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PubMed1985

Rats homozygous for the grc complex have defective transport of androgen-binding protein to the epididymis, but normal secretion into the blood.

Male rats homozygous for the growth and reproduction complex (grc), which is closely linked to the major histocompatibility complex, are sterile due to a uniform arrest of spermatogenesis after formation of primary spermatocytes. The present study was conducted to determine whether grc influenced the secretion of the Sertoli cell product androgen-binding protein (rABP) in two F2 hybrid crosses. One was an F2 hybrid of the R10 and ACP strains and the other was an F2 hybrid population derived from the R16 and BI strains. These crosses were chosen because they allowed examination of the effects of grc on rABP secretion when this gene complex was associated with two different major histocompatibility complex backgrounds. In both crosses the rABP content of testes and epididymides from rats homozygous for grc were markedly reduced whereas heterozygotes were normal. By contrast, the concentration of rABP in plasma was normal or near normal at all ages. These observations are compatible with the hypothesis that grc influences the secretion of rABP from the apical portion of Sertoli cells into the tubular lumen and its subsequent transport to the epididymis, but has little or no effect on rABP secretion from the base of Sertoli cells into the blood. In other studies we have shown that infertile animals that have received prenatal x-ray or that are heterozygous for the Hre gene have patterns of rABP secretion similar to that of grc/grc homozygotes. It is therefore possible that the abnormal rABP secretion into tubular lumen in these animals is secondary to germ cell depletion, which is a factor common to grc/grc, Hre/+, and x-ray-treated rats.

Androgen-Binding Protein↗
PubMed1983

Minimal change glomerulonephritis and focal glomerulosclerosis markers and 'in vitro' activity of peripheral blood mononuclear cell.

Serum concentrations of IgG, IgA and IgM and PBMC were investigated in 11 prevalently adult patients with idiopathic glomerulonephritis, five minimal change glomerulonephritis (MCGN) and six focal glomerulosclerosis (FGS) and nephrotic syndrome. Among the peripheral blood mononuclear cells (PBMC) E rosette forming cells (T-cells), surface immunoglobulin bearing cells (B-cells), and T-cells with IgG Fc receptor (T gamma) were determined. In the culture supernatants of PBMC stimulated with PWM, the concentration of secreted IgG, IgA and IgM was determined by a solid phase immunofluorescence assay. After stimulation with PWM and PHA, the 3H-TdR uptake from PBMC was evaluated. In patients only the serum values of IgG were found significantly decreased. No difference was observed between patients and healthy age and sex matched controls, in percentage and absolute number of T and B-cells, whereas an increased number of T gamma was present in patients. In these patients after stimulation with PWM the production of IgG and IgA, but not IgM, and the 3H-TdR incorporation, were significantly lower than in healthy controls. These results suggest an imbalance in the cellular co-operation or an intrinsic B-cell defect in the synthesis or secretion of Ig in MCGN and FGS.

Adolescent↗
PubMed1993

An inborn error in epidermal growth factor prohormone metabolism in a mouse model of autosomal recessive polycystic kidney disease.

The C57BL/6J CPK heterozygous breeders secrete in urine a variant EGF-prohormone with a molecular mass of 154 kDa in addition to the normal 165 kDa EGF-prohormone. The 154 kDa prohormone is secreted as a heterodimer with the normal 165 kDa prohormone. The phenotypically normal littermates, like their parents, secrete the 154 and 165 kDa EGF-prohormones in urine while their cystic siblings secrete neither protein. Examination of renal extracts of normal littermates revealed the presence of the 165 kDa but not the 154 kDa EGF-prohormone; renal extracts of cystic siblings contained neither protein. Cyst fluid, however, contained 56 and 49 kDa EGF-immunoreactive proteins in high concentrations. The data suggest that in the absence of normal 165 kDa prohormone, the 154 kDa EGF-prohormone undergoes proteolysis and that the resultant fragments function as cystogens. Since normal siblings do not acquire renal cystic disease despite expressing the variant 154 kDa EGF-prohormone while the affected littermates, which lack the normal 165 kDa EGF-prohormone, manifest renal cystic disease, we suggest that congenital polycystic kidney disease is due to an inborn defect in the synthesis and secretion of the normal 165 kDa renal EGF-prohormone.

