A novel neuroendocrine cell surface glycoprotein: identification, isolation, and initial characterization

Murine monoclonal antibodies (MAbs) HISL-5, -9, and -14, generated after immunization of mice with human pancreatic islet cell preparations, recognize a differentiation antigen expressed by the pancreatic islet cells. These MAbs react strongly with all endocrine cell subtypes of human pancreatic islets, but minimally if at all with the exocrine acinar cells, vascular cells, and stromal connective tissue cells of the pancreas. The antigen is located on the cell surface (plasma membranes), as indicated by immunofluorescence staining of viable cell preparations. Besides the pancreatic islets, HISL-5, -9, and -14 antigenic determinants are also expressed by thyroid follicular cells, parathyroid chief cells, and anterior pituitary cells, other commonly involved targets in organ-specific autoimmune disorders. Preliminary biochemical findings indicated that the MAb-defined epitope(s) is trypsin sensitive and resistant to periodate oxidation and exposure to chloroform-methanol. Further biochemical studies, including single step MAb immunoaffinity chromatographic purification, indicate that the antigen recognized by the MAbs HISL-5, -9, and -14 is a 100 K glycoprotein.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes

We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh- primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh- untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).     

Demonstration of reversible priming of human neutrophils using platelet- activating factor

Exposure of neutrophils to agents such as lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major upregulation of subsequent agonist-induced NADPH oxidase activation. This priming effect is a prerequisite for neutrophil-mediated tissue damage and has been widely considered to be an irreversible process. We have investigated the potential for neutrophils to recover from a priming stimulus by studying the effects of platelet-activating factor (PAF). PAF did not stimulate respiratory burst activity directly, but caused a rapid (maximal at 10 minutes) and concentration-dependent (EC50 50.2 nmol/L) increase in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated superoxide anion release. At time-points > 10 minutes, this priming effect spontaneously declined, with return to basal levels of fMLP- stimulated superoxide anion generation by 120 minutes. An identical priming time-course was observed with N-methyl carbamyl PAF, a nonmetabolizable analogue of PAF, indicating that the transient nature of PAF-induced priming was not secondary to PAF metabolism. Two structurally diverse PAF receptor antagonists (UK-74,505 and WEB 2086), added 10 minutes after PAF addition, increased the rate of decay of the priming effect. In contrast, TNF-alpha-induced priming, which was of a similar magnitude to that observed for PAF, was slower to evolve (maximal at 30 minutes) and remained constant for at least 120 minutes. The reversible nature of PAF-induced priming was confirmed by demonstrating that PAF-, but not TNF-alpha-, induced cell polarization (shape change) and CD11b-dependent neutrophil binding of albumin-coated latex beads was also transient, with return to basal, unstimulated levels by 120 minutes. Furthermore, cells that had spontaneously deprimed following PAF exposure retained their capacity to be fully reprimed by a subsequent addition of either PAF or TNF-alpha. These data imply that neutrophil priming is not an irreversible event: the demonstration of a cycle of complete priming, depriming, and repriming offers the potential for functional recycling of neutrophils at sites of inflammation.     

Molecular and cellular effects of antisickling concentrations of alkylureas

Alkylureas are capable of inhibiting sickling in vitro and the gelation of solutions of hemoglobin S at concentrations between 0.05 and 0.1 M with increasing effectiveness that is directly proportional to the length of the alkyl chain (butyl greater than propyl greater than ethyl greater than methyl). 6The inhibitory effect is independent of pH between 6.5 and 7.5 and is a process driven by entropy. The alkylureas at concentrations of 0.1 M have minimal effects on several erythrocyte functions. Oxygen equilibria, osmotic fragility, reduced glutathione content, and glutathione reductase activity are totally unaffected, while pyruvic kinase activity is decreased only by butylurea by about 20%, and glucose-6-phosphate dehydrogenase activity is decreased progressively to a maximum of 30% in direct proportion to the length of the alkyl chain. Alkylureas not only inhibit sickling but are also capable of desickling erythrocytes that have been maintained in the deoxygenated state. They have little effect on several erythrocyte functions at antisickling concentrations, but their toxicity must be evaluated before they can be examined as potential therapeutic agents for the treatment or prevention of acute episodes in sickle cell anemia.     

