Biomedical subjects
Publications and source records attributed to M Raftery.
A strain of Listeria monocytogenes recovered from blood and cerebrospinal fluid had no detectable catalase activity, a characteristic used for primary identification. The sporadic occurrence of pathogenic catalase-negative strains highlights the need for a reconsideration of diagnostic criteria and questions the role of catalase in the pathogenesis of listeria infection.
AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.
The initial stage of oxidation of high density lipoproteins (HDL) is accompanied by the lipid hydroperoxide-dependent, selective oxidation of two of the three Met residues of apolipoprotein A-I (apoA-I) to Met sulfoxides (Met(O)). Formation of such selectively oxidized apoA-I (i.e. apoA-I(+32)) may affect the antiatherogenic properties of HDL, because it has been suggested that Met(86) and Met(112) are important for cholesterol efflux and Met(148) is involved in the activation of lecithin:cholesterol acyl transferase (LCAT). We therefore determined which Met residues were oxidized in apoA-I(+32) and how such oxidation of apoA-I affects its secondary structure, the affinity for lipids, and its ability to remove lipids from human macrophages. We also assessed the capacity of discoidal reconstituted HDL containing apoA-I(+32) to act as substrate for LCAT, and the dissociation of apoA-I and apoA-I(+32) from reconstituted HDL. Met(86) and Met(112) were present as Met(O), as determined by amino acid sequencing and mass spectrometry of isolated peptides derived from apoA-I(+32). Selective oxidation did not alter the alpha-helicity of lipid-free and lipid-associated apoA-I as assessed by circular dichroism, and the affinity for LCAT was comparable for reconstituted HDL containing apoA-I or apoA-I(+32). Cholesteryl ester transfer protein mediated the dissociation of apoA-I more readily from reconstituted HDL containing apoA-I(+32) than unoxidized apoA-I. Also, compared with native apoA-I, apoA-I(+32) had a 2- to 3-fold greater affinity for lipid (as determined by the rate of clearance of multilamellar phospholipid vesicles) and its ability to cause efflux of [(3)H]cholesterol, [(3)H]phospholipid, and [(14)C]alpha-tocopherol from lipid-laden human monocyte-derived macrophages was significantly enhanced. By contrast, no difference was observed for cholesterol and alpha-tocopherol efflux to lipid-associated apolipoproteins. Together, these results suggest that selective oxidation of Met residues enhances rather than diminishes known antiatherogenic activities of apoA-I, consistent with the overall hypothesis that detoxification of lipid hydroperoxides by HDL is potentially antiatherogenic.
For millions of years viruses have adapted strategies to interfere with the immune defense of the host, which in turn has to deal with this challenge. In general the antiviral defense remains one step behind the pathogen. To achieve this strategic advantage large DNA-containing Viruses encode cellular homologues that mimic or counteract key molecules of the host immune system. Understanding how these cellular homologues enable the viruses to evade the antiviral defense and persist in the host for the lifetime will ultimatively lead also to a better understanding of the principle functions of the immune system. In this review we focused on cellular homologues encoded by human herpesviruses and discuss the functional consequences of their expression.
Viral IL-10 (vIL-10), the IL-10 homologue of Epstein-Barr virus, has so far been described as a cytokine that solely inhibits T cell function. Here we show in vivo and in vitro that after long-term secretion vIL-10 has a stimulatory effect on T cells. For this purpose we employed transfectants derived from a mastocytoma cell line (P815 cells, H-2(d)) constitutively secreting vIL-10 (P815-vIL-10) or expressing the co-stimulatory molecule B7-1 (P815-B7). After in vitro stimulation of splenocytes from syngeneic DBA/2 mice for 7 days in the presence of P815-vIL-10 cells we could detect a marked reduction of proliferation as well as cytotoxicity against P815 target cells. However, this inhibitory effect was reversed when stimulation with P815-vIL-10 cells was extended to 14 days. In vivo P815-vIL-10 cells were rejected whereas P815 cells transfected with a control plasmic were tumorigenic after injection into syngeneic DBA/2 mice. Furthermore, this stimulatory effect of constitutive vIL-10 secretion could be exploited to irradicate already established P815 tumors which were smaller than 5 x 5 mm. In contrast, paracrine vIL-10 secretion for a limited time period of 8 - 9 days was associated with inhibitory effects in vivo: P815-B7 cells, which are normally eliminated in DBA/2 mice, could grow if exposed to temporarily secreted vIL-10. These time-dependent immunomodulatory effects have to be considered in potential therapeutic applications of vIL-10.
