Occupational risk of human Cytomegalovirus and Parvovirus B19 infection in female day care personnel in the Netherlands; a study based on seroprevalence.

Cytomegalovirus (CMV) and Parvovirus B19 infections acquired during pregnancy may result in developmental disabilities of the foetus. This study evaluates the occupational risk of these infections in female day care personnel. IgG seroprevalence was determined in 310 Dutch day care workers and 158 nursing school students. CMV seroprevalence was age-related, starting at 21% in those <20 years and reaching 65% in those >35 years. Between the ages of 20 and 24 years the CMV prevalence was higher in day care personnel than in controls, 50% versus 31% (p = 0.03). In the first 2 years of employment the risk of attracting CMV was significantly increased (OR(adj) = 3.80; p < 0.001) and the occupational risk was also increased (OR(adj) 2.19; p < 0.001). Parvovirus seropositivity (71-77%) was not related to age or working at a day care centre. In conclusion, an occupational risk was observed for CMV, but not for Parvovirus infection in female day care personnel.

Evaluation of a rapid one-step immunochromatographic test and two immunoenzymatic assays for the detection of anti-Treponema pallidum antibodies.

BACKGROUND: The control of syphilis depends on screening of the population at risk and is usually performed using the Treponema pallidum particle agglutination test (TPPA). Outside Europe the rapid plasma reagin test (RPR) or venereal disease research laboratory test is most often used for screening purposes. Because of the drawbacks in current diagnostic procedures, ie, long turnaround time, the need is felt for a rapid and simple test that can potentially be performed on whole blood. OBJECTIVE AND STUDY DESIGN: In this study a one-step immunochromatographic test (Biorapid Syphilis) and two ELISA, the Bioelisa Syphilis 3.0 and ETI-Treponema Plus, were evaluated. METHODS: Serum samples were collected between February 2000 and May 2006 at the University Hospital in Maastricht, The Netherlands. 145 TPPA-positive sera, confirmed by fluorescent treponemal antibody absorption (FTA-Abs, treponemal test) and/or RPR (non-treponemal) were included. Furthermore, 41 sera from healthy controls and 144 TPPA-negative sera from controls with underlying conditions that might interfere with T pallidum serology were collected. RESULTS: The sensitivity and specificity of the Biorapid Syphilis, Bioelisa Syphilis 3.0 and ETI-Treponema Plus were 92% and 79%, 100% and 100% and 100% and 100%, respectively, with our selected sera. CONCLUSIONS: The performance of both ELISA was excellent in our study and is favoured over the TPPA because of its ability to be run on an automated system. The sensitivity and specificity of the Biorapid Syphilis were considered too low to implement the test in a hospital laboratory in a developed country but it might be useful in primary healthcare settings in developing countries.

Comparison of five different immunoassays for the detection of Borrelia burgdorferi IgM and IgG antibodies.

The performances of five commercially available enzyme immunoassays were compared for the detection of Borrelia burgdorferi IgM and IgG antibodies. Sensitivity was assessed with European serum samples collected from 45 patients with clinically defined Lyme disease in conjunction with a positive immunoblot (n = 44) or other serological test (n = 1). Sensitivities for the detection of IgM and IgG with each test were: Dako IgM 64%; Dako IgG 53%; Serion IgM 89%; and Serion IgG 88%. The Immunetics assay makes no distinction between IgM and IgG antibodies and had a sensitivity of 91%. Specificity was calculated by testing a control group comprising 40 patients with acute Epstein-Barr virus infection, cytomegalovirus infection, syphilis or rheumatoid factor positivity. The specificities achieved for each test were: Dako IgM 78%; Dako IgG 100%; Serion IgM 52%; Serion IgG 92%; and Immunetics 92%. The discriminatory power between control and patient samples appeared highest for the Immunetics assay. Between-run variation was comparable for the five tests and did not exceed 13%. When the Immunetics assay was used as an initial screening test, with low-titre positive results confirmed by an immunoblot, a sensitivity of 91% and a specificity of 100% were achieved. To attain maximal sensitivity, the Serion IgM and IgG tests were also performed on samples with negative Immunetics results. All positive Serion IgM and IgG results were also confirmed by immunoblot. In conclusion, the Immunetics assay, based on a synthetic C6 peptide, can be used reliably as an initial screening test for the serodiagnosis of Lyme disease.

Interleukin-1beta induces ornithine decarboxylase activity in insulin-producing cells.

