HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype

Some rare HIV-1-infected individuals, referred to as HIV controllers (HIC), have persistently undetectable plasma viral load in the absence of therapy. This control of HIV-1 replication has been associated with a strong, multifunctional specific CD8(+) T cell response. However, no direct link between this immune response and the control of viremia has so far been provided. We investigated parameters of specific CD8(+) T cell response and in vitro susceptibility to HIV-1 infection in 11 HIC. We found high frequencies of HIV-specific CD8(+) T cells. Interestingly, these cells expressed the activation marker HLA-DR but not CD38. This unique phenotype differentiates HIV-specific CD8(+) T cells from HIC and noncontroller subjects and likely reflects a high potential to expand upon exposure to antigen and a capacity to exert effector functions. Accordingly, although CD4(+) T cells from HIC were fully susceptible to HIV-1 superinfection, their CD8(+) T cells effectively suppressed HIV-1 infection. Remarkably, this potent anti-HIV activity was observed without prior stimulation of CD8(+) T cells. This activity was not mediated by secreted inhibitory factors but was due to the elimination of infected CD4(+) T cells and was observed only with autologous CD4(+) T cells, indicating an HLA-restricted cytotoxic mechanism. This constitutive antiviral capacity of CD8(+) T cells could account for the control of viral replication in HIC.     

Regulators of G-protein signaling 4 in adrenal gland: localization, regulation, and role in aldosterone secretion

Regulators of G-protein signaling (RGS proteins) interact with Galpha subunits of heterotrimeric G-proteins, accelerating the rate of GTP hydrolysis and finalizing the intracellular signaling triggered by the G-protein-coupled receptor (GPCR)-ligand interaction. Angiotensin II (Ang II) interacts with its GPCR in adrenal zona glomerulosa cells and triggers a cascade of intracellular signals that regulates steroidogenesis and proliferation. On screening for adrenal zona glomerulosa-specific genes, we found that RGS4 was exclusively localized in the zona glomerulosa of the rat adrenal cortex. We studied RGS4 expression and regulation in the rat adrenal gland, including the signaling pathways involved, as well as the role of RGS4 in steroidogenesis in human adrenocortical H295R cells. We reported that RGS4 mRNA expression in the rat adrenal gland was restricted to the adrenal zonal glomerulosa and upregulated by low-salt diet and Ang II infusion in rat adrenal glands in vivo. In H295R cells, Ang II caused a rapid and transient increase in RGS4 mRNA levels mediated by the calcium/calmodulin/calmodulin-dependent protein kinase and protein kinase C pathways. RGS4 overexpression by retroviral infection in H295R cells decreased Ang II-stimulated aldosterone secretion. In reporter assays, RGS4 decreased Ang II-mediated aldosterone synthase upregulation. In summary, RGS4 is an adrenal gland zona glomerulosa-specific gene that is upregulated by aldosterone secretagogues, in vivo and in vitro, and functions as a negative feedback of Ang II-triggered intracellular signaling. Alterations in RGS4 expression levels or functions may be involved in deregulations of Ang II signaling and abnormal aldosterone secretion.     

Angiotensin II-mediated protein kinase D activation stimulates aldosterone and cortisol secretion in H295R human adrenocortical cells

Protein kinases are important mediators in intracellular signaling. Angiotensin II is the most important modulator of adrenal zona glomerulosa cell physiology. Angiotensin II regulates steroidogenesis and proliferation among many other metabolic processes. H295R human adrenal cells are a widely used experimental model to study adrenal cell physiology and metabolism. We screened for protein kinase expression levels using the Kinetwork system in H295R cells after 3 h angiotensin II treatment. Protein kinase D (PKD) was the protein kinase that suffers the most dramatic changes. PKD is a member of a new class of serine/threonine protein kinases that is activated by phosphorylation. Our studies indicated that angiotensin II time- and dose-dependently increased PKD phosphorylation, which occurred within 2 min of angiotensin II treatment and at concentrations as low as 1 nm. PKD phosphorylation was also dose-dependently increased by the PKC activator phorbol 12-myristate 13-acetate. Angiotensin II-mediated PKD phosphorylation was blocked by several PKC inhibitors. Furthermore, PKCepsilon translocation inhibitor peptide decreased angiotensin II-mediated PKD phosphorylation, and PKCepsilon down-regulation by RNA interference also decreased PKD phosphorylation mediated by angiotensin II. Cotransfection of constitutively active PKD mutant constructs up-regulated aldosterone synthase and 11beta-hydroxylase expression in reporter assays. Constitutively active PKD mutants increased aldosterone and cortisol secretion under angiotensin II stimulatory conditions. This study reveals that PKD is an intracellular signaling mediator of angiotensin II regulation of steroidogenesis in human adrenal cells. These data provide new insights into the molecular mechanisms involved in angiotensin II-induced physiological and pathophysiological events in adrenal cells.     

