In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs). Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur. These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.
Although the involvement of T helper (Th1) cells is central to protection against intracellular bacteria, including Mycobacterium tuberculosis, the involvement of Th2 cells, characterized by potent interleukin (IL)-4 secretion in mycobacterial infection is still unclear. In order to clarify the role of IL-4 in murine tuberculosis, IL-4-deficient mutant mice, IL-4 knockout (IL-4 KO) mice, were utilized. The mice were infected with H37Rv, Kurono or BCG Pasteur via an airborne infection route by placing them in the exposure chamber of a Middlebrook airborne infection apparatus. Their capacity to control mycobacterial growth, granuloma formation, cytokine secretion, and nitric oxide (NO) production were examined. These mice developed large granulomas, but not necrotic lesions in the lungs, liver or spleen (P<0.05). This was consistent with a significant increase in lung colony-forming units (CFU). Compared with levels in wild-type mice, upon stimulation with mycobacteria, splenic IL-10 levels were low and IL-6 levels were intermediate, but interferon (IFN)-γ and IL-12 levels were significantly higher. IL-18 levels were within the normal range. The level of NO production by alveolar macrophages of the IL-4 KO mice was similar to that of the wild-type mice. Granulomatous lesion development by IL-4 KO mice was inhibited significantly by treatment with exogenous recombinant IL-4. These findings were not specific to the IL-4 KO mice used. Our data show that IL-4 may play a protective role in defense against mycobacteria, although IFN-γ and TNF-α play major roles in it. Our data do not rule out an IFN-γ-independent function of IL-4 in controlling tuberculosis.
Serological variations were examined among 12 type or reference strains and 91 oral isolates of vitamin B6-dependent Abiotrophia and Granulicatella spp. Rabbits were immunized with whole cells of 12 selected strains and 10 typing antisera were obtained, which were unreactive with the Lancefield group A to G antigen preparations. The reactivity of the antisera and autoclaved cell surface antigen extracts was tested by double diffusion in agar gel and a capillary precipitin test. These typing antisera categorized all Abiotrophia defectiva strains, all except one Granulicatella elegans strain, three-quarters of the Granulicatella adiacens, and half of the Granulicatella paraadiacens into 8 serotypes and 2 subserotypes. The Granulicatella balaenopterae type strain was unserotypable. All A. defectiva strains were serotype I, some of which were divided into subserotype I-1 and/or I-5. The G. adiacens strains generally belonged to serotype II or III, and the G. paraadiacens strains to serotype IV, V or VI. All G. adiacens or G. paraadiacens serotype II strains were also subserotype I-5. The G. elegans strains were serotype VII or VIII. These Abiotrophia and Granulicatella serotypes were undetectable among 33 strains of the other 11 species including the bacteriolytic enzyme-producing but vitamin B6-independent strains of Streptococcus, Enterococcus, Dolosigranulum and Aerococcus. The proposed serotyping system for Abiotrophia and Granulicatella spp. would be helpful in the identification and classification of these unique coccal isolates in ecological and epidemiological studies.
Salmonella species represent a leading cause of gastroenteritis worldwide. More recently, they have been proposed as putative vaccine delivery vehicles in humans. Oral infection with Salmonella leads to invasion of the intestinal epithelial barrier and subsequent interaction with mucosal macrophages. In this study, we investigated the fate of Salmonella typhimurium-infected human macrophages differentiated from blood monocytes by GM-CSF. Wild type S. typhimurium strain SL1344 induced macrophage surface blebbing and caused the release of host cytoplasmic lactate dehydrogenase beginning 30min post-infection. Three hours later more than 80% of the macrophages in the culture were killed. In contrast, during the same period, macrophages infected with the non-invasive S. typhimurium strain BJ66 remained viable. Chromatin fragmentation is a hallmark of cells undergoing apoptosis. Using TUNEL analysis, we observed chromatin fragmentation in macrophages infected with SL1344 but not in BJ66 infected cells. Consistent with this observation, we found that pretreatment of human macrophages with an inhibitor of caspase-3, a member of the pro-apoptotic enzyme family shown to be involved in S. typhimurium-induced killing of mouse macrophages, reduced SL1344-mediated cytotoxicity by 40%. Our study provides the first evidence that invasive S. typhimurium induces apoptosis in human macrophages that were differentiated from blood monocytes by GM-CSF, and that cell death is a caspase-dependent phenomenon.
