Immunofluorescence technique using HeLa cells expressing recombinant nucleoprotein for detection of immunoglobulin G antibodies to Crimean-Congo hemorrhagic fever virus

A HeLa cell line continuously expressing recombinant nucleoprotein (rNP) of the Crimean-Congo hemorrhagic fever virus (CCHFV) was established by transfection with an expression vector containing the cDNA of CCHFV NP (pKS336-CCHFV-NP). These cells were used as antigens for indirect immunofluorescence (IF) to detect immunoglobulin G antibodies to CCHFV. The sensitivity and specificity of this IF technique were examined by using serum samples and were compared to those of the IF technique using CCHFV-infected Vero E6 cells (authentic antigen). Staining of the CCHFV rNP expressed in HeLa cells showed a unique granular pattern similar to that of CCHFV-infected Vero E6 cells. Positive staining could easily be distinguished from a negative result. All 13 serum samples determined to be positive by using the authentic antigen were also determined to be positive by using CCHFV rNP-expressing HeLa cells (recombinant antigen). The 108 serum samples determined to be negative by using the authentic antigen were also determined to be negative by using the recombinant antigen. Thus, both the sensitivity and the specificity of this IF technique were 100% compared to the IF with authentic antigen. The novel IF technique using CCHFV rNP-expressing HeLa cells can be used not only for diagnosis of CCHF but also for epidemiological studies on CCHFV infections.     

Increased matrix metalloproteinase-9 activity in human ovarian cancer cells cultured with conditioned medium from human peritoneal tissue

Ovarian cancer cells disseminate by attachment to the peritoneal mesothelial cell surface of the abdominal cavity. We therefore investigated the influence of conditioned medium (CM) from human peritoneal tissues and mesothelial cells on the secretion of matrix metalloproteinases (MMPs) by ovarian cancer cells. The molecular weights of MMPs stimulating factors derived from human peritoneal tissues and mesothelial cells were estimated using microconcentrators with various cut-off membranes. Human peritoneal tissues were obtained from 12 surgical patients, and mesothelial cells were isolated from three peritoneal specimens. Exposure to CM from peritoneal tissue caused a concentration-dependent increase of the MMP-2 and MMP-9 bands in CM from NOM1 ovarian cancer cells, as shown by zymography. There was a significant difference in the increase of MMP-2 and MMP-9 (2.46-fold and 7.14-fold, respectively, at 0.4mg/ml protein; P < 0.005). CM from mesothelial cells also significantly increased the secretion of MMP-9 by NOM1 cells. The molecular size of possible MMP-9-stimulating factors secreted by peritoneal tissues and mesothelial cells was above M 100000. Further, CM of peritoneal tissues and mesothelial cells also induced the invasiveness of NOM1 cells. These findings suggest that mesothelial cells may secrete some factors which predominantly induce the MMP-9 production and increase invading cell numbers.     

Toxoplasma gondii infection inhibits the development of lupus-like syndrome in autoimmune (New Zealand Black x New Zealand White) F1 mice

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies and lupus nephritis. In the present study using New Zealand Black (NZB) x New Zealand White (NZW) F1 (NZBW F1) mice, we planned to investigate the effects of Toxoplasma gondii infection on the progress of lupus nephritis. Female NZBW F1 mice at the age of 2 months were perorally infected with T. gondii. The T. gondii infection reduced the number of mice developing proteinuria and immune complex deposits in their kidneys and prolonged their life span. A marked decrease in the levels of IgM and IgG anti-DNA antibodies, especially IgG2a and IgG3 subclasses, was observed in T. gondii-infected NZBW F1 mice at 9 months of age. The level of anti-HSP70 IgG autoantibody in the sera of NZBW F1 mice was significantly higher than that in control mice at 9 weeks after T. gondii infection. Moreover, NZBW F1 mice treated with anti-self heat shock protein 70 (HSP70) monoclonal antibody were substantially protected against the onset of glomerulonephritis. Further, down-regulation of intracellular expression of IFN-gamma and IL-10 was shown in spleen cells of T. gondii-infected NZBW F1 mice. This was consistent with the previous data indicating the involvement of Th1-type and Th2-type cytokines in the development of lupus-like nephritis. These results suggest that T. gondii infection is capable of preventing the development of autoimmune renal disorder in NZBW F1 mice.     

Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.     

Fine structure and mineral components of fibrous stonelike masses obtained from the human mesenteries

We investigated the fine structure and mineral components of 29 stonelike masses obtained from the mesenteries of four adult cadavers, using optical microscopy, backscattered electron imaging, scanning electron microscopy, energy-dispersive X-ray microanalysis, and X-ray diffraction. Although the overall appearance of the stonelike masses measuring about 5–20mm in diameter and 0.06–3.1g in dry weight was roughly grouped into smooth bulb- and uneven bulk-shaped types, all the calcified masses basically consisted of core and mantle regions. The smooth bulb-shaped masses had a broad mantle with many concentric rings, whereas the uneven bulk-shaped masses contained a large core. In their core regions, spherulitic and short bundle-shaped deposits composed of needle-shaped apatite crystals were mainly found among loose collagen fibers. Their mantle regions, on the other hand, showed the concentric structures of dense collagen fibers in the intra- and/or extrafibrous calcification with fine sandy grain-shaped deposits. The mineral elements were mainly Ca and P, and the major crystals were hydroxyapatite. Hexahedral whitlockite containing Mg was a minor component. The fiber-rich mantle regions showed lower calcification and lesser crystallization than the fiber-poor core region. When necrotic or some tumor adipose tissues and necrotic lymphoid tissues that might have been caused by some digestive diseases are recognized as foreign matter, their tissues occasionally will be calcified and grow into stonelike masses. These stonelike masses tend to occur more often in women than in men.     

WSX-1 is required for resistance to Trypanosoma cruzi infection by regulation of proinflammatory cytokine production

WSX-1 is a class I cytokine receptor with homology to the IL-12 receptors and is essential for resistance to Leishmania major infection. In the present study, we demonstrated that WSX-1 was also required for resistance to Trypanosoma cruzi. WSX-1-/- mice exhibited prolonged parasitemia, severe liver injury, and increased mortality over wild-type mice. WSX-1-/- splenocytes produced enhanced levels of Th2 cytokines, which were responsible for the prolonged parasitemia. Massive necroinflammatory lesions were observed in the liver of infected WSX-1-/- mice, and IFN-gamma that was overproduced in WSX-1-/- mice compared with wild-type mice was responsible for the lesions. In addition, vast amounts of various proinflammatory cytokines, including IL-6 and TNF-alpha, were produced by liver mononuclear cells in WSX-1-/- mice. Thus, during T. cruzi infection, WSX-1 suppresses liver injury by regulating production of proinflammatory cytokines, while controlling parasitemia by suppression of Th2 responses, demonstrating its novel role as an inhibitory regulator of cytokine production.