Cytokines might play a role in the development of insulin-dependent diabetes. Interleukin 1beta (IL-1beta) has been shown to alter the functional state of insulin-producing beta cells. This effect appears mediated through induction of certain proteins and suppression of others. The present study demonstrates that the ornithine decarboxylase (ODC) activity of rat beta cells and insulin-producing rat insulinoma (RIN) cells increases more than three-fold within 2 h of IL-1beta exposure. Both basal and IL-1 beta induced ODC activities completely disappear following a 2 h block of protein translation. In Western blot analysis, a 51-kDa protein varies in parallel with the ODC-activity. Exposure to IL-1beta increases the 51-kDa band through an effect at the transcriptional level. The higher cellular ODC activity in IL-1beta treated RIN cells is associated with an increased cellular content of its enzymatic product putrescine, but not of putrescine-derived products such as polyamines and GABA. The 30-fold lower ODC activity in rat beta cells forms a technical obstacle to studies on the regulation and functional significance of this enzyme in normal cells. The present findings list ODC-activity as an early response to the effect of interleukin 1beta on the transcriptional activity in insulin-producing cells. Further work is needed to identify whether ODC activation contributes to the previously described functional changes in pancreatic beta cells.

Interleukin 1beta increases arginine accumulation and activates the citrulline-NO cycle in rat pancreatic beta cells.

Nitric oxide (NO) may contribute to pancreatic beta cell damage during the development of type 1 diabetes. Its formation can be triggered by cytokines which induce the expression of the inducible form of nitric oxide synthase (iNOS) in pancreatic islets. In the iNOS-catalyzed reaction, arginine is converted into citrulline and NO. Cellular NO formation may be regulated by the availability of arginine. Arginine can be provided extracellularly, entering the cell mainly through the cationic amino acid transporter system y+CAT, and intracellularly, by protein degradation or synthesis from citrulline (the citrulline-NO cycle). This study demonstrates for the first time that the citrulline-NO cycle is induced in FACS-purified rat beta cells exposed to interleukin-1beta(IL-1beta) and that extracellular arginine or citrulline is required for NO production by beta cells. Moreover, the accumulation of arginine was higher in IL-1beta-treated beta cells than in control cells.beta cells expressed mRNAs for the two y+CAT transporters CAT-2A and CAT-2B with no change in transporter expression after exposure to IL-1beta. It is concluded that the activation of the citrulline-NO cycle and an increase in arginine accumulation may be adaptive responses in cytokine-exposed beta-cells to assure an adequate arginine supply for continuous NO production in the presence of low extracellular arginine levels which may prevail during insulitis.

Radioimmunoassay for glutamic acid decarboxylase-65.

BACKGROUND: Glutamic acid decarboxylase-65 (GAD65), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA), is the major autoantigen in both type 1 (insulin-dependent) diabetes and stiff-man syndrome (SMS). The observation that GAD65 autoantibodies may be present for years before the clinical onset of diabetes raises the question of when GAD65 is available to initiate an immune response to allow the formation of autoantibodies. In order to address this question it will be necessary to measure GAD65 in tissue, cells, and plasma. METHODS: A radioimmunoassay (RIA) was developed for GAD65 based on the use of a polyclonal rabbit antiserum directed to the N-terminus of GAD65. RESULTS: Using the GAD65 RIA, we have determined the GAD65 content in a human GAD65 gene transfected cell line and in beta-cell preparations from different species. The assay detects an increase of immunoreactive GAD65 after glucose-stimulation and GAD65 that is discharged from rat beta cells after their exposure to the diabetogenic agent streptozotocin. It also measures good recovery of GAD65 added to human plasma samples. CONCLUSIONS: The GAD65 RIA makes it possible to determine both cellular and extracellular levels of GAD65; this might be useful in investigating the mechanisms leading to the formation of GAD65 autoantibodies in type 1 diabetes and SMS patients.

Intercellular differences in interleukin 1beta-induced suppression of insulin synthesis and stimulation of noninsulin protein synthesis by rat pancreatic beta-cells.

