Analysis of Host Cells Associated with the Spv-Mediated Increased Intracellular Growth Rate of Salmonella typhimurium in Mice

The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P. A. Gulig and T. J. Doyle, Infect. Immun. 61:504–511, 1993). The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae. To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice. Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv⁺ or Spv⁻ S. typhimurium strains at 5 days postinoculation. When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv⁺ versus Spv⁻ S. typhimurium was not changed after oral inoculation. In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv⁺ and Spv⁻ salmonellae. To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv⁺ and Spv⁻ S. typhimurium strains. InvA⁻ Spv⁺ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA⁻ Spv⁻ salmonellae were only slightly attenuated relative to InvA⁺ Spv⁻ salmonellae. Invasion-defective salmonellae still exhibited the Spv phenotype. Therefore, infection of nonphagocytes is not involved with the Spv virulence function. Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv⁻ S. typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.     

Developmental switch from GABA to glycine release in single central synaptic terminals

Early in postnatal development, inhibitory inputs to rat lateral superior olive (LSO) neurons change from releasing predominantly GABA to releasing predominantly glycine into the synapse. Here we show that spontaneous miniature inhibitory postsynaptic currents (mIPSCs) also change from GABAergic to glycinergic over the first two postnatal weeks. Many 'mixed' mIPSCs, resulting from co-release of glycine and GABA from the same vesicles, are seen during this transition. Immunohistochemistry showed that a large number of terminals contained both GABA and glycine at postnatal day 8 (P8). By P14, both the content of GABA in these mixed terminals and the contribution of GABA to the mixed mIPSCs had decreased. The content of glycine in terminals increased over the same period. Our results indicate that switching from GABAergic to glycinergic inputs to the LSO may occur at the level of a single presynaptic terminal. This demonstrates a new form of developmental plasticity at the level of a single central synapse.     

Beneficial effect of amrinone on murine cardiac allograft survival.

Amrinone is a non-glycoside positive inotropic agent with an inhibitory effect on a cyclic adenosine monophosphate (AMP) phosphodiesterase isoenzyme. In the present study, we examined the immunosuppressive action of amrinone, since several other cyclic AMP-elevating agents have been shown to suppress T lymphocyte activation. First, the in vivo effects of amrinone were investigated. Oral amrinone treatment, at 40 mg/kg per day, significantly prolonged median cardiac allograft survival compared with non-treated controls (22.0 days versus 10.5 days, P < 0.01) when DBA/2 mouse hearts (H-2d) were heterotopically transplanted into C57B1/6 mice (H-2b). Histopathological examination showed that there was less prominent cellular infiltration in the amrinone-treated than in the non-treated allografts. Plasma amrinone concentrations of mice after a single oral dose of 40 mg/kg were within the range of clinical relevance. To clarify the mechanism of action, in vitro studies were done. The generation of specific cytotoxic T lymphocytes after mixed lymphocyte culture was significantly suppressed by addition of amrinone to the culture medium at 5 micrograms/ml. The production of IL-2 and the interferon-gamma during mixed lymphocyte culture was also suppressed by amrinone at 5 micrograms/ml. However, the level of intracellular cyclic AMP in mouse splenic lymphocytes was not affected significantly by the same dose of amrinone. In conclusion, amrinone has immunosuppressive actions at the therapeutic doses, and it may be a beneficial agent for therapy against acute cardiac allograft rejection.     

Lon,a stress-induced ATP-dependent protease,is critically important for systemic Salmonella enterica serovar typhimurium infection of mice

Studies on the pathogenesis of Salmonella enterica serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics-the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of S. enterica serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of S. enterica serovar Typhimurium in mice. Determination of 50% lethal doses for the lon disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the lon disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the lon disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with S. enterica serovar Typhimurium, which can be fatal. Of further interest is the finding that the lon disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.     

Clinical heterogeneity of neonatal intrahepatic cholestasis caused by citrin deficiency: case reports from 16 patients

A deficiency of citrin, which is encoded by the SLC25A13 gene, causes both adult-onset type II citrullinemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). We analyzed 16 patients with NICCD to clarify the clinical features of the disease. Severe intrahepatic cholestasis with fatty liver was the most common symptom, but the accompanying clinical features were variable, namely; suspected cases of neonatal hepatitis or biliary atresia, positive results from newborn screening, tyrosinemia, failure to thrive, hemolytic anemia, bleeding tendencies and ketotic hypoglycemia. Laboratory data showed elevated serum bile acid levels, hypoproteinemia, low levels of vitamin K-dependent coagulation factors, and hypergalactosemia. Hypercitrullinemia was detected in 11 out of 15 patients examined. Most of the patients were given a lactose-free and/or medium chain triglycerides-enriched formula and lipid-soluble vitamins. The prognosis of the 16 patients is going fairy well at present, but we should observe these patients carefully to see if they manifest any symptom of CTLN2 in the future.     

Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases.

A new monoclonal antibody (MoAb) HA58 (IgG1) was prepared, which recognizes the binding site on the intercellular adhesion molecule-1 (ICAM-1) antigen to the lymphocyte function-associated antigen-1 (LFA-1). The double-determinant immunoassay (DDIA) was established with use of MoAb HA58 and another anti-ICAM-1, MoAb CL207, to detect the soluble, shedding ICAM-1 antigen. Human recombinant interferon-gamma (IFN-gamma) induced not only the expression of cell surface ICAM-1, but also the shedding ICAM-1 antigen in an IFN-gamma concentration-dependent and incubation-time-dependent manner. DDIA was applied to detect the shedding ICAM-1 antigen in the sera of patients with malignant or benign diseases. The incidence of positivity for ICAM-1 antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM-1 antigen. These findings suggest that serum ICAM-1 antigen may be a useful marker to monitor tumor burden in cancer patients.     

Comparative studies of commercially available particle agglutination assay and enzyme-linked immunosorbent assay for screening of human T-cell leukemia virus type I antibodies in blood donors.

Human T-cell leukemia virus type I (HTLV-I) transmission during blood transfusion can be prevented by screening for and eliminating blood containing anti-HTLV-I antibodies. For this purpose, we evaluated two commercial test kits for HTLV-I antibodies, Serodia-ATLA (particle agglutination assay [PA]) and Eitest-ATL (enzyme-linked immunosorbent assay [ELISA]), by using serum samples from Japanese blood donors. Of 2,316 serum samples, 39 (1.7%), 34 (1.5%), and 28 (1.2%) were positive for the antibody by PA, ELISA, and immunofluorescence (IF), respectively. The coincidence rate for antibody-positive and antibody-negative results was 99.5% between PA and IF, whereas it was 99.6% between ELISA and IF. The serum samples which were positive by IF were also positive by PA, whereas 2 of 26 IF-positive samples were negative by ELISA. All the samples positive by both PA and ELISA were shown to be positive by IF unequivocally. The samples positive by only one of the two methods (PA or ELISA) were different. Eleven and eight serum samples that were negative by IF but positive by PA and ELISA, respectively, were further studied for HTLV-I antibody by radioimmunoprecipitation. Three of the former but none of the latter were found to be positive for antibody. Moreover, a PA-positive but IF-negative serum sample was shown to have significantly decreased PA titers after 2-mercaptoethanol treatment. The PA was further shown to detect not only IgG antibody but also IgM antibody to HTLV-I after separation of this serum sample by high-performance liquid chromatography.     

Lectin histochemical study on the olfactory organ of the newt,Cynops pyrrhogaster,revealed heterogeneous mucous environments in a single nasal cavity

Expression patterns of glycoconjugates were examined by lectin histochemistry in the nasal cavity of the Japanese red-bellied newt, Cynops pyrrhogaster. Its nasal cavity consisted of two components, a flattened chamber, which was the main nasal chamber (MNC), and a lateral diverticulum called the lateral nasal sinus (LNS), which communicated medially with the MNC. The MNC was lined with the olfactory epithelium (OE), while the diverticulum constituting the LNS was lined with the vomeronasal epithelium (VNE). Nasal glands were observed beneath the OE but not beneath the VNE. In addition, a secretory epithelium was revealed on the dorsal boundary between the MNC and the LNS, which we refer to as the boundary secretory epithelium (BSE) in this study. The BSE seemed to play an important role in the construction of the mucous composition of the VNE. Among 21 lectins used in this study, DBA, SBA and Jacalin showed different staining patterns between the OE and the VNE. DBA staining showed remarkable differences between the OE and the VNE; there was intense staining in the free border and the supporting cells of the VNE, whereas there was no staining or weak staining in the cells of the OE. SBA and Jacalin showed different stainings in the receptor neurons for the OE and the VNE. Furthermore, UEA-I and Con A showed different stainings for the nasal glands. UEA-I showed intense staining in the BSE and in the nasal glands located in the ventral wall of the MNC (VNG), whereas Con A showed intense staining in the BSE and in the nasal glands located in the dorsal and medial wall of the MNC (DMNG). The DMNG were observed to send their excretory ducts into the OE, whereas no excretory ducts were observed from the VNG to the OE or the VNE. These results suggested that the secretion by the supporting cells as well as the BSE and the DMNG establishes that there are heterogeneous mucous environments in the OE and the VNE, although both epithelia are situated in the same nasal cavity.