The β-lactamase inhibitor, sulbactam, was tested for β-lactamase inhibitory activity in Pseudomonas aeruginosa cells producing various levels of both the MexAB-OprM efflux pump and β-lactamase. We found that sulbactam lowered the MICs of cefoperazone and piperacillin by inhibiting the β-lactamase 8-fold in the cell producing a constitutively high level of AmpC-type β-lactamase and a wild-type level of MexAB-OprM pump compared with that without sulbactam. The MICs of cefoperazone and piperacillin in the cell producing a constitutively high level of both the efflux pump and β-lactamase under the presence of sulbactam were 8 and 4 times, respectively, lower than that without sulbactam. The MICs of sulbactam in the cell producing a constitutively high and a wild-type level of the efflux pump were 16- and 8-fold higher, respectively, than that in the mutant lacking the efflux pump. We concluded that sulbactam exerts potent β-lactamase inhibitory activity in the cell producing a high level of efflux pump, in spite of the fact that sulbactam serves as a substrate of the MexAB-OprM pump. Increasing amounts of sulbactam over the weight of β-lactams further strengthen the effect of β-lactam antibiotics.
Eleven pure cultures of Borrelia from 3 species of wild rodents (Apodemus agrarius, Mus formosanus, Rattus losea) captured in Taichung, located in the center of Taiwan island, and on Kinmen Island were characterized. Five isolates showed restriction fragment length polymorphism (RFLP) patterns of 5S-23S rRNA gene intergenic spacer sequences identical to those of strains 5MT and 10MT, identified as Borrelia valaisiana, which were isolated in the southern tip of South Korea. Although the remaining six isolates showed novel RFLP patterns, these isolates showed more similarity to members of B. valaisiana from Korea, Japan and Europe based on 16S rRNA gene and flagellin gene sequences. This led us to speculate that transmission and proliferation of this type of borrelia occurred between Taiwan and the southern part of South Korea.
Human cytomegalovirus (CMV) is a β-herpesvirus that causes a chronic subclinical infection in healthy man. The immune system is unable to eliminate the virus completely, allowing virus to persist in a latent state. In the immunocompromised host, this equilibrium is disturbed, resulting in a clinical infection. In immunocompromised rats, clinical CMV infection is associated with an increase in NK cells and CD8+ T cells, including a phenotypically aberrant CD8+ T cell population. Using flow cytometry, we examined the effect of acute CMV infection on the composition of leukocyte subsets in immunocompromised patients. Therefore, we used peripheral blood of CMV seronegative patients receiving a kidney from a seronegative (control group) or a seropositive donor. Of the patients receiving a seropositive kidney, only the patients undergoing acute CMV infection were included (experimental group). Special attention was paid to the phenotype of the cytotoxic T cells. The development of acute CMV infection resulted in an increased NK cell number and an activation of both CD4+ and CD8+ T cells, as determined by HLA-DR expression. An aberrant CD8+ T cell subset with decreased expression of CD8 and TCRαβ appeared in the infected patients. Furthermore, the size of this subpopulation of CD8+ T cells is positively correlated with the viral load.
Attempts were made to infect human vascular smooth muscle cells derived from the pulmonary artery (hPASMC) with two different human immunodeficiency virus (HIV) vector systems. ADA/Luc or HXB2/Luc were generated by cotransfection of luciferase reporter gene vector, pNL4-3-Luc-E-R-, and one of two envelope expressing vectors, pSMADA (R5) or pSMHXB2 (X4). The VSV-G/Luc or VSV-G/GFP were produced by a three-plasmid expression system which consisted of vesicular stomatitis virus G protein (VSV-G) expressing vector, packaging plasmid, and one of two reporter genes (pHR'-CMV-Luc or pHR'-CMV-GFP). We used hPASMC, U87.CD4.CCR5 and U87.CD4.CXCR4 for infection. Neither ADA/Luc nor HXB2/Luc could infect hPASMC, though they could infect U87.CD4 with corresponding coreceptors. On the other hand, the transduction of both VSV-G/Luc and VSV-G/GFP to hPASMC was remarkable. At day 3, the relative proportion of positive cells of hPASMC infected with VSV-G/GFP was 15%. The above finding indicates a direct role of HIV-1 infection in pulmonary hypertension 'a rare complication of HIV-1 infection' and HIV-based vectors could introduce foreign genes into hPASMC for gene therapy of pulmonary hypertension.