Adult↗
PubMed2001

Nutrient-secretion coupling in the pancreatic islet beta-cell: recent advances.

Insulin secretion from pancreatic islet beta-cells is a tightly regulated process, under the close control of blood glucose concentrations, and several hormones and neurotransmitters. Defects in glucose-triggered insulin secretion are ultimately responsible for the development of type II diabetes, a condition in which the total beta-cell mass is essentially unaltered, but beta-cells become progressively "glucose blind" and unable to meet the enhanced demand for insulin resulting for peripheral insulin resistance. At present, the mechanisms by which glucose (and other nutrients including certain amino acids) trigger insulin secretion in healthy individuals are understood only in part. It is clear, however, that the metabolism of nutrients, and the generation of intracellular signalling molecules including the products of mitochondrial metabolism, probably play a central role. Closure of ATP-sensitive K+(K(ATP)) channels in the plasma membrane, cell depolarisation, and influx of intracellular Ca2+, then prompt the "first phase" on insulin release. However, recent data indicate that glucose also enhances insulin secretion through mechanisms which do not involve a change in K(ATP) channel activity, and seem likely to underlie the second, sustained phase of glucose-stimulated insulin secretion. In this review, I will discuss recent advances in our understanding of each of these signalling processes.

Adenosine Triphosphate↗
PubMedDate not supplied

Turner's syndrome and carbohydrate metabolism. I. Impaired insulin secretion after tolbutamide and glucagon stimulation tests: evidence of insulin deficiency.

Tolbutamide (25 mg/kg: maximum 1 mg) intravenously (IV) and glucagon (0.03 mg/kg; maximum 1 mg) intramuscularly (IM) were given sequentially to 12 untreated girls with XO-Turner's syndrome (ages 6.5 to 17.0 years) and to ten female siblings (ages 8.0 to 16.7 years) to evaluate blood sugar (BS), plasma free fatty acids (FFA), serum immunoreactive insulin (IRI), and growth hormone (IRGH) responses to these insulinogenic secretagogues in order to appreciate any differences of genotypes on carbohydrate metabolism within identical family backgrounds. Seven of 12 patients with Turner's syndrome (58%) but none of the siblings were 20% or more overweight for height. There was a family history of diabetes mellitus in 7 to 12 patients (58%). The results showed significant elevations of mean FFA levels and decreased mean IRI responses to both insulinogenic stimuli without differences in mean BS or serum IRGH responses in the Turner's syndrome patients when compared to the controls. Three of 12 patients (25%) had abnormally elevated and prolonged blood sugar responses to IM glucagon. These findings show a significant incidence of abnormal carbohydrate and lipid metabolism and insulin deficiency in untreated patients with XO-Turner's syndrome when compared to normal female siblings and implicate this chromosomal defect in the impaired insulin secretion.

Adolescent↗
PubMed2001

Heritability of insulin secretion and insulin action in women with polycystic ovary syndrome and their first degree relatives.

Polycystic ovary syndrome (PCOS), one of the most common endocrine disorders of reproductive age women, is associated with an increased risk of type 2 diabetes mellitus. Defects in both insulin action and insulin secretion contribute to this predisposition to diabetes, but the extent to which these defects are heritable among PCOS families has not been examined. In the present study we used the frequently sampled iv glucose tolerance test to quantitate insulin secretion (AIRg), insulin action (Si), and their product (AIRg x Si) among women with PCOS (n = 33) and their nondiabetic first degree relatives (n = 48). We then quantitated the heritability of these measures from familial correlations estimated within a genetic model. Familial (spousal, rhoMF; parent-offspring, rhoPO; and sibling, rhoSS) correlations were derived for log-transformed body mass index (BMI) as well as for AIRg, Si, and AIRg x Si, the latter three of which were adjusted for BMI. There was no evidence of significant heritability for either lnBMI or lnSi in these families. In contrast, the sibling correlation (rhoSS = 0.74) for lnAIRg was highly significant (chi(2) = 7.65; 1 df; P = 0.006). In addition, the parameter quantitating insulin secretion in relation to insulin sensitivity [i.e. ln(AIRg x Si)] was significant among siblings (rho(SS) = 0.74; chi(2) = 4.32; 1 df; P = 0.04). In summary, the results of the present study indicate that there is an heritable component to beta-cell dysfunction in families of women with PCOS. We conclude that heritability of beta-cell dysfunction is likely to be a significant factor in the predisposition to diabetes in PCOS.

