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Biomedical subjects

Robert C Read

Publications and source records attributed to Robert C Read.

36 records · Page 2Linked to original sources
PubMed2004

Mannose-binding lectin enhances phagocytosis and killing of Neisseria meningitidis by human macrophages.

Deficiency of mannose-binding lectin (MBL) is probably the most common human immunodeficiency and is associated with an increased risk of mucosally acquired infections including meningococcal disease. Tissue macrophages are an important component of mucosal defense, and so we determined the effect of MBL on uptake of meningococci by human monocyte-derived macrophages. Opsonization with MBL significantly increased the capture and doubled the amount of internalization of Neisseria meningitidis. Inhibition of f-actin polymerization indicated that MBL exerted this effect by a dose-dependent acceleration of uptake into phagosomes, which was maximal within the normal physiological concentration of MBL (1.5 microg/ml) and was independent of scavenger receptors. MBL accelerated the acquisition and subsequent loss of the early endosome marker, early endosomal antigen-1, and enhanced the acquisition of the late endosomal marker, lysosome-associated membrane protein-1. MBL reduced the survival of meningococci within macrophages by more than half, despite the increased uptake of organisms, and significantly reduced the number of viable extracellular bacteria by 80%. We conclude that MBL is a dependent opsonin able to accelerate microbial uptake and killing. These results suggest that MBL could modify disease susceptibility by modulating macrophage interactions with mucosal organisms at the site of initial acquisition.

Cells, Cultured↗
PubMed2004

The glycopeptide vancomycin does not enhance toll-like receptor 2 (TLR2) activation by Streptococcus pneumoniae.

OBJECTIVES: The exposure of Streptococcus pneumoniae to cell-wall-active antibiotics in vivo and in vitro results in the release of bacterial components that can induce proinflammatory activation of human cells via toll-like receptor 2 (TLR2). The aim of this study was to compare the activation of human TLR2 pathways after exposure of S. pneumoniae to faropenem, cefotaxime and vancomycin. MATERIALS AND METHODS: Streptococcus pneumoniae D39 was exposed to cefotaxime, faropenem or vancomycin for 6 h during lag or early log phase growth. IL-8 promoter activity of HeLa cells was measured using a dual luciferase reporter plasmid system. HeLa cells were transfected with an expression vector containing TLR2/CD14, or empty vector/CD14 and IL-8 promoter activity was measured using luminescence. Cells were stimulated with antibiotic-treated bacteria, untreated bacteria or medium-only controls. RESULTS: Lag phase S. pneumoniae treated at sub-MIC (1/8 MIC) cefotaxime or faropenem induced 11-fold and 8-fold increases, respectively, in TLR2-mediated IL-8 promoter activity when compared with untreated bacteria. Early log MIC cefotaxime or faropenem-treated bacteria also enhanced TLR2 activation by 3-fold and 4-fold, respectively, when compared with untreated bacteria. Vancomycin treatment had no effect on TLR2 induction at any growth stage or MIC ratio tested. CONCLUSIONS: beta-Lactam antibiotics induce surface changes and release of cell wall structures from bacteria that are proinflammatory via TLR2, but the glycopeptide vancomycin does not.

Anti-Bacterial Agents↗
PubMed2004

Nitric oxide levels regulate macrophage commitment to apoptosis or necrosis during pneumococcal infection.

Macrophages are resistant to constitutive apoptosis, but infectious stimuli can induce either microbial or host-mediated macrophage apoptosis. Phagocytosis and killing of opsonized pneumococci by macrophages are potent stimuli for host-mediated apoptosis, but the link between pneumococcal killing and apoptosis induction remains undefined. We now show phagocytosis of pneumococci by differentiated human monocyte-derived macrophages (MDM) results in up-regulation of inducible nitric oxide synthase (iNOS) and increased production of NO and reactive nitrogen species. NO accumulation in macrophages initiates an apoptotic program that involves NO-dependent mitochondrial membrane permeabilization, Mcl-1 down-regulation, and caspase activation and results in nuclear condensation and fragmentation. An inhibitor of mitochondrial permeability transition, bongkrekic acid, decreases pneumococcal-associated macrophage apoptosis. Conversely, inhibition of NO production using iNOS inhibitors decreases bacterial killing and shifts the cell death program from apoptosis to necrosis. Pneumolysin contributes to both NO production and apoptosis induction. After initial microbial killing, NO accumulation switches the macrophage phenotype from an activated cell to a cell susceptible to apoptosis. These results illustrate important roles for NO in the integration of host defense and regulation of inflammation in human macrophages.