The normal pancreatic beta-cell population exhibits intercellular differences in its responsiveness to glucose. This cellular heterogeneity allows glucose to regulate, in a dose-dependent manner, total rates of insulin synthesis and release. It may also predispose to intercellular differences in susceptibility to dysregulating agents. The present study examines whether this is the case for interleukin 1beta (IL-1beta), which is known to suppress glucose-induced insulin synthesis and release. The effects of the cytokine were compared on beta-cell subpopulations with, respectively, high and low sensitivity to glucose. These subpopulations were separated on the basis of differences in the cellular metabolic responsiveness to an intermediate glucose concentration (7.5 mmol/liter) and then cultured for 20 h at 5 or 20 mmol/liter with or without IL-1beta. The suppressive action of IL-1beta (0.1 ng/ml) occurred predominantly in glucose-activated beta cells, reducing their high rates of insulin synthesis and release by more than 80%. Glucose-unresponsive cells became subject to a similar inhibition after their activation during culture at 20 mmol/liter glucose. On the other hand, IL-1beta induced or enhanced the expression of several noninsulin proteins in both subpopulations. The IL-1beta-stimulated expression of inducible nitric oxide synthase (iNOS) and heat shock protein 70 was more marked in the glucose-responsive subpopulation; that of heme oxygenase and Mn superoxide dismutase was comparable in the two subpopulations. Exposure to IL-1beta resulted in 10-fold higher medium nitrite levels in both subpopulations; this effect was prevented by the iNOS blocker, N(G)-methyl-L-arginine, which also prevented the IL-1beta-induced suppression in the glucose-responsive subpopulation. This study demonstrates that the cellular heterogeneity in glucose responsiveness predisposes to intercellular differences in the IL-1-induced suppression of insulin synthesis and release. While the cytokine induces the expression of noninsulin proteins such as iNOS in both glucose responsive and unresponsive cells, the subsequent nitric oxide production appears to predominantly affect glucose-stimulated functions in the glucose-activated cells.

Nutrient regulation of gamma-aminobutyric acid release from islet beta cells.

Glutamate decarboxylase (GAD) of pancreatic beta cells seems to be involved in the development of autoimmune reactivities which occur in insulin-dependent diabetes mellitus. Little is known about the regulation and role of the GAD activity in normal beta cells. In the betaTC6 line, the enzymatic product, gamma-aminobutyric acid (GABA) was reported to be released under glucose stimulation, thus supporting the concept that GABA transmits a suppressive action of glucose-stimulated beta cells on neighbouring alpha cells. In this study GABA was found to be released from normal rat beta cells. Over 24-h culture periods, the released amounts represented a constant fraction (25% per h) of the cellular GABA content. Cellular GABA content and release were dose-dependently increased by the glutamine concentration in the medium; both values decreased following a sustained (24 h) glucose activation (culture at 10 or 20 mmol/l glucose instead of 3 mmol/l). The variations in the medium GABA content did not parallel the changes in insulin release, indicating that both beta-cell secretory products follow different routes of storage and release. We suggest that beta cells can discharge GABA via exocytosis of microvesicles storing GABA as well as via direct transport from the cytoplasmic pool of newly formed product. Variations in GABA production result in parallel changes in extracellular GABA concentration; the high fractional release of GABA makes it also a likely parameter of the cellular GAD activity. Since chronically elevated glucose levels result in a reduced GABA discharge from the beta cells, it is conceivable that the subsequent decrease in GABA-mediated suppression of the alpha cells is responsible for a higher glucagon release, as observed in diabetes.

Damaged rat beta cells discharge glutamate decarboxylase in the extracellular medium.

In type I (insulin-dependent) diabetes, destruction of pancreatic beta cells has been associated with the presence of circulating antibodies against glutamate decarboxylase (GAD), a GABA (gamma-aminobutyric acid) synthesizing enzyme which is located in the beta cells. We examined whether destruction of islet beta cells can lead to discharge of GAD in the extracellular medium, making it a potential autoantigen. Rat islet beta cells were first exposed for 1 hour to streptozotocin and then cultured for 4 to 24 hours before cellular and medium GAD activities were measured. After 24 hours culture, 70 percent of streptozotocin-treated beta cells were disintegrated whereas the number of control cells remained unchanged. Control cells exhibited a stable cellular GAD activity over the 24 hour period with no enzyme activity detectable in their culture medium. The cells recovered 24 hours after streptozotocin treatment exhibited 10-fold lower levels of GAD-activity and of GABA; their culture medium contained GAD, its enzymatic activity reaching peak values after 10 hours. The beta-cell enzymes glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were not detectable in the medium of control or streptozotocin-treated cells. Similar observations were made when beta cells had been exposed to cytotoxic concentrations of alloxan. It is concluded that damage to rat islet beta cells results in transient discharge of GAD in the extracellular medium making this enzyme a candidate extracellular marker for beta cell toxic processes and a potential autoantigen for immune reactivity.