Development of a panel of monoclonal antibodies against the mineralocorticoid receptor

Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1-18, 64-82, 79-97, and 365-381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.     

Urinary 18-hydroxycortisol and its relationship to the excretion of other adrenal steroids

The urinary excretion of 18-hydroxycortisol was recently reported to be increased in patients with primary aldosteronism who have an adrenal adenoma and in those with glucocorticoid-suppressible aldosteronism. A direct RIA for 18-hydroxycortisol in urine and plasma has been described, and we here report our experience using a similar direct RIA and a more elaborate RIA which includes a preliminary high pressure liquid chromatography (HPLC) purification step. The urinary excretion of 18-hydroxycortisol was compared with that of other adrencorticoids. The urinary excretion of 18-hydroxycortisol in 37 normal subjects using the direct RIA was 112 +/- 49 (+/- SD) microgram/24 h, and that with the HPLC-RIA method was 63 +/- 36 micrograms/24 h. The accuracy and specificity of the HPLC-RIA assay method were confirmed by measuring the steroid after the HPLC step as the glycolic acid ester derivative. The urinary excretion of 18-hydroxycortisol correlated with that of cortisol (r = 0.36; P less than 0.01), 18-oxocortisol (r = 0.42; P less than 0.01), and 19-nordeoxycortisosterone (r = 0.71; P less than 0.001), but did not correlate with the excretion of aldosterone 18-oxoglucuronide (r = 0.25; P = 0.15942). Dexamethasone administration to five normal subjects significantly decreased 18-hydroxycortisol excretion from 81 +/- 47 to 23 +/- 8 micrograms/24 h. ACTH infusion in these subjects receiving dexamethasone significantly raised 18-hydroxycortisol excretion to 147 +/- 37 micrograms/24 h. Five days of a sodium-restricted diet (10 mmoles/day) resulted in a significant (P less than 0.02) increase in 18-hydroxycortisol excretion, but two of eight subjects had decreased excretion, although urinary aldosterone excretion increased, as expected. These studies demonstrate that the direct RIA significantly overestimates urinary 18-hydroxycortisol excretion. These studies also demonstrate that the major factor resulting 18-hydroxycortisol excretion is ACTH. However, since 18-hydroxycortisol excretion may increase during sodium depletion, angiotensin or other factors may also regulate its secretion.     

Increased diagnosis of primary aldosteronism, including surgically correctable forms, in centers from five continents

Primary aldosteronism (PA) is a common form of endocrine hypertension previously believed to account for less than 1% of hypertensive patients. Hypokalemia was considered a prerequisite for pursuing diagnostic tests for PA. Recent studies applying the plasma aldosterone/plasma renin activity ratio (ARR) as a screening test have reported a higher prevalence. This study is a retrospective evaluation of the diagnosis of PA from clinical centers in five continents before and after the widespread use of the ARR as a screening test. The application of this strategy to a greater number of hypertensives led to a 5- to 15-fold increase in the identification of patients affected by PA. Only a small proportion of patients (between 9 and 37%) were hypokalemic. The annual detection rate of aldosterone-producing adenoma (APA) increased in all centers (by 1.3-6.3 times) after the wide application of ARR. Aldosterone-producing adenomas constituted a much higher proportion of patients with PA in the four centers that employed adrenal venous sampling (28-50%) than in the center that did not (9%). In conclusion, the wide use of the ARR as a screening test in hypertensive patients led to a marked increase in the detection rate of PA.     

