| 1. |
- Janson, Håkan, et al.
(författare)
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Protein D, the glycerophosphodiester phosphodiesterase from Haemophilus influenzae with affinity for human immunoglobulin D, influences virulence in a rat otitis model
- 1994
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Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 62:11, s. 4848-4854
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Tidskriftsartikel (refereegranskat)abstract
- A mutant lacking the ability to express the surface-exposed lipoprotein protein D was constructed by linker insertion and deletion mutagenesis of a cloned DNA insert containing the protein D structural gene from a nontypeable Haemophilus influenzae strain (NTHi). An isogenic NTHi mutant was isolated after transformation of genetically competent bacteria. The transformant was unreactive to a protein D-specific monoclonal antibody in a colony immunoassay. In addition, the mutant lacked the ability to synthesize detectable levels of protein D by protein staining, immunoblot methods, glycerophosphodiester phosphodiesterase activity, and binding studies of radiolabelled immunoglobulin D. The isogenic protein D-deficient mutant was compared with its parental strain for its ability to induce experimental otitis media in rats challenged with bacteria. An approximately 100-times-higher concentration of the mutant compared with that of the wild-type strain was required in order to cause otitis among all rats challenged with that given dose. The protein D mutant exhibited a generation time that was equal to that of the wild-type strain in complex broth medium. No difference in lipopolysaccharide expression was found between the mutant and the parental strain. These results suggest that protein D may influence the pathogenesis of NTHi in the upper respiratory tract.
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| 2. |
- Jonsson, Maria, et al.
(författare)
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Heterogeneity of outer membrane proteins in Borrelia burgdorferi : comparison of osp operons of three isolates of different geographic origins.
- 1992
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 60:5, s. 1845-53
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical and immunochemical studies of the outer membrane proteins of Borrelia burgdorferi have shown that the OspA and OspB proteins from strains of different geographic origins may differ considerably in their reactivities with monoclonal antibodies and in their apparent molecular weights. To further characterize this variation in Osp proteins between strains, the osp operons and deduced translation products from two strains, one from Sweden (ACAI) and one from eastern Russia (Ip90), were studied. Polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses confirmed differences between ACAI, Ip90, and the North American strain B31 in their Osp proteins. The sequences of the ospA and ospB genes of ACAI and Ip90 were compared with that of the previously studied osp operon of B31 (S. Bergström, V. G. Bundoc, and A. G. Barbour, Mol. Microbiol. 3:479-486, 1989). The osp genes of ACAI and Ip90, like the corresponding genes of B31, were found on plasmids with apparent sizes of about 50 kb and are cotranscribed as a single unit. Pairwise comparisons of the nucleotide sequences revealed that the ospA genes of ACAI and Ip90 were 85 and 86% identical, respectively, to the ospA gene of strain B31 and 86% identical to each other. The ospB sequences of these two strains were 79% identical to the ospB gene of B31 and 81% identical to each other. There was significantly greater similarity between the ospA genes of the three different strains than there was between the ospA and ospB genes within each strain. These studies suggest that the duplication of osp genes in B. burgdorferi occurred before the geographical dispersion of strains represented by ACAI, Ip90, and B31.
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| 3. |
- Katouli, Mohammad, et al.
(författare)
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Composition and diversity of intestinal coliform flora influence bacterial translocation in rats after hemorrhagic stress
- 1994
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 2:11, s. 4768-4774
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Tidskriftsartikel (refereegranskat)abstract
- Coliform bacteria are the most frequently reported bacteria to translocate afterhemorrhage. We investigated the correlation between composition and diversity of the cecal coliform flora and the degree of translocation in a rat model of hemorrhagic stress. Two groups of nine rats each were bled to 60 and 50 mm Hg mean arterial blood pressure, respectively. A sham-operated group without bleeding (n = 9) and a noninstrumented group (n = 6) served as controls. From each rat, 40 coliform isolates from the cecum and up to 16 from positive mesenteric lymph node (MLN) cultures were tested with an automated biochemical fingerprinting method. The phenotypic diversity of coliforms in each cecal sample was calculated as Simpson's diversity index (DI), and similarities between bacterial types in different samples were calculated as population similarity coefficients. Three rats in the sham-operated group and seven in each of the bled groups showed bacterial translocation. Of the different biochemical phenotypes (BPTs) found in the cecum of bled rats (mean, 6.5 BPTs), only a few were detected in MLNs (mean, 1.9 BPTs per MLN), with Escherichia coli being the dominant species. The translocating E. coli strains were mainly of two BPTs. Rats showing no translocation either did not carry these strains or had a high diversity of coliforms in the cecum. Furthermore, translocation of these coliform types was independent of their proportion in the cecum. In bled rats, the diversityof coliforms (mean DI, 0.53) was significantly higher than that in control groups (mean DI, 0.30; P = 0.004), suggesting that hemorrhage stimulates an increase in diversity of cecal coliforms. Rats with similar coliform flora and subjected to the same treatment showed similar patterns of translocation. Our results suggest that the composition of the coliformflora is an important factor in translocation and that certain coliform strains have the ability to translocate and survive in MLNs more easily than others.