Adolescent↗
PubMed2003

Mitochondrial functional state in clonal pancreatic beta-cells exposed to free fatty acids.

Excessive free fatty acid (FFA) exposure represents a potentially important diabetogenic condition that can impair insulin secretion from pancreatic beta-cells. Because mitochondrial oxidative phosphorylation is a main link between glucose metabolism and insulin secretion, in the present work we investigated the effects of the FFA oleate (OE) on mitochondrial function in the clonal pancreatic beta-cell line, MIN6. Both the long term (72 h) and short term (immediately after application) impact of OE exposure on beta-cells was investigated. After 72 h of exposure to OE (0.4 mm, 0.5% bovine serum albumin) cells were washed and permeabilized, and mitochondrial function (respiration, phosphorylation, membrane potential formation, production of reactive oxygen species) was measured in the absence or presence of OE. MIN6 cells exposed to OE for 72 h showed impaired glucose-stimulated insulin secretion and decreased cellular ATP. Mitochondria in OE-exposed cells retained normal functional characteristics in FFA-free medium; however, they were significantly more sensitive to the acute uncoupling effect of OE treatment. The mitochondria of OE-exposed cells displayed increased depolarization caused by acute OE treatment, which is attributable to the elevation in the FFA-transporting function of uncoupling protein 2 and the dicarboxylate carrier. These cells also had an increased production of reactive oxygen species in complex I of the mitochondrial respiratory chain that could be activated by FFA. A high level of reduction of respiratory complex I augmented acute FFA-induced uncoupling in a way compatible with activation of mitochondrial uncoupling protein by intramitochondrial superoxide. A stronger augmentation was observed in OE-exposed cells. Together, these events may underlie FFA-induced depression of the ATP/ADP ratio in beta-cells, which accounts for the defective glucose-stimulated insulin secretion associated with lipotoxicity.

Animals↗
PubMed1998

HIV-induced IL-6/IL-10 dysregulation of CD4 cells is associated with defective B cell help and autoantibody formation against CD4 cells.

To analyse CD4 cell cytokine secretion and helper/suppressor function at a clonal level we established 446 CD4+ T cell clones (TCC) in four healthy controls, three HIV- haemophilia patients, four CDC II,III and four CDC IV patients. Spontaneous TCC secretion of Th1 cytokines (IL-2, interferon-gamma (IFN-gamma)) and Th2 cytokines (IL-4, IL-6, IL-10) was determined by ELISA. TCC helper and suppressor functions were tested in a pokeweed mitogen (PWM)-stimulated allogeneic co-culture system using a reverse haemolytic plaque assay for assessment of B cell responses. There was a significant association of TCC surface marker expression (Leu-8, CD45RA) with TCC IL-6 secretion in healthy controls (P < 0.01), HIV- patients (P < or = 0.001) and CDC II,III patients (P < or = 0.01) but not in CDC IV patients. Likewise, TCC expression of Leu-8 and CD45RA was significantly associated with TCC suppressor function in healthy controls (P < or = 0.0005) but not in HIV-infected patients. A reduced TCC helper frequency (< or = 10% of TCC) and an enhanced TCC suppressor frequency (> 80% of TCC) were detected only in those HIV-infected patients who showed an excessively increased TCC IL-6 secretion (> 70% of TCC) together with a significantly diminished TCC IL-10 secretion (< or = 10% of TCC). CD4 cell autoantibodies also were found only in patients with this type of cytokine dysregulation. These data indicate that CD4 cell surface markers lose their functional relevance in HIV-infected patients. HIV-induced IL-6/IL-10 dysregulation of CD4+ T cells, i.e. the up-regulation of spontaneous IL-6 and down-regulation of spontaneous IL-10 secretion, appears to be involved in inducing CD4 helper defects and may promote autoantibody formation against CD4 cells.