Acute phase response in acute pancreatitis: a comparison with abdominal sepsis

BACKGROUND: Increased knowledge on the underlying pathophysiological mechanisms in acute pancreatitis (AP) and abdominal sepsis (AS) is essential, not least for the development of novel ways of treatment. The present study aims at determining dynamic changes in the systemic inflammatory response in AP and AS. METHODS: AP was induced by the intraductal injection of sodium taurodeoxycholate in the rat, while AS was induced by caecal ligation and puncture. The animals were killed 1, 3, 6 and 9 h after challenge. Plasma exudation of radiolabelled albumin, myeloperoxidase (MPO), TNF-alpha, MCP-1, superoxide and hydrogen peroxide was measured. RESULTS: Leakage index of human serum albumin showed a significant increase early (I h) after induction of AP and later (9h) in AS compared to controls (P < 0.05). Hydrogen peroxide generation by circulating monocytes/macrophages was high early (1 h) in AP and after 3 and 6h in AS. Superoxide generation increased by time after both challenges. MPO activity increased significantly, starting at 3 h in both AP and AS (P < 0.05). TNF-alpha increased significantly at 6 and 9 h in both models. CONCLUSION: Our results indicate differences in the release of systemic inflammatory mediators and cellular response in AP and AS. However, potential similarities in end-effect, such as the development of remote organ injury (lungs), may exist as implied by the MPO results. Further investigations of the mechanisms are crucial.     

Autoantibodies against recombinant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17alpha-hydroxylase in Addison's disease and autoimmune polyendocrine syndrome type III

OBJECTIVE: To evaluate the frequency of autoantibodies (Ab) against 21 hydroxylase (21OH), side-chain cleavage (SCC) and 17alpha-hydroxylase (17OH), in Addison's disease (AD) and autoimmune polyendocrine syndrome type III (APSIII). DESIGN AND METHODS: We used radiobinding assays and in vitro translated recombinant human (35)S-21OH, (35)S-SCC or (35)S-17OH and studied serum samples from 29 AD (18 idiopathic, 11 granulomatous) and 18 APSIII (autoimmune thyroid disease plus type 1 diabetes mellitus, without AD) patients. Results were compared with those of adrenocortical autoantibodies obtained with indirect immunofluorescence (ACA-IIF). RESULTS: ACA-IIF were detected in 15/18 (83%) idiopathic and in 1/11 (9%) granulomatous AD subjects. 21OHAb were found in 14/18 (78%) idiopathic and in the same (9%) granulomatous AD subject. A significant positive correlation was shown between ACA-IIF and 21OHAb levels (r(2)=0.56, P<0.02). The concordance rate between the two assays was 83% (24/29) in AD patients. SCCAb were found in 5/18 (28%) idiopathic (4 of whom were also positive for 21OHAb) and in the same (9%) granulomatous AD subject. 17OHAb were found in only 2/18 (11%) idiopathic and none of the granulomatous AD patients. Two APSIII patients were positive for ACA-IIF, but only one was positive for 21OHAb and SCCAb. 17OHAb were found in another two APSIII patients. CONCLUSIONS: Measurement of 21OHAb should be the first step in immune assessment of patients with AD and individuals at risk for adrenal autoimmunity, in addition to ACA-IIF. Due to their low prevalence in AD, measurement of SCCAb and 17OHAb should be indicated only for 21OHAb negative patients and/or for those with premature ovarian failure, regardless of ACA-IIF results.     

Association of the subtype 2 of the Epstein-Barr virus with T-cell non- Hodgkin's lymphoma of the midline granuloma type [see comments]

Lethal midline granuloma (LMG) is associated with Epstein-Barr virus (EBV). The latter has at least two subtypes with different biological properties. The subtypes can be identified by their genomic configuration. Using EBV-RNA (EBER) in situ hybridization and EBV polymerase chain reaction (PCR), we have looked for the presence of EBV in six LMGs and six non-Hodgkin's lymphomas (NHLs) located in the nasopharyngeal region, and determined the subtype of EBV. Six of six LMGs were positive by PCR and EBER in situ hybridization, whereas NHLs were either negative or, in three of six cases, showed few EBER- positive cells considered to be nonneoplastic lymphocytes. The subtype 2 was found in LMG lesions of three of six patients; the remaining three of six patients with LMG had the generally occurring subtype 1. The results indicate that the association of EBV with NHL may depend more on tumor type than on its localization. The occurrence of the rare subtype 2 in LMG may relate to a covert immune defect.