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| 4. |
- Majeed, Meytham, et al.
(författare)
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Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells
- 1993
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 61:4, s. 1406-1414
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Tidskriftsartikel (refereegranskat)abstract
- The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.
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| 5. |
- Sadziene, Ariadna, et al.
(författare)
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A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein
- 1994
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Ingår i: Infection and Immunity. - : American Society for Microbiology (ASM). - 0019-9567 .- 1098-5522. ; 62:5, s. 2037-2045
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Tidskriftsartikel (refereegranskat)abstract
- Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.
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| 6. |
- Agace, W., et al.
(författare)
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Selective cytokine production by epithelial cells following exposure to Escherichia coli
- 1993
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Ingår i: Infection and Immunity. - 0019-9567. ; 61:2, s. 602-609
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Tidskriftsartikel (refereegranskat)abstract
- This study compared the repertoire of cytokines produced by epithelial cell lines and human peripheral blood monocytes in response to Escherichia coli. The A-498 and J82 urinary tract epithelial cell lines and human peripheral blood monocytes were exposed to E. coli Hu734. The cytokine content of single cells was detected by indirect immunofluorescence using monoclonal antibodies to interleukin-1α (IL-1α), IL-1β, tumor necrosis factor alpha (TNF-α), TNF-β, IL-6, IL-8, and granulocyte macrophage- colony-stimulating factor, and the number of positive cells was used to quantitate the response. The J82 bladder cell line stained positive for IL- 6, IL-8, and IL-1α. The IL-8 and IL-6 response peaked at 2 h, while the number of IL-1α-positive cells reached a peak 6 h after E. coli stimulation. The A-498 kidney cell line stained for IL-8 with a peak at 2 h and IL-6 with a peak at 6 h after E. coli stimulation. Peripheral blood monocytes stained for the cytokines IL-1α, IL-1β, IL-8, IL-6, and TNF-α but not for TNF-β and granulocyte macrophage-colony-stimulating factor after stimulation with E. coli. The results demonstrated that bacteria activated a cytokine response in the epithelial cell lines and monocytes. The epithelial cell lines had a more limited cytokine response profile than circulating monocytes, which may serve to limit the consequences of microbial exposure at the mucosal surface and help maintain the integrity of other tissue compartments.
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| 7. |
- Baker, N, et al.
(författare)
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Glycosphingolipid receptors for Pseudomonas aeruginosa.
- 1990
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Ingår i: Infection and immunity. - 0019-9567. ; 58:7, s. 2361-6
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Tidskriftsartikel (refereegranskat)abstract
- The binding of Pseudomonas aeruginosa to glycosphingolipids and to buccal and bronchial epithelial cells was analyzed. Three independently expressed specificities were found by bacterial binding to glycosphingolipids separated by thin-layer chromatography. All strains bound gangliotria- and gangliotetrasylceramide. All but one of the strains bound sialic acid-containing glycosphingolipids and lactosylceramide. The latter two specificities could be separated in that the lactosylceramide binding was retained and the sialic acid binding was suppressed when bovine serum albumin was used as a blocking agent in the thin-layer chromatography assay. The attachment to buccal epithelial cells, like the binding to sialylated compounds and lactosylceramide, was abolished by Formalin treatment of the bacteria, suggesting the importance of these specificities for cell adherence. In contrast, the binding to gangliotria- and gangliotetraosylceramide was retained by nonattaching Formalin-treated bacteria.
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| 8. |
- Hedges, S., et al.