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An expansion of CD8+ lymphocytes associated with a monoclonal rearrangement of the T-cell receptor gamma locus was found in a woman with HIV-1 infection. A subpopulation of HIV-positive patients display an unusual response to HIV infection characterized by a persistent marked CD8+ lymphocytosis, the presence of which appears to be associated with an improved long-term prognosis. This condition is thought to represent a florid immune response to an ongoing viral infection which may be HIV itself, and suggests that monoclonal proliferation of CD8+ lymphocytes does not imply the presence of an underlying malignant process.
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The lipids of high density lipoproteins (HDL) are initially oxidized in preference to those in low density lipoprotein when human plasma is exposed to aqueous peroxyl radicals. In this work we report on the relative susceptibility of HDL protein and lipid to oxidation and on the role HDL's alpha-tocopherol (alpha-TOH) plays in modulating protein oxidation. Exposure of isolated HDL to either low fluxes of aqueous peroxyl radicals, Cu2+ ions, or soybean lipoxygenase resulted in the oxidation of apoAI and apoAII during the earliest stages of the reaction, i.e. after consumption of ubiquinol-10 and in the presence of alpha-TOH. Hydro(pero)xides of cholesteryl esters and phospholipids initially accumulated together with specific oxidized forms of apoAI and apoAII, separated by high pressure liquid chromatography. The specific oxidized forms of apoAI were 16 and 32 mass units heavier than those of the native apolipoproteins and contained 1 and 2 methionine sulfoxide residues per protein, respectively. The third methionine residue in apoAI, as well as Trp residues, remained unoxidized during the earliest stages of HDL oxidation examined. Exposure of isolated apoAI to peroxyl radicals, Cu2+, or soybean lipoxygenase resulted in nonspecific (for peroxyl radicals) or no discernible protein oxidation (Cu2+ and soybean lipoxygenase). This indicated that the formation of the specific oxidized forms of apoAI observed with native HDL was not the result of direct reaction of these oxidants with the apolipoprotein. In vitro and in vivo enrichment of HDL with alpha-TOH resulted in a dose-dependent increase in the extent of peroxyl radical-induced formation of HDL cholesteryl ester hydroperoxides (r = 0.96) and cholesteryl ester hydroxides (r = 0. 92), as well as the loss of apoAI (r = 0.96) and apoAII (r = 0.94). alpha-TOH enrichment also enhanced HDL lipid and protein oxidation induced by Cu2+ or soybean lipoxygenase. These results indicate that the earliest stages of HDL oxidation are accompanied by the oxidation of specific methionine residues in apoAI and apoAII and that in the absence of co-antioxidants, alpha-TOH can promote this process.
The Epstein-Barr virus (EBV) encodes an open reading frame with significant homology to the cellular IL-10 gene. This viral IL-10 (vIL-10) might enable EBV to evade antiviral T cells. We employed transfectants of a murine tumor cell line (P815) to investigate whether vIL-10 interferes with the first (antigenic) or second (co-stimulatory) signal of T cell activation. Untransfected P815 cells caused tumors in syngeneic DBA/2 mice after s.c. inoculation. In contrast, transfectants that provided either a strong antigenic stimulus (P815-Kb cells) or a strong co-stimulatory signal (P815-B7 cells) were rejected. Injection of double-transfected P815 cells expressing Kb and secreting high levels of vIL-10 (P815-Kb-vIL-10) did not result in tumor growth. We then investigated whether vIL-10 could paralyse co-stimulation by B7 under the same conditions. Therefore P815-B7 cells were mixed with vIL-10-secreting P815-Kb cells and co-injected into DBA/2 animals. Most of these mice developed a tumor. Explanted tumor cells expressed the B7 molecule but not the Kb antigen. These observations in vivo were mirrored by experiments in vitro: vIL-10 could induce T cell tolerance towards P815-B7 cells but not P815-Kb cells. Taken together our results suggest that vIL-10 acts directly on T cells to inhibit co-stimulatory signals mediated via B7 receptors such as CD28 or CTLA-4.