Autoantibodies↗
PubMed1999

The proopiomelanocortin system.

POMC (31,000 MW) is localized to the pituitary, brain, skin, and other peripheral sites. The particular enzyme profile present within a cell dictates the nature of the hormonal ligand (melanocortin) synthesized and secreted: melanotropic peptides (alpha-MSH beta-lipotropin, lambda-MSH), corticotropin (ACTH), several endorphins (e.g., met-enkephalin). These POMC-derived peptides mediate their actions through typical seven-spanning membrane receptors (MCRs; MCR1, 2, 3, 4, and 5). A specific melanocortin acting on a specific MCR regulates a particular biological response; for example, alpha-MSH on MCR1 increases melanogenesis within melanocytes, ACTH on MCR2 increases cortisol production within adrenal zona fasciculata cells. Within the brain melanocortins regulate satiety (MCR4) and erectile activity (MCR?). MCRs have been localized by melanocortin macromolecular probes, for example, fluorescent to human epidermal melanocytes and also to keratinocytes, suggesting that systemic melanocortins or localized POMC products might regulate these integumental cellular elements in synchrony to enhance skin pigmentation and/or immunological responses. Superpotent, prolonged acting melanotropic peptides have been synthesized and their application in clinical medicine has been demonstrated. MCR antagonists have been used to discover and further delineate other roles of melanocortin ligands. For example, melanocortin-induced satiety can be antagonized by a melanocortin antagonist. Defects in melanocortin ligand biosynthesis, secretion, and melanocortin receptor function can lead to a diverse number of pathological states.

Adrenocorticotropic Hormone↗
PubMed1997

Functional defects in lysosomal enzymes in autosomal dominant polycystic kidney disease (ADPKD): abnormalities in synthesis, molecular processing, polarity, and secretion.

The phenotype of autosomal dominant polycystic kidney disease (ADPKD) is characterized by basement membrane abnormalities, hyperproliferation, and alterations in epithelial cell polarity. Since proteinases have been implicated in matrix degradation and growth factor activation, lysosomal enzymes were compared in normal and ADPKD tissues and cell cultures. Acidic proteolytic activity (azocasein) was reduced in ADPKD, and specific enzymatic assays detected disease-dependent decreases in the specific activities of beta-galactosidase, beta-hexosaminidase, and cathepsins, B, L, and H. Cathepsin D-specific activities were unchanged. Lucifer yellow fluorescence in ADPKD cells was consistent with an alteration in heterogeneity of lysosomal enzyme content in ADPKD rather than a decrease in total lysosomal number. Western analysis, metabolic labeling, and immunoprecipitation analysis confirmed decreases in the expression and synthesis of the major normal molecular immunoreactive species of beta-galactosidase and cathepsins B and H in ADPKD tissue and cells but no changes in cathepsin D. In addition, ADPKD-specific high-molecular-weight species of cathepsin H were seen and abnormal forms of cathepsin B and beta-galactosidase were common in ADPKD, suggesting abnormal molecular processing and posttranslational modifications. In addition, immunolocalization studies showed abnormal apical plasma-membrane localization of cathepsins B and H in ADPKD cyst epithelial cells, consistent with a protein sorting defect in ADPKD. Increased extracellular secretion of lysosomal enzymes was also measured in ADPKD cultured cells and in filter-grown epithelia shown to be predominantly directed to the basal compartment. These results demonstrate that lysosomal enzyme alterations in ADPKD may play a role in aberrant processing of the basement membrane. Alterations in the polarized secretion of lysosomal enzymes by ADPKD epithelia in vitro were also detected. Whereas all normal epithelia cells secreted lysosomal enzymes predominantly to the apical medium compartments, basally directed secretion was increased in all ADPKD epithelia and attained an overall reversal of polarity for cathepsins B + L. It is concluded that alterations in lysosomal enzyme function in ADPKD are the result of alterations in synthesis, molecular processing, and polarized secretion of specific enzymes and may have impact on proliferative and basement membrane abnormalities in this genetic disease. These results are consistent with a fundamental defect in protein processing sorting, and trafficking in ADPKD.