Adult↗
PubMed2004

Community-acquired pneumonia mortality: a potential link to antibiotic prescribing trends in general practice.

BACKGROUND: Community prescribing of antibiotics has decreased substantially in the UK in recent years. We examine the association between pneumonia mortality and recent changes in community-based antibiotic prescribing for lower respiratory tract infections (LRTI). METHODS: Retrospective analysis of aggregated data for pneumonia mortality, influenza incidence, and antibiotic prescribing for LRTI in England and Wales during 12-week winter periods between 1993/94 and 1999/2000. RESULTS: Winter antibiotic prescribing for LRTI showed a 30.0% decline since 1995/96. Over the same period, there was a 50.6% increase in winter excess pneumonia mortality adjusted for influenza incidence. Negative binomial regression analysis showed that the incidence of influenza alone had a significant association with winter pneumonia mortality (P<0.001). The analysis also showed the reduction in antibiotic prescribing had a small but significant association with mortality (P<0.001), when simultaneously modelling for influenza incidence. CONCLUSIONS: Our findings suggest an association between recent reductions in antibiotic prescribing for LRTI in general practice and an increase in pneumonia mortality in England and Wales. This retrospective study of aggregate data represents the first attempt to assess the effect of limiting antibiotic prescribing on patient outcomes, and highlights the need to identify which patients benefit from antibiotic treatment for LRTI.

Anti-Bacterial Agents↗
PubMed2004

Post-transcriptional regulation of bacterial motility by aconitase proteins.

Escherichia coli and Bacillus subtilis aconitases can act as iron and oxidative stress-responsive post-transcriptional regulators. Here, it is shown that a Salmonella enterica serovar Typhimurium LT2 acnB mutant exhibits impaired binding to the surface of J774 macrophage-like cells. Proteomic analyses were used to investigate further the binding defect of the acnB mutant. These revealed that the levels of the flagellum protein FliC were much lower for the acnB mutant. This strain was correspondingly less motile and possessed fewer flagella than either the parental strain or the acnA and acnAB mutants. The acnB lesion did not alter fliC transcription, nor did apo-AcnB select the fliC transcript from a library of S. enterica transcripts; thus, the effect of AcnB on FliC is indirect. Evidence is presented to show that apo-AcnB regulates FliC synthesis via interaction with the ftsH transcript to decrease the intracellular levels of FtsH. The lower levels of FtsH protease activity then influence sigma32, DnaK and, ultimately, FliC production.

ATP-Dependent Proteases↗
PubMed2003

Streptococcus pneumoniae-associated human macrophage apoptosis after bacterial internalization via complement and Fcgamma receptors correlates with intracellular bacterial load.

Opsonization enhances Streptococcus pneumoniae-induced human monocyte-derived macrophage (MDM) apoptosis. Both depletion of complement and immunoglobulin from opsonizing serum and blockade of the macrophages CR1, CR3, FcgammaRII, and FcgammaRIII partially decreased MDM apoptosis after S. pneumoniae phagocytosis, and these effects correlated with reduced numbers of internalized bacteria. Chloramphenicol inhibition of protein synthesis by opsonized S. pneumoniae down-regulated subsequent MDM apoptosis. Phagocytosis of an unencapsulated mutant of S. pneumoniae resulted in increased MDM apoptosis, in association with enhanced internalization. Caspase inhibition was associated with decreased killing of bacteria. Enhanced induction of apoptosis by opsonized S. pneumoniae is the result of increased intracellular burden of bacteria, rather than of a specific pattern of engagement of complement receptor or FcgammaR. A dynamic interaction between live intracellular bacteria and the host cell is necessary for induction of apoptosis in MDMs, and induction of apoptosis contributes to the host defense against S. pneumoniae.

