Low oxygen tension enhances hepatitis C virus replication.

Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infection-based assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-α) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCV-host cell interaction.

Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.

Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R.

Evaluation of the efficacy of pelvic shielding in preadolescent girls.

SUMMARY: A standing anteroposterior pelvic radiograph with gonadal shielding is used as a screening tool for all patients evaluated for intoeing at our institution. Sixty-two normal consecutive screening pelvic radiographs obtained in 61 female patients between the ages of 4 and 6 years were evaluated. Radiographs were evaluated for the adequacy to assess the hips as well as the protection afforded the ovaries from radiation exposure. Radiographs were judged to be inadequate because the shield covered essential landmarks in at least one hip in eight radiographs (13%). Five radiographs (8%) covered >50% of the area of both ovaries, and only one radiograph covered >75% of the area of both ovaries. Standard techniques of positioning gonadal shields in preadolescent girls are inadequate and provide minimal protection with a high rate of interference with vital landmarks. We no longer advocate using gonadal shields on initial screening radiographs of preadolescent girls.

Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Sequencing of the CYP11B1 gene showed two new base substitutions, a conservative 954 G-->C transversion at the last base of exon 5 (T318T), and a IVS8 + 4A-->G transition in intron 8. In addition, two polymorphisms were found in exons 1 and 2. The genetically female patient was raised as a male because of severe pseudohermaphroditism. Glucocorticoid-suppressive treatment encountered difficulties in equilibration and compliance, resulting in uncontrolled hypertension with pronounced hypertrophic cardiomyopathy. At 42 yr of age the occurrence of central retinal vein occlusion with permanent loss of left eye vision led to the decision to perform bilateral laparoscopic adrenalectomy. Surgery was followed by normalization of blood pressure and good compliance with glucocorticoid and androgen substitutive therapies. In vitro, adrenal cells in culture and isolated mitochondria showed extremely low 11beta-hydroxylase activity. Analysis of adrenal CYP11B1 messenger ribonucleic acid (mRNA) by RT-PCR and sequencing showed the expression of a shorter mRNA that lacked exon 8 and did not contain either the exon 5 mutation or the exon 1 and 2 polymorphisms. This suggested that one CYP11B1 allele carried the intron 8 mutation, responsible for skipping exon 8. The other allele carried the exon 5 mutation, and its mRNA was not detectable. Western blot analysis showed weak expression of a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus, bilateral adrenalectomy in this patient allowed effective treatment of severe hypertension and helped in understanding the mechanisms and physiopathological consequences of two novel mutations of CYP11B1.

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Expression and regulation of melanocortin receptor-5 (MC5-R) in the bovine adrenal cortex.

Among the five members of the melanocortin receptor (MC-R) family, MC2 and MC5 are expressed in peripheral tissues. The receptor MC2 (ACTH receptor) almost exclusively expressed in the adrenal cortex whereas MC5-R is expressed in several organs including the adrenal cortex. Both receptors bind ACTH and activate adenylate cyclase. The aim of this work was to study the spatial distribution of MC5-R among the different zones of the bovine adrenal cortex and to analyze the regulation of its expression by its own ligands, ACTH and alpha-MSH and by angiotensin II (AII). Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and RNase protection assay, MC5-R was detected only in the glomerulosa zone whereas MC2-R was present in both glomerulosa and fasciculata zones of adult adrenal cortex. Treatments by ACTH, alpha-MSH, or AII increased the MC5-R mRNA level in glomerulosa cells by factors 7, 5, and 4.5, respectively. However, although potentially regulated by hormones, MC5-R is expressed at a level at least 100 times less than MC2-R, suggesting that MC5-R expression might only be at trace levels in grown adults, but could be much higher during embryogenesis.

Cushing's syndrome due to a gastric inhibitory polypeptide-dependent adrenal adenoma: insights into hormonal control of adrenocortical tumorigenesis.

We studied a patient with food-induced, ACTH-independent, Cushing's syndrome and a unilateral adrenocortical adenoma. In vivo cortisol secretion was stimulated by mixed, glucidic, lipidic, or proteic meals. Plasma ACTH levels were undetectable, but iv injection of ACTH stimulated cortisol secretion. Unilateral adrenalectomy was followed by hypocortisolism with loss of steroidogenic responses to both food and ACTH. In vitro, cortisol secretion by isolated tumor cells was stimulated by the gut hormone gastric inhibitory polypeptide (GIP) and ACTH, but not by another gut hormone, glucagon-like peptide-1 (GLP-1). Both peptides stimulated the production of cAMP but not of inositol 1,4,5-trisphosphate. In quiescent cells, GIP and ACTH stimulated [3H]thymidine incorporation and p42-p44 mitogen-activated protein kinase activity. GIP receptor messenger ribonucleic acid (RNA), assessed by RT-PCR, was highly expressed in the tumor, whereas it was undetectable in the adjacent hypotrophic adrenal tissue, in two adrenal tumors responsible for food-independent Cushing's syndrome, and in two hyperplastic adrenals associated with ACTH hypersecretion. In situ hybridization demonstrated that expression of GIP receptor RNA was confined to the adrenocortical tumor cells. Low levels of ACTH receptor messenger RNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase in cAMP that may participate in the stimulation of both cortisol secretion and proliferation of the tumor cells.

Expression of ACTH receptors (MC2-R and MC5-R) in the glomerulosa and the fasciculata-reticularis zones of bovine adrenal cortex.