Adult↗
PubMed1983

Cytotoxicity and inhibition of normal collagen synthesis in mouse fibroblasts by lipoteichoic acid from Streptococcus pyogenes type 12.

The toxicity of lipoteichoic acid (LTA) from Streptococcus pyogenes type 12 was investigated by using mouse fibroblasts in culture in the absence of serum. Morphologically, while low concentrations of LTA elicited a subtle effect characterized by progressive cellular degeneration with practically no release of protein, larger concentrations (greater than 50 micrograms/ml) of this amphiphile resulted in rapid death of cell monolayers. Metabolic studies utilized a concentration of LTA (17.5 micrograms/ml) which caused the smallest change in cell morphology in the least number of mouse fibroblast cells per monolayer. Under these conditions, cell monolayers showed an increase of 450% in their content of collagenous protein after exposure to LTA. However, the amount of such material secreted remained unchanged. Also, changes in the type of collagenous protein formed were observed after exposure to LTA. Collagenous protein accumulating intracellularly was found to be practically hydroxyproline-free. However, collagenous protein secreted by this cell line showed a significantly reduced content of hydroxyproline as compared with control cells unexposed to this coccal membrane component. Column chromatographic studies confirmed that the collagenous protein secreted by monolayers exposed to LTA was defective (under hydroxylated). It was concluded that LTA does not affect the amount of collagenous protein secreted. However, it does increase the amount of this protein formed and retained by this cell line as well as causing a reduction in the hydroxylation of proline in both intracellular and secreted collagenous material. A possible relationship between abnormal basement membrane morphology and disturbed collagen synthesis in post-streptococcal glomerulonephritis as related to LTA is discussed.

Animals↗
PubMed1998

The Caulobacter crescentus paracrystalline S-layer protein is secreted by an ABC transporter (type I) secretion apparatus.

Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3' of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of Pseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. The prtB and aprA genes were introduced into C. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.

ATP-Binding Cassette Transporters↗
PubMed1976

Renal hydrogen ion secretion after release of unilateral ureteral obstruction.

The effect of 24 h of unilateral ureteral obstruction on HCO3 reabsroption and urinary acidification was studied in dogs. The postobstructed kidney (EK) had a significantly lower glomerular filtration rate and renal plasma flow than the contralateral kidney (CK). Urinary pH prior to HCO3 loading was significantly higher in the EK as was maximal HCO3 reabsorption. Saline loading depressed HCO3 reabsorption to the same degree in both kidneys. Urinary PCO2, during HCO3 loading, and during phosphate infusion, was significantly lower in the EK than the CK. Fractional Na excretion was significantly higher in the EK than the CK after deoxycorticosterone acetate administration. Na2SO4 administration enhanced acid excretion only in the CK. K excretion was significantly lower in the EK than the CK both during HCO3 loading and Na2SO4 administration. There was redistribution of cortical blood flow from the outer cortex toward the inner cortex in the EK as compared to the CK. There was no difference in plasma renin activity from both renal veins. These data demonstrate enhanced proximal H+ secretion (which is abolished by volume expansion) and impaired distal H+ secretion by the postobstructed kidney. The distal defect is likely an effect of a generalized disorder of distal transport in that both K secretion and steroid-responsive Na reabsorption were impaired in the postobstructed kidney.

Animals↗
PubMed1998

Cushing's disease: a comparison of pituitary corticotroph microadenomas and macroadenomas.