Apoptosis↗
PubMed2003

Penicillin enhances the toll-like receptor 2-mediated proinflammatory activity of Streptococcus pneumoniae.

The Streptococcus pneumoniae cell-wall components peptidoglycan and lipoteichoic acid activate Toll-like receptor 2 (TLR2), which transduces an inflammatory response. After exposure to penicillin, type 2 S. pneumoniae strain D39, but not the isogenic autolysin-deficient mutant AL2, induced significantly enhanced interleukin-8 promoter activity in TLR2-transfected HeLa cells. Lag-phase D39 exhibited enhanced TLR2 activation after exposure to penicillin at levels below the minimum inhibitory concentration (MIC); in contrast, early log-phase S. pneumoniae were most active when exposed to the MIC. This enhancement was not ablated by heat treatment but was attenuated by autolysin inhibitors. The antimicrobial activity of moxifloxacin and erythromycin was not associated with TLR2 activation by S. pneumoniae. These data show that penicillin treatment of S. pneumoniae releases proinflammatory cell-wall components that activate TLR2 and that this activity is dependent on autolysin, the growth phase of the organism, and the antibiotic concentration.

Anti-Infective Agents↗
PubMed2003

Pulmonary immunoglobulin responses to Streptococcus pneumoniae are altered but not reduced in human immunodeficiency virus-infected Malawian adults.

We tested the hypothesis that human immunodeficiency virus (HIV)-infected adults have a specific defect in anti-pneumococcal capsular polysaccharide (Pn-specific) immunoglobulin (Ig) in fluid obtained from the lower respiratory tract. Higher levels of total IgG and IgM were present in bronchoalveolar lavage samples from HIV-infected subjects than in those from HIV-uninfected subjects. Pn-specific IgG and IgM in bronchoalveolar lavage samples were not significantly different between HIV-infected and -uninfected subjects. After pneumococcal infection, HIV-infected patients had higher bronchoalveolar lavage levels of Pn-specific IgG than HIV-infected patients without recent infection (geometric means, 387 vs. 30 ng/mL, P=.001).

AIDS-Related Opportunistic Infections↗
PubMed2003

Variation within genes encoding interleukin-1 and the interleukin-1 receptor antagonist influence the severity of meningococcal disease.

BACKGROUND: Genetically determined variation in proinflammatory cytokine release influences severity of meningococcal disease and other serious infections. OBJECTIVE: To ascertain the relative frequencies of single nucleotide polymorphisms within the interleukin-1 gene locus among patients who survived and those who died of meningococcal disease and a control population of blood donors. DESIGN: Association study. SETTING: England and Wales. PATIENTS: 1106 consecutively received blood samples from persons with microbiologically confirmed meningococcal disease and 839 samples from blood donors. MEASUREMENTS: Patient demographic and outcome data, infecting meningococcal serogroups, and genotype at the IL1B(-511) and IL1RN(+2018) loci of patients and blood donor controls. RESULTS: Genotype frequency did not differ between patients with meningococcal disease and blood donor controls. Logistic regression analysis revealed that the likelihood of death was significantly influenced by age but not socioeconomic status and was higher in patients who were infected with serogroup C (odds ratio for survival, 0.50 [95% CI, 0.33 to 0.78]). Patients carrying the common allele at IL1B(-511) were more likely to survive (odds ratio, 2.01 [CI, 1.11 to 3.79]). Patients with this allele were less likely to survive if they also carried the rare allele at IL1RN(+2018) (odds ratio, 0.61 [CI, 0.38 to 0.993]). CONCLUSION: Genotype at the interleukin-1 gene locus influences likelihood of survival of meningococcal disease but has no effect on susceptibility to the infection. Increasing age and infection with serogroup C also influence the likelihood of death.