The activity of mononuclear cells to inhibit plaque formation of varicella-zoster virus (VZV) was investigated by an in vitro infectious center assay. Peripheral blood mononuclear cells (PBMC) inhibited VZV plaque formation by co-cultivation with VZV-infected fibroblasts. As compared to mononuclear cells from normal individuals, mononuclear cells from umbilical cord blood and from patients receiving corticosteroids showed a significant decrease in the ability to inhibit viral replication. This ability was significantly increased for mononuclear cells collected during the acute phase of varicella. PBMC obtained from patients in the acute phase of varicella produced significantly higher amounts of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-12 in the supernatant compared with those of healthy individuals. These data suggest that the cytokines have an important role in the inhibition of the spread of VZV at an early stage of varicella. Th1 type adaptive immunity might play a major role in VZV infection.
In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-γ, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.
In the present study, we examined whether natural killer (NK) cells have direct fungicidal activity against Cryptococcus neoformans. Splenic NK cells were obtained from SCID mice and stimulated with a combination of interleukin (IL)-12 and IL-18 in flat culture plates or round tubes. They were then or at the same time cultured with the yeast cells and the number of viable yeast cells was examined. We could not detect direct fungicidal activity by NK cells under any culture condition, although they produced a large amount of IFN-γ and exerted marked cytotoxic activity against YAC-1 cells. On the other hand, NK cells significantly potentiated the nitric oxide-mediated cryptococcocidal activity of thioglycolate-elicited peritoneal macrophages obtained from SCID mice upon stimulation with IL-12 and IL-18. The culture supernatants of NK cells stimulated with IL-12 and IL-18 provided similar results when used in place of NK cells. The induction of macrophage anticryptococcal activity by NK cells and NK cell culture supernatants were both mediated by IFN-γ because the specific mAb almost completely abrogated such effect. Considered collectively, our results suggested that NK cells may play a regulatory role in potentiating macrophage-mediated fungicidal mechanisms in host resistance to infection with C. neoformans rather than exerting a direct killing activity against the fungal pathogen.
The nature of target molecules of natural killer (NK) cell-mediated lysis remains to be elucidated. As we previously reported, mAb 109 recognizes one of the tumor-associated antigens, designated as 109 antigen (Ag), expressed on the cell surface of rat fibrosarcomas W31 and W14, which are transformants of WFB (rat fetal fibroblast cell line) with H-ras oncogene. 109Ag was thought to be a target structure of NK cells since mAb 109 inhibited NK cell-mediated lysis against W31 and W14. Here, we demonstrate by molecular cloning that 109Ag is identical to rat CD44. Immunoprecipitation and immunoblotting studies also showed that mAb 109 and anti-rat CD44 mAb OX-50 recognize the same protein of W31 cell lysates with an 86kDa molecular size. CD44 was suggested to be a target structure of NK cell-mediated lysis; however, rat CD44 cDNA transfection alone into CD44 null cell lines did not result in up-regulation of target cell susceptibility to NK cell-mediated lysis. Our results therefore indicated that CD44 may play a crucial role as one of the target structures in our rat fibrosarcoma system though the cell surface expression of CD44 alone does not affect NK susceptibility of the target cells.
The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon γ. Interferon γ-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.
A rapid and efficient method of purification of staphylococcal exfoliative toxin A, the causative agent of staphylococcal scalded skin syndrome (SSSS), has been developed. It is based on ammonium sulfate precipitation of the culture supernatant and hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B. This procedure results in 87-fold purification of this toxin, which appears as a single band in sodium dodecyl sulfate-polyacrylamide gels.
Borreliae have genomes composed of both linear and circular replicons. We have characterized the organization of linear DNA molecules from the Borrelia duttonii strain Ly. It contains a linear one megabase chromosome and 12 linear plasmids of 11 to 200kb in size. A variant of the strain obtained after successive in vitro cultivation in BSKII medium had a 69kb molecule instead of the 44kb linear plasmid. No detectable differences in the growth rates and cellular structures were found. Southern hybridization using the vsp33 gene sequence from Borrelia hermsii as a probe showed that both plasmids (69 and 44kb molecules) contained a similar part of the sequence. The spirochetes of the parental strain cause erythrocytes to aggregate in mice blood, but the variant did not form such aggregates and seemed to have lost its infectivity in mice. Size conversion of the linear plasmid may be associated with the host-parasite relationship in mammals.
To investigate the influence of corticosteroid administration on the serum level of macrophage migration inhibitory factor (MIF), sera obtained from 9 patients with Vogt-Koyanagi-Harada's disease who had been treated with high-dose corticosteroid were analyzed. The serum MIF levels of most patients were prominently increased on day 7 and/or day 14 after corticosteroid treatment. No TNF-α was detected in the sera. The average serum MIF level of nine patients at the highest stages after corticosteroid administration was significantly higher than that before the corticosteroid treatment. It seems that MIF is a unique cytokine and acts together with corticosteroid to regulate inflammation and immunity.