Cushing's disease appears as a functionally heterogeneous disease, but criteria that are able to distinguish between different clinical forms remain elusive. We compared two subgroups of patients with proven Cushing's disease according to the size of the pituitary adenoma, evaluated by computed tomography or magnetic resonance imaging. Our series comprised 11 patients with a microadenoma and 10 with a macroadenoma (median volumes (range): 173 (13-270) and 3022 (500-10312) mm3 respectively; P < 0.0001). The clinical presentation was similar in the two groups, but the time elapsed before diagnosis was longer, and visual impairment was less frequent in the patients with a microadenoma (1.5+/-0.8 years and 0%) than in those with a macroadenoma (0.7+/-0.6 years and 40%; P < 0.05). Morning and evening peripheral concentrations of ACTH were greater in patients with macroadenoma (134+/-78 and 130+/-7 ng/l respectively) than in those with microadenoma (52+/-28 and 56+/-19 ng/l, P < 0.05). Hypokalaemia and lymphopenia were also more pronounced in patients with macroadenoma (3.4+/-0.3 mmol/l and 1273+/-401 lymphocytes/mm3) than in those with microadenoma (3.8+/-0.3 mmol/l and 1852+/-668 lymphocytes/mm3 P < 0.05), although morning and evening plasma cortisol concentrations were similar in both groups. In patients with macroadenoma, there was less relative nycthemeral variation of ACTH concentrations (28+/-24%, compared with 62+/-39% in those with microadenoma; P < 0.05), less suppression of plasma cortisol by high doses of dexamethasone (-30+/-14%, compared with -61+/-25%; P < 0.05), and a reduced concentration ratio of mean basal cortisol to ACTH (7+/-3, compared with 12+/-5; P < 0.05). Plasma IGF-I concentration and the TSH peak response to TRH were significantly lower in patients with macroadenoma than in those with microadenoma (0.4+/-0.2 x 10(3) IU/I and 2.3+/-1.8 mIU/I, compared with 1.8+/-0.6 x 10(3) IU/I and 5.2+/-1.6 mUI/l; P < 0.05). Thus, in comparison with microadenomas, corticotroph macroadenomas are characterized by a greater and more autonomous ACTH secretion, inducing more pronounced biological signs of hypercorticism, and are more often accompanied by visual field defects and impairment of other pituitary hormonal secretions.

Adenoma↗
PubMed2006

Association of the Leu72Met polymorphism of the ghrelin gene with the risk of Type 2 diabetes in subjects with impaired glucose tolerance in the Finnish Diabetes Prevention Study.

AIMS: Ghrelin is a gut-brain regulatory peptide stimulating appetite and controlling energy balance. In previous studies, the Leu72Met polymorphism of the ghrelin gene has been associated with obesity and impaired insulin secretion. We investigated whether the Leu72Met polymorphism is associated with the incidence of Type 2 diabetes in subjects with impaired glucose tolerance (IGT) participating in the Finnish Diabetes Prevention Study (DPS). METHODS: DPS was a longitudinal intervention study carried out in five participating centres in Finland. A total of 522 subjects with IGT were randomized into either an intervention or a control group and DNA was available from 507 subjects. The Leu72Met polymorphism was screened by the restriction fragment length polymorphism method. RESULTS: There were no differences in clinical and anthropometric characteristics among the genotypes at baseline. IGT subjects with the Met72 allele were at higher risk of developing Type 2 diabetes than subjects with the Leu72Leu genotype (P = 0.046). Our data also demonstrated that IGT subjects with the common Leu72Leu genotype developed Type 2 diabetes less frequently under intervention circumstances than subjects with the Met72 allele (OR = 0.28, 95% CI 0.10-0.79; P = 0.016). CONCLUSIONS: Subjects with the Leu72Leu genotype had a lower risk for the development of Type 2 diabetes. This was observed particularly in the study subjects who underwent an intensive diet and exercise intervention. Defective first-phase insulin secretion related to the Met72 allele might be one factor contributing to the conversion to Type 2 diabetes.