Adolescent↗
PubMed2003

Activation of toll-like receptor 2 (TLR2) and TLR4/MD2 by Neisseria is independent of capsule and lipooligosaccharide (LOS) sialylation but varies widely among LOS from different strains.

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N. meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N. meningitidis and N. gonorrhoeae) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria.

Bacterial Capsules↗
PubMed2002

Choline-binding protein A of Streptococcus pneumoniae elicits chemokine production and expression of intercellular adhesion molecule 1 (CD54) by human alveolar epithelial cells.

Recruitment of neutrophils into alveolar air spaces is an early event in the pathogenesis of pneumonia due to Streptococcus pneumoniae. This results from chemokines released by activated endothelial and epithelial cells and alveolar macrophages. Culture supernatants of 6 wild-type strains of S. pneumoniae, shown to contain choline-binding protein A (CbpA; clades A and B), induced release of chemokine CXCL8 from the human alveolar epithelial cell line A549, whereas a CbpA deletion mutant elicited significantly reduced CXCL8 release, compared with that of its isogenic parent (P<.01). Recombinant CbpA up-regulated expression of messenger RNA of CXCL8 and CCL2 but not of XCL1, CXCL10, CCL1, CCL3, CCL4, or CCL5 in A549 cells and induced increased secretion of CXCL8, CCL2, CXCL1, and CXCL5 in a dose- and time-dependent manner. CbpA also increased the expression of intercellular adhesion molecule 1 (CD54) by A549 cells. Thus, CbpA of S. pneumoniae induces the transcription and release of proinflammatory molecules by human alveolar epithelial cells.

Antigens, Bacterial↗
PubMed2002

Pneumococcal disease in HIV-infected Malawian adults: acute mortality and long-term survival.

OBJECTIVE: HIV-infected patients in Africa are vulnerable to severe recurrent infection with Streptococcus pneumoniae, but no effective preventive strategy has been developed. We set out to determine which factors influence in-hospital mortality and long-term survival of Malawians with invasive pneumococcal disease. DESIGN, SETTING AND PATIENTS: Acute clinical features, inpatient mortality and long-term survival were described among consecutively admitted hospital patients with S. pneumoniae in the blood or cerebrospinal fluid. Factors associated with inpatient mortality were determined, and patients surviving to discharge were followed to determine their long-term outcome. RESULTS: A total of 217 patients with pneumococcal disease were studied over an 18-month period. Among these, 158 out of 167 consenting to testing (95%) were HIV positive. Inpatient mortality was 65% for pneumococcal meningitis (n = 64), 20% for pneumococcaemic pneumonia (n = 92), 26% for patients with pneumococcaemia without localizing signs (n = 43), and 76% in patients with probable meningitis (n = 17). Lowered consciousness level, hypotension, and age exceeding 55 years at presentation were associated with inpatient death, but not long-term outcome in survivors. Hospital survivors were followed for a median of 414 days; 39% died in the community during the study period. Outpatient death was associated with multilobar chest signs, oral candidiasis, and severe anaemia as an inpatient. CONCLUSION: Most patients with pneumococcal disease in Malawi have HIV co-infection. They have severe disease with a high mortality rate. At discharge, all HIV-infected adults have a poor prognosis but patients with multilobar chest signs or anaemia are at particular risk.

Age Factors↗
PubMed2002

Interaction of the Salmonella typhimurium transcription and virulence factor SlyA with target DNA and identification of members of the SlyA regulon.

