FEMS immunology and medical microbiology最新文献

We have previously shown that matrix metalloproteinase-9 (MMP-9) activity is greatly enhanced within the active chronic inflammation of Helicobacter pylori infected individuals, of which a major fraction derives from macrophages in the tissue. Here, we have investigated the ability of macrophages to secrete MMPs in response to H. pylori. Human macrophages secrete MMP-9 in response to live and inactivated H. pylori, as well as to specific bacterial products. Protein kinase C, phosphatiolylinositol 3-kinase and calcium uptake channels all play a role in MMP-9 secretion, whereas neither tumour necrosis factor alpha, interleukin-8, nor interleukin-1β autocrine stimulation appear to contribute. We conclude that human macrophages have the ability to react directly against several H. pylori derived factors, utilising several signalling pathways.

Mucosal secretions contain a range of defense effector molecules including antimicrobial peptides and proteinase inhibitors. These molecules play a central role in host defense against infection, and in a variety of immune and inflammatory reactions. The aim of this study was to analyze the levels of neutrophil defensins, the cathelicidin hCAP-18/LL-37, and the proteinase inhibitors secretory leukocyte proteinase inhibitor, SKALP/elafin and cystatin M/E in various mucosal secretions and urine. We show here that especially seminal plasma is characterized by high concentrations of hCAP-18/LL-37, SLPI, SKALP/elafin and cystatin M/E. The results of this study demonstrate that each mucosal secretion is characterized by a unique profile of effector molecules, which may supply individual mucosal secretions with specific properties related to the control of local infection and inflammation.

Immunostimulatory DNA sequences and their synthetic oligonucleotide analogs (CpG-ODN) activate innate immunity and can stimulate antibacterial effects against numerous intracellular pathogens. While it has been shown previously that CpG-ODN inhibit growth of Mycobacterium avium in murine and human macrophages, we now report that Mycobacterium tuberculosis growth can be inhibited by CpG-ODN treatment of human monocyte-derived macrophages (hMDM). This inhibitory effect was reversed by IFN-γ, which has been shown repeatedly to enhance the growth of virulent M. tuberculosis in cultured hMDM. The antibacterial effect of CpG-ODN in human macrophages was specific for M. tuberculosis when compared to other intracellular pathogens including Listeria monocytogenes and Salmonella enterica serovar Dublin. These data indicate that CpG-ODN can improve the ability of hMDM to contain growth of virulent M. tuberculosis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 μg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 °C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.

Control of infection by Chlamydia trachomatis usually requires the production of interferon-γ. Whilst this can be produced by CD4+ and CD8+ T lymphocytes, natural killer (NK) cells are another important source of this cytokine, and are known to be recruited early to the infected genital tract. We show that both IL-12 and IL-18, which synergise to stimulate NK cells to produce interferon-γ, are produced following the infection of dendritic cells and epithelial cells respectively, since supernatants from infected cells could substitute for recombinant cytokines. These results suggest that conditions, which lead to NK cell production of interferon-γ will be present at the site of infection, where epithelial cells are the primary targets of infection and dendritic cells within the epithelium can also access the bacterium.

Clostridium histolyticum culture supernatant contains numerous enzymes, which exert a cytotoxic effect on host cells. This includes lethal toxin, clostripain and high-potassium-sensitive toxin. Since the number of C. histolyticum infections increased during the last several years, it seems worthwhile to evaluate whether protease inhibitors, used for the treatment of many diseases, could influence toxicity, and thus, pathogenicity of C. histolyticum. In this study we evaluated in vitro the influence of four common protease inhibitors: aprotinin, phenylmethylsulphonyl fluoride (PMSF), l-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone-HCl (TLCK) and chymostatin on the toxicity of C. histolyticum supernatant towards human epithelial HeLa cells. We show that aprotinin has no effect, while PMSF, TLCK and chymostatin potentiate the cytotoxic activity of C. histolyticum, probably by hindering natural defence mechanisms of cells. In addition, PMSF and TLCK block clostripain enzymatic activity, while chymostatin leaves it intact. Elevated cytotoxicity of the supernatant is not related to the quantity of high-potassium-sensitive toxin, as was reported previously, since desalted supernatant still exerted its strong toxic effect. Our results show that addition of protease inhibitors for treating diseases complicated by concurrent C. histolyticum infection must require special attention.