Diabetes Mellitus, Type 2↗
PubMedDate not supplied

Impaired leptin response to glucocorticoid as a chronic complication of diabetes.

Leptin has anorectic, anti-obesity, and insulin-sensitizing properties. We recently reported subnormal responses to the leptin secretagogue dexamethasone in diabetes (DM). To determine whether this defect precedes or follows the occurrence of diabetes, we have studied 37 adults: 11 with type 2 DM diagnosed within 6 months prior to study, 16 with chronic (> or =20 years) DM, and 10 healthy controls. After baseline measurements, subjects ingested dexamethasone (4 mg), followed by blood sampling 16 and 40 h later. Nadir plasma cortisol levels (<2.5 mg/dl) occurred 16 h after dexamethasone ingestion in all study groups; this period of maximal biological action of dexamethasone was associated with peak plasma leptin levels. The peak dexamethasone-stimulated plasma leptin responses (% baseline, +/-SEM) were 188+/-18.7% among healthy controls, 180+/-13.8% among new DM patients, and 127+/-10.5% (P<0.01) in chronic DM patients. Following dexamethasone ingestion, plasma glucose remained stable in the control and new DM groups but increased by 240% in the chronic DM patients; in contrast, plasma insulin increased significantly in controls and new DM patients but not in patients with chronic DM. These results indicate that plasma leptin responses to secretagogue are preserved in newly diagnosed diabetes patients but markedly attenuated in patients with long-standing diabetes, who also were unable to augment insulin secretion during glucocorticoid treatment. Thus, defective glucocorticoid augmentation of plasma leptin, probably related to beta-cell failure, may be a novel chronic complication of diabetes. Theoretically, such a defect could contribute to the obesity and insulin resistance associated with diabetes.

Adult↗
PubMed2006

Prolongation of renal allograft survival in rats by replication-defective recombinant adenovirus-mediated coexpression of CD40L and CTLA4Ig.

BACKGROUND: Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. METHODS: We constructed Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. RESULTS: Successful construction of Adv-shCD40L-IRES2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2%+/-0.6% and 42.1%+/-0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES2-CTLA4Ig resulted in further inhibition of MLR to 22.0%+/-0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8+/-2.5 days and 27.3+/-3.6 days, respectively, as compared to vehicle-treated controls (7.8+/-0.3 days). Cotransfection of both genes extended graft survival to 41.8+/-3.7 days. CONCLUSIONS: Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.

Abatacept↗
PubMed2001

Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island: essential genes for CagA translocation in host cells and induction of interleukin-8.

Helicobacter pylori (Hp) carries a type IV secretion system encoded by the cag pathogenicity island (cag-PAI), which is used to: (i) translocate the bacterial effector protein CagA into different types of eukaryotic cells; and (ii) induce the synthesis and secretion of chemokines, such as interleukin-8 (IL-8). The cag-PAI in Hp 26695 consists of 27 putative genes, six of which were identified as homologues to the basic type IV secretion system represented by the Agrobacterium tumefaciens virB operon. To define the role and contribution of each of the 27 genes, we applied a precise deletion/insertion mutagenesis procedure to knock out each individual gene without causing polar effects on the expression of downstream genes. Seventeen out of 27 genes were found to be absolutely essential for translocation of CagA into host cells and 14 out of 27 for the ability of Hp fully to induce transcription of IL-8. The products of hp0524 (virD4 homologue), hp0526 and hp0540 are absolutely essential for the translocation of CagA, but not for the induction of IL-8. In contrast, the products of hp0520, hp0521, hp0534, hp0535, hp0536 and hp0543 are not necessary for either translocation of CagA or for IL-8 induction. Our data argue against a translocated IL-8-inducing effector protein encoded by the cag-PAI. We isolated a variant of Hp 26695, which spontaneously switched off its capacity for IL-8 induction and translocation of CagA, but retained the complete cag-PAI. We identified a point mutation in gene hp0532, causing a premature translational stop in the corresponding polypeptide chain, providing a putative explanation for the defect in the type IV secretion system of the spontaneous mutant.

Amino Acid Sequence↗