The SlyA protein from Salmonella typhimurium is a transcription factor that contributes to virulence. It is shown that a slyA mutant is attenuated in the presence of murine macrophages compared with the parent strain. Moreover, after growth in minimal medium, survival of the slyA mutant was reduced. Altered levels of flagellin (fliC), PagC, IroN, and outer membrane proteins suggest that the slyA mutation affects the surface properties of Salmonella. The isolated SlyA protein is a cofactor-free homodimer that recognizes five sites within the promoter region of the slyA gene. One of these sites contained a near perfect inverted repeat TTAGCAAGCTAA. The other four sites contained related sequences. Occupation of the SlyA sites in the slyA promoter prevented open-complex formation, consistent with the pattern of slyA::lacZ expression parental and slyA mutant strains. By combining the footprinting data with potential SlyA binding sites recovered from a pool of random DNA sequences, a consensus was defined and used to probe the NIH Salmonella unfinished genomes data base. These searches revealed the presence of consensus SlyA sites upstream of omp, ispA, xseB, slyA, and a gene encoding a protein with homology to a hemagglutinin. Accordingly, transcription of an omp::lacZ fusion was reduced in a slyA mutant. Given the difficulties in obtaining a comprehensive picture of intracellular gene expression, the definition of the DNA sequence recognized by a transcription factor (SlyA) that is essential for survival in the macrophage environment should allow a complete regulon of genes with altered expression upon exposure to macrophages to be determined once the S. typhimurium genome annotation is complete.

Animals↗
PubMed2002

Invasion by Neisseria meningitidis varies widely between clones and among nasopharyngeal mucosae derived from adult human hosts.

Colonization of the human nasopharynx is a feature of some species of Neisseria, and is a prerequisite of invasive meningococcal disease. The likelihood of colonization by Neisseria meningitidis varies widely between humans, and very few develop invasive disease. Explants of nasal mucosa derived from adult patients with non-allergic nasal obstruction were infected experimentally with Neisseria spp. At intervals over 18 h incubation, washed explants were homogenized, and viable bacteria were counted. To estimate bacterial invasion of mucosa, explants were exposed to 0.25% sodium taurocholate for 30 s prior to homogenization. N. meningitidis was recovered from the mucosa and the organism invaded and replicated within the tissue, in contrast to N. lactamica and N. animalis (n=9, P<0.008). N. meningitidis isolates of clones ET-5, ET-37 and lineage III were recovered from and invaded tissue, but strains of clones A4, A:subgroup I, A:subgroup III and A:subgroup IV-1 did not invade (n=6). To measure host variation, survival of N. meningitidis within nasal mucosa of 40 different human donors was measured. Intra-class correlation of replicates was 0.97, but the coefficient of variation of recovered viable counts was 1335% after 4 h and 77% after 18 h incubation. It is concluded that the distinctive colonization and disease potential of Neisseria spp. may be partly a consequence of their ability to invade and survive within human nasopharyngeal mucosa, but that this is influenced greatly by genetic or environmental factors operating on the host mucosa. This is consistent with the unpredictable epidemiology of meningococcal disease.

Adult↗
PubMed2002

Flavohemoglobin Hmp protects Salmonella enterica serovar typhimurium from nitric oxide-related killing by human macrophages.

Survival of macrophage microbicidal activity is a prerequisite for invasive disease caused by the enteric pathogen Salmonella enterica serovar Typhimurium. Flavohemoglobins, such as those of Escherichia coli, Salmonella, and yeast, play vital roles in protection of these microorganisms in vitro from nitric oxide (NO) and nitrosative stress. A Salmonella hmp mutant defective in flavohemoglobin (Hmp) synthesis exhibits growth that is hypersensitive to nitrosating agents. We found that respiration of this mutant exhibited increased inhibition by NO, whereas wild-type cells pregrown with sodium nitroprusside or S-nitrosoglutathione showed enhanced tolerance of NO. Most significantly, hmp mutants internalized by primary human peripheral monocyte-derived macrophages survived phagocytosis relatively poorly compared with similarly bound and internalized wild-type cells. That the enhanced sensitivity to macrophage microbicidal activity is due primarily to the failure of Salmonella to detoxify NO was suggested by the ability of L-N(G)-monomethyl arginine-an inhibitor of NO synthase-to eliminate the difference in killing between wild-type and hmp mutant Salmonella cells. These observations suggest that Salmonella Hmp contributes to protection from NO-mediated inhibition by human macrophages.

Bacterial Adhesion↗
PubMed2002

Nitric oxide metabolism in Neisseria meningitidis.

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.

Aerobiosis↗