The recent cloning of a family of melanocortin receptors (MC-R) has identified five distinct G protein- and adenylate cyclase-coupled receptors. The MC2-receptor (MC2-R) preferentially binds ACTH. It is expressed in the adrenal cortex and is hence considered to be the ACTH receptor. The MC5-receptor (MC5-R) binds ACTH and alpha-MSH and is more widely expressed. The aim of this work was to study the sites of MC5-R expression in the bovine adrenal cortex and to compare the regulation of the expression of MC2-R and MC5-R in bovine adrenocortical cells in primary culture. Analysis of the expression of MC5-R was obtained by RT-PCR, using total RNA purified from glomerulosa and fasciculata zones of bovine adrenocortical tissue. MC5-R expression could be detected in RNA from the glomerulosa zone but was undetectable in the fasciculata zone. In bovine adrenocortical cells in culture, ACTH stimulates MC5-R expression in the glomerulosa and fasciculata cells. A DNA fragment, was obtained using primers based on the bovine ACTH receptor (MC2-R) sequence. This fragment was detected in RNA from the two zones. The probe was used to quantify MC2-R by Ribonuclease Protection assay and we observed that MC2-R mRNA is 3.6-fold more abundant in glomerulosa than in fasciculata-reticularis cells.

Gastric inhibitory polypeptide (GIP) stimulates cortisol secretion, cAMP production and DNA synthesis in an adrenal adenoma responsible for food-dependent Cushing's syndrome.

We studied in vitro an adrenal tumor responsible for food-dependent, ACTH independent, Cushing's's syndrome. Cortisol secretion by isolated tumor cells was stimulated by GIP and ACTH, but not by the gut hormone glucagon-like peptide-1 (GLP-1). Both GIP and ACTH stimulated production of cAMP but not inositol 1,4,5-trisphosphate IP3). In quiescent tumor cells, GIP and ACTH stimulated [3H]-thymidine incorporation and p42-p44 MAP kinase activity. In normal human adrenocortical cells cortisol secretion and [3H]-thymidine incorporation were stimulated by ACTH but not by GIP. GIP receptor mRNA, assessed by RT-PCR, was highly expressed in the tumor, but undetectable in the adjacent hypotrophic adrenal tissue, in a normal adrenal, in two adrenal tumors responsible for food-independent Cushing's syndrome and in two hyperplastic adrenals associated with ACTH hypersecretion. Low levels of ACTH receptor mRNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase of cAMP that may participate in stimulation of both cortisol secretion and proliferation of the tumor cells.

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells.

Angiotensin II (ANG II) has long been known for its pressor and growth-promoting effects, which are both mediated by the AT1 receptor. By contrast, the AT2 receptor has recently been reported to mediate inhibition of proliferation through as yet undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the cells in G1 phase. Consistent with this, ANG II inhibits cyclin D1 expression and cyclin D1-associated kinase activity. The antimitogenic effect of ANG II is partly mimicked by the AT2-selective agonist CGP-42112. It is also blocked partly and in an additive fashion by the AT1- and AT2-selective antagonists losartan and PD-123319, indicating the contribution of both receptor subtypes to this response. AT1-dependent antiproliferation is selectively blocked by the cyclooxygenase inhibitor indomethacin and restored by prostaglandin E2, whereas AT2-receptor-mediated inhibition of growth is suppressed by the tyrosine phosphatase inhibitors orthovanadate and bpV(pic). Both pathways are, however, pertussis toxin sensitive. We hypothesize that, in fasciculata cells, the AT1 receptor inhibits bFGF-induced proliferation by stimulating prostaglandin synthesis, whereas the AT2 receptor mediates its effect through a pathway that requires protein tyrosine phosphatase activation.

Extensive nodular sarcoidosis in the hand.

We report an atypical case of nodular sarcoidosis involving both hands. The pattern of extensive involvement of all digits with lesions extending into the pulp spaces has not been reported previously. The diagnosis of sarcoidosis should be considered even in patients presenting with clinically uncharacteristic manifestations.

ACTH angiotensin II and TGF beta participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells.

Bovine zona glomerulosa cells, on the first day of culture, produce aldosterone as their major steroid with no detectable cortisol secretion. Continuous incubation with ACTH had no effect on aldosterone production nor on aldosterone synthase activity. This treatment resulted in a dose and time dependent rise in 17 alpha-hydroxylase activity, in parallel with an increase in cytochrome P-450(17 alpha) (CYP17) protein and mRNA. We have previously shown that TGF beta 1 is a potent inhibitor of differentiated functions of bovine fasciculata-reticularis cells and that CYP17 and AII receptors are the major targets explaining this effect. The present study examined whether 17 alpha-hydroxylase activity in glomerulosa cells could be regulated by angiotensin II (AII) and transforming growth factor-beta 1 (TGF beta 1). AII inhibits the induction of CYP17 by ACTH in a dose dependent manner. TGF beta 1 also blocks almost completely the stimulatory effect of ACTH. In order to suppress the endogenous action of TGF beta 1, incubations were performed with an anti-TGF beta antibody. This specific antibody induces the expression of CYP17 resulting in increased activity and mRNA levels. These results show that AII is able to modulate the expression of CYP17 in adrenal glomerulosa cells following ACTH stimulation. Furthermore, TGF beta 1 exerts an autocrine effect on the differentiation of glomerulosa cells through a regulatory loop repressing CYP17 activity.