(författare)
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Uroepithelial cells are part of a mucosal cytokine network
- 1994
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Ingår i: Infection and Immunity. - 0019-9567. ; 62:6, s. 2315-2321
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Tidskriftsartikel (refereegranskat)abstract
- This study compared the cytokine production of uroepithelial cell lines in response to gram-negative bacteria and inflammatory cytokines. Human kidney (A498) and bladder (J82) epithelial cell lines were stimulated with either Escherichia coli Hu734, interleukin 1α (IL-1α), or tumor necrosis factor alpha (TNF-α). Supernatant samples were removed, and the RNA was extracted from cells at 0, 2, 6, and 24 h. The secreted cytokine levels were determined by bioassay or immunoassay; mRNA was examined by reverse transcription-PCR. The two cell lines secreted IL-6 and IL-8 constitutively. IL-6 and IL-8 mRNA were constitutively produced in both cell lines; IL-1β mRNA was detected in J82 cells. IL-1α induced significantly higher levels of IL-6 secretion than did E. coli Hu734 or TNF-α. IL-1α and TNF-α induced significantly higher levels of IL-8 secretion than did E. coli Hu734. Secreted IL-1β was not detected; IL-1α and TNF-α were not detected above the levels used for stimulation. IL-1α, IL-1β, IL-6, and IL-8 mRNAs were detected in both cell lines after exposure to the stimulants. TNF-α mRNA was occasionally detected in the J82 cell line after TNF-α stimulation. Cytokine (IL-6 and IL-8) and control (glyceraldehyde 3-phosphate dehydrogenase [G3PDH] and β-actin) mRNA concentrations were quantitated with internal PCR standards. Cytokine mRNA levels relative to β-actin mRNA levels were the highest in E. coli- stimulated cells. In comparison, the cytokine mRNA levels relative to G3PDH mRNA levels were the highest in IL-1α-stimulated cells. β-Actin mRNA levels decreased after bacterial stimulation but not after cytokine stimulation, while G3PDH mRNA levels increased in response to all of the stimulants tested. These results suggested that E. coli Hu734 lowered the β-actin mRNA levels in uroepithelial cells, thus distorting the IL-6 and IL-8 mRNA levels relative to this control. In summary, E. coli IL-1α and TNF-α were found to activate the de novo synthesis and secretion of IL-6 and IL-8 in uroepithelial cells. These results emphasize the role of epithelial cells in cytokine-mediated responses during the early stages of infection.
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| 9. |
- Janson, Håkan, et al.
(författare)
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Limited diversity of the protein D gene (hpd) among encapsulated and nonencapsulated Haemophilus influenzae strains
- 1993
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Ingår i: Infection and Immunity. - 1098-5522. ; 61:11, s. 4546-4552
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Tidskriftsartikel (refereegranskat)abstract
- Protein D is a surface-exposed lipoprotein of the gram-negative bacterium Haemophilus influenzae with affinity for human immunoglobulin D myeloma protein. The gene encoding protein D (hpd) in a serotype b strain of H. influenzae was cloned. Escherichia coli carrying the hpd gene bound human myeloma immunoglobulin D. Nucleotide sequence analysis identified an 1,092-bp open reading frame that was more than 99% identical to the hpd gene from a nontypeable H. influenzae strain. In the deduced amino acid sequences for protein D, only 2 of 364 amino acid residues differed. The restriction fragment length polymorphism of the hpd region in different strains was analyzed by Southern blot analyses of PstI- or EcoRI-digested genomic DNA from 100 H. influenzae strains. The analysis was performed by using isolated fragments of the cloned hpd gene, originating from the nontypeable H. influenzae 772, as probes. All strains tested had DNA sequences with a high degree of homology to the hpd probes. The analysis also showed that restriction endonuclease sites within the gene were more conserved than sites adjacent to the hpd gene. An interesting difference between type b strains and unencapsulated strains was observed. The majority of type b strains seem to have a 1.4-kbp DNA fragment upstream of the hpd gene that is absent in nontypeable strains. On the basis of the high degree of conservation of the hpd gene among H. influenzae strains, we conclude that protein D is a possible vaccine candidate.
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| 10. |
- Janson, Håkan, et al.
(författare)
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Protein D, an immunoglobulin D-binding protein of Haemophilus influenzae: cloning, nucleotide sequence, and expression in Escherichia coli
- 1991
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Ingår i: Infection and Immunity. - 1098-5522. ; 59:1, s. 119-125
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Tidskriftsartikel (refereegranskat)abstract
- The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli from an endogenous promoter, and the gene product has an apparent molecular weight equal to that of H. influenzae protein D (42,000). The complete nucleotide sequence of the gene for protein D was determined, and the deduced amino acid sequence of 364 residues includes a putative signal sequence of 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Cys, for bacterial lipoproteins. The sequence of protein D shows no similarity to those of other immunoglobulin-binding proteins. Protein D is the first example of immunoglobulin receptors from gram-negative bacteria that has been cloned and sequenced.
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