We evaluated a cytomegalovirus (CMV) 24-hour shell vial assay (SVA), the Murex Hybrid Capture CMV DNA assay (HCA), and a CMV plasma PCR for the detection of CMV viremia in renal and bone marrow transplant recipients and human immunodeficiency virus-infected patients. CMV viremia was detected by at least one method in 125 of 317 evaluable samples (39.4%) from 78 patients and was detected in 19.8% of samples by SVA, 26.8% by HCA, and 32.2% by plasma PCR. There was moderate to substantial agreement between the results of the different tests (kappa coefficient = 0.415 to 0.631). However, HCA and plasma PCR were significantly more sensitive than SVA (P = 0.001 and P < 0.0001, respectively; McNemar's test), and plasma PCR was more sensitive than HCA (P = 0.031; McNemar's test). HCA and plasma PCR were more consistently positive than SVA during viremic episodes (P = 0.0002 and P < 0.0001, respectively; McNemar's test). The use of HCA or plasma PCR may therefore improve the diagnosis and management of CMV disease in susceptible patient groups.
Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms which have not been elucidated. Since multinucleated cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the bone-resorbing multinucleated cells and the distinct mononuclear stromal cells from these tumors. We have found that these mononuclear cells produce an activity which stimulates both giant cells from giant cell tumors and rodent osteoclasts to resorb bone in vitro. We have identified the activity and found that it represents several products of the 5-lipoxygenase pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the leukotrienes. These data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear stromal cells in human giant cell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption.
To determine if the short- and long-term prognosis of renal micropolyarteritis (including Wegener's granulomatosis) had improved in our Unit in the last 5 years compared to our previous series from 1965 to 1980, we examined data from 26 recent patients, 1981-1986. Twenty were admitted during acute phase and six after a mean of 7.1 months of disease. The acute phase showed mild to severe impairment of renal function, and renal biopsies showed necrotising glomerulitis in all acute patients with extensive crescents in 67%. Acute patients (20) were treated with 'aggressive' immunosuppression including i.v. methylprednisolone (15), plasmapheresis (seven) and cyclophosphamide (ten). Chronic immunosuppressive treatment was continued in most patients. In the 20 acute-onset patients, life survival at 2 and 5 years was 77%; four of five deaths occurred during the acute phase in aged patients with severe renal failure. The notable improvement in life and kidney survival (only one patient underwent chronic dialysis) probably resulted from greater awareness of vasculitis in general practice with early referral of patients to specialised units, and to more aggressive treatment.
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A series of synthetic 1,n-bis(3-aminopyridinio)-alkane fluorescent probes have been used to determine the ligand binding properties of the acetylcholine receptor purified from Torpedo californica electroplax. At equilibrium, the probes bound to a single class of sites. The binding affinity of the fluorescent decamethonium analogues increased progressively as the number of methylene groups (n) increased from 4 to 12 and decreased in the range of 16--18 such groups. The receptor bound 1,12-bis(3-aminopyridinio)dodecane and 1,14-bis(3-aminopyridinio)tetradecane with the highest affinity while related monofunctional probes such as 1-(3-amino-pyridinio)propane were bound with a substantially lower affinity. The data indicate that the receptor interacts strongly with both ends of a bifunctional probe such as 1,14-bis(3-aminopyridinio)tetradecane. Also, competition between bifunctional fluorescent probe binding and the binding of conventional cholinergic ligands, was investigated and led to the conclusion that the probes, which are antagonists, form ternary complexes in the presence of acetylcholine.