Culture of autologous bone marrow in diffusion chambers: effect of host irradiation

Autologous bone marrow (BM) cells were cultured in diffusion chambers (DC) implanted into whole-body irradiated, non-irradiated, or sham- irradiated goats. Proliferation was apparent in DC implanted in both irradiated and nonirradiated goats. However, cells in DC cultured in irradiated hosts increased in number beginning earlier, proceeded at a faster rate, and reached higher numbers than in DC in nonirradiated hosts. Growth enhancement could not have occurred as a result of radiation-induced immunosuppression in autologous hosts. The nonirradiated "target cells" in the DC therefore constituted an indicator system for stimulatory or inhibitory substances in the host. The simultaneous increase in the number of granulocytes in peripheral blood and in DC of irradiated hosts was paralleled by an initial rise in serum colony-stimulating factor (CSF). A second, prolonged period of severe granulocytopenia following irradiation of the host correlated with high levels of serum CSF. Increased numbers of megakaryocytes were seen in DC as thrombocytopenia developed in the irradiated host. DC erythropoiesis disappeared rapidly in nonirradiated goats; however, in DC of irradiated goats, the number of erythrocytic precursors increased exponentially during ablation of host erythroid marrow. Anemia did not develop in the host during the culture period. Proliferation of mononuclear cells in DC was markedly stimulated by irradiation of the host. Proliferation of macrophages appeared independent of host treatment. These observations provide strong evidence for diffusion of specific and/or nonspecific humoral hematopoietic stimulators from the host into the DC. This stimulation appears to be elicited and/or intensified by irradiation of the host.     

Intraductal papillary-mucinous tumors of the pancreas: Clinicopathologic features, outcome, and nomenclature. Members of the Pancreas Clinic, and Pancreatic Surgeons of Mayo Clinic

BACKGROUND & AIMS: Intraductal papillary-mucinous tumor (IPMT) of the pancreatic ducts is increasingly recognized. This study investigated if clinical, imaging, or, histological features predicated outcome, formulated a treatment algorithm, and clarified relationships among IPMT, mucinous cystic neoplasms of the pancreas (MCN), and chronic pancreatitis. METHODS: The medical records, radiographs, and pathological specimens of 15 patients with IPMT (dilated main pancreatic duct or branch ducts with mucin overproduction) who were evaluated between October 1983 and January 1994 were reviewed. RESULTS: One patient had hepatic metastases. Fourteen underwent an operation (6 distal pancreatectomy, 4 total pancreatectomy, and 4 pancreaticoduodenectomy); all had dysplastic intraductal epithelium and chronic pancreatitis, whereas 3 had invasive adenocarcinoma. After a median of 25 months, 10 patients were alive; 3 of 4 with malignant and 2 of 11 with benign IPMT died (P < 0.05). Patients with or without carcinoma had similar clinical and radiographic features. A clinical diagnosis of chronic pancreatitis had been made in 9 patients with benign IMPT and in none with malignant IPMT (P < 0.05). CONCLUSIONS: IPMT is a dysplastic and likely precancerous lesion that is frequently diagnosed as chronic pancreatitis and is separate from MCN. Because it is not possible to distinguish noninvasive from invasive IPMT preoperatively, complete surgical excision of the dysplastic process is our treatment of choice whenever appropriate. (Gastroenterology 1996 Jun;110(6):1909-18)     

The Potassium Channel, Kir3.4 Participates in Angiotensin II-Stimulated Aldosterone Production by a Human Adrenocortical Cell Line

Angiotensin II (A-II) regulation of aldosterone secretion is initiated by inducing cell membrane depolarization, thereby increasing intracellular calcium and activating the calcium calmodulin/calmodulin kinase cascade. Mutations in the selectivity filter of the KCNJ5 gene coding for inward rectifying potassium channel (Kir)3.4 has been found in about one third of aldosterone-producing adenomas. These mutations result in loss of selectivity of the inward rectifying current for potassium, which causes membrane depolarization and opening of calcium channels and activation of the calcium calmodulin/calmodulin kinase cascade and results in an increase in aldosterone secretion. In this study we show that A-II and a calcium ionophore down-regulate the expression of KCNJ5 mRNA and protein. Activation of Kir3.4 by naringin inhibits A-II-stimulated membrane voltage and aldosterone secretion. Overexpression of KCNJ5 in the HAC15 cells using a lentivirus resulted in a decrease in membrane voltage, intracellular calcium, expression of steroidogenic acute regulatory protein, 3-β-hydroxysteroid dehydrogenase 3B2, cytochrome P450 11B1 and cytochrome P450 11B2 mRNA, and aldosterone synthesis. In conclusion, A-II appears to stimulate aldosterone secretion by depolarizing the membrane acting in part through the regulation of the expression and activity of Kir3.4.