariate analysis. In a multivariate logistic regression analysis, we isolated intraventricular hemorrhage, acute hydrocephalus, and angiographic vasospasm as independent, significant risk factors for shunt dependency. The meta-analysis, including the current data, revealed a significantly higher risk for shunt dependency after coiling than after clipping (P = 0.01). CONCLUSION: Clipping of a ruptured aneurysm may be associated with a lower risk for developing shunt dependency, possibly by clot removal. This might influence long-term outcome and surgical decision making.