Plasmid pSET152 is a broad host range mobilizable vector which integrates into streptomyces chromosome utilizing att site and int function of ∅C31. Transformation of this plasmid into Mycobacterium smegmatis mc² 155 SMR5 gave stable transformants carrying the pSET152 as an integrated copy. Integration occurred at the cross over sequence 5′TTG disrupting the gatA gene (Glu-tRNA^Gln amidotransferase subunitA), which is non-essential under conditions used. Recombinant pSET152 plasmids carrying mce1 locus of Mycobacterium leprae were used to construct M. smegmatis transformants carrying the mce1 locus in their chromosome. RT-PCR analysis revealed specific transcripts of M. leprae mce in M. smegmatis. The transcribed mRNA carried intergenic regions between genes of mce1 locus indicating that mce1 locus is an operon. Examination of M. leprae specific mRNA from lepromatous leprosy patient’s biopsy showed that mce locus is transcribed as an operon in the pathogen also.

Nontypeable Haemophilus influenzae (NTHI) are a major cause of human infections. We previously demonstrated high affinity and high specificity binding of NTHI to minor gangliosides of human respiratory (HEp-2) cells and macrophages, but not to brain gangliosides. We further identified the NTHI-binding ganglioside of human macrophages as α2,3-sialylosylparagloboside (IV³NeuAc-nLcOse₄Cer, nLM1), which possesses a neolacto core structure that is absent in brain gangliosides. This supported a hypothesis that lacto/neolacto core carbohydrates are critical for NTHI-ganglioside binding. To investigate, we determined the core carbohydrate structure of NTHI-binding gangliosides of HEp-2 cells, through multiple approaches, including specific enzymatic degradation, mass spectral analysis and gas–liquid chromatography. Our analyses denote the following critical structural attributes of NTHI-binding gangliosides: (1) a conserved lacto/neolacto core structure; (2) requisite sialylation, which may be either internal or external, with α2,3 (human macrophages) or α2,6 (HEp-2 cells) anomeric linkages; (3) internalized galactose residues. Mass spectral and gas chromatographic analyses confirm that NTHI-binding gangliosides of HEp-2 cells possess lacto/neolacto carbohydrate cores and identify the structure of the major peak as NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer (α2,6-sialosylparagloboside, nLM1). Collectively, our studies denote NTHI-binding gangliosides as lacto/neolacto series structures.

The aim of the study was to evaluate intracellular interferon-γ (IFN-γ), and interleukin-4 (IL-4) levels in pre- and post-treatment periods of brucellosis patients and to determine the relationship between these parameters and patients’ clinical findings. Twenty-five patients diagnosed as brucellosis and 11 aged-matched healthy volunteers were included in the study. CD3⁺CD4⁺ T lymphocytes levels were significantly lower in patients with brucellosis as compared to the control group. CD3⁺CD8⁺ T lymphocytes and CD3⁺IFN-γ⁺ levels were increased in brucellosis patients compared with the control group. CD4⁺IFN-γ⁺ and CD4⁺IL-4⁺ levels were no different between patients and healthy individuals. CD3⁺IL-4⁺ levels decreased in patients compared with healthy controls. Pre-treatment CD3⁺IFN-γ⁺ levels dramatically increased in patients responsive to management compared with the unresponsive ones. In responsive cases, CD3⁺IFN-γ⁺ levels decreased statistically after the treatment while in unresponsive cases no meaningful change was observed with respect to treatment. Adding IFN-γ to the treatment for improving the depleted levels of IFN-γ can be beneficial in patients with brucellosis who shows a tendency to chronicity or patients who do not respond to the treatment.

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P−) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD₅₀ of P+ strain and those with 1/10 LD₅₀ of P− strain produced a high level of gamma interferon (IFN-γ) upon stimulation with heat-killed bacteria, and CD4⁺ T cells were exclusively responsible for IFN-γ production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-γ response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-γ-producing CD8⁺ T cells as well as the increase in IFN-γ-producing CD4⁺ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.