Biomedical subjects

Neil Hunter

Publications and source records attributed to Neil Hunter.

25 records · Page 2Linked to original sources

PubMed2003

Porphyrin-mediated cell surface heme capture from hemoglobin by Porphyromonas gingivalis.

The porphyrin requirements for growth recovery of Porphyromonas gingivalis in heme-depleted cultures are investigated. In addition to physiologically relevant sources of heme, growth recovery is stimulated by a number of noniron porphyrins. These data demonstrate that, as for Haemophilus influenzae, reliance on captured iron and on exogenous porphyrin is manifest as an absolute growth requirement for heme. A number of outer membrane proteins including some gingipains contain the hemoglobin receptor (HA2) domain. In cell surface extracts, polypeptides derived from HA2-containing proteins predominated in hemoglobin binding. The in vitro porphyrin-binding properties of a recombinant HA2 domain were investigated and found to be iron independent. Porphyrins that differ from protoporphyrin IX in only the vinyl aspect of the tetrapyrrole ring show comparable effects in competing with hemoglobin for HA2 and facilitate growth recovery. For some porphyrins which differ from protoporphyrin IX at both propionic acid side chains, the modification is detrimental in both these assays. Correlations of porphyrin competition and growth recovery imply that the HA2 domain acts as a high-affinity hemophore at the cell surface to capture porphyrin from hemoglobin. While some proteins involved with heme capture bind directly to the iron center, the HA2 domain of P. gingivalis recognizes heme by a mechanism that is solely porphyrin mediated.

Adhesins, Bacterial↗

PubMed2002

Physical and functional interactions among basic chromosome organizational features govern early steps of meiotic chiasma formation.

Analysis of meiotic recombination by functional genomic approaches reveals prominent spatial and functional interactions among diverse organizational determinants. Recombination occurs between chromatin loop sequences; however, these sequences are spatially tethered to underlying chromosome axes via their recombinosomes. Meiotic chromosomal protein, Red1, localizes to chromosome axes; however, Red1 loading is modulated by R/G-bands isochores and thus by bulk chromatin state. Recombination is also modulated by isochore determinants: R-bands differentially favor double-strand break (DSB) formation but disfavor subsequent loading of meiotic RecA homolog, Dmc1. Red1 promotes DSB formation in both R- and G-bands and then promotes Dmc1 loading, specifically counteracting disfavoring R-band effects. These complexities are discussed in the context of chiasma formation as a series of coordinated local changes at the DNA and chromosome-axis levels.

Cell Cycle Proteins↗

PubMed2002

Microvasculature in gingivitis and chronic periodontitis: disruption of vascular networks with protracted inflammation.

Gingivitis occurring when bacterial plaque accumulates in the gingival crevice provides a convenient and interesting model for chronic inflammation in humans. In some patients, gingivitis progresses to the destructive lesion of periodontitis, involving the formation of periodontal pockets. The basis for pocket formation and progression is not as yet clear, although neutrophilic polymorphonuclear leukocytes (PMN) appear to play a protective role. Vascular changes appear to either facilitate or inhibit PMN function with the effect of either protecting from, or stimulating, periodontitis. Contrary to most circumstances, high endothelial cells in periodontitis are involved with PMN rather than lymphocyte emigration. Expansion of the microvasculature through increased vascular diameter and tortuosity as well as the development of high endothelial cells appears to protect from periodontitis by increasing the supply of both plasma defense factors and PMN to the tissues. Vascular changes that may oppose this and promote periodontitis are the formation of perivascular hyaline material and accumulation of basement membrane rests. The inadequate tissue turnover that accumulation of these vascular products represents can be argued as a vascular response to a chronic inflammation that has failed to eliminate the irritant. It is suggested that these vascular changes may account for the highly localized and burst-like pattern of pocket formation in periodontitis. Finally, it is possible that the recent observation that periodontitis is an independent risk factor for systemic vascular disease may reflect stimulation of acute phase protein synthesis by cytokines released by periodontal high endothelial cells.

Chronic Disease↗

PubMed2002

Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

Bacteria, Aerobic↗

PubMed2002

Modulation of an interleukin-12 and gamma interferon synergistic feedback regulatory cycle of T-cell and monocyte cocultures by Porphyromonas gingivalis lipopolysaccharide in the absence or presence of cysteine proteinases.

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-gamma) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-gamma have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogen Porphyromonas gingivalis stimulated monocytes primed with IFN-gamma to release IL-12, thereby enhancing IFN-gamma accumulation in T-cell populations. P. gingivalis LPS was shown to enhance IL-12 induction of IFN-gamma in T cells in a manner independent from TNF-alpha contribution. The levels of T-cell IL-12 receptors were not affected by P. gingivalis LPS and played only a minor role in the magnitude of the IFN-gamma response. These data suggest that LPS from P. gingivalis establishes an activation loop with IL-12 and IFN-gamma with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-gamma accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-gamma levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) of P. gingivalis could lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.

Adhesins, Bacterial↗

PubMed2002

Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis.

The bacteria found in carious dentine were correlated with the tissue response of the dental pulps of 65 teeth extracted from patients with advanced caries and pulpitis. Standardized homogenates of carious dentine were plated onto selective and nonselective media under anaerobic and microaerophilic conditions. In addition, real-time PCR was used to quantify the recovery of anaerobic bacteria. Primers and fluorogenic probes were designed to detect the total anaerobic microbial load, the genera Prevotella and Fusobacterium, and the species Prevotella melaninogenica, Porphyromonas endodontalis, Porphyromonas gingivalis, and Micromonas (formerly Peptostreptococcus) micros. The pulpal pathology was categorized according to the cellular response and degenerative changes. Analysis of cultured bacteria showed a predominance of gram-positive microorganisms, particularly lactobacilli. Gram-negative bacteria were also present in significant numbers with Prevotella spp., the most numerous anaerobic group cultured. Real-time PCR analysis indicated a greater microbial load than that determined by colony counting. The total number of anaerobes detected was 41-fold greater by real-time PCR than by colony counting, while the numbers of Prevotella and Fusobacterium spp. detected were 82- and 2.4-fold greater by real-time PCR than by colony counting, respectively. Real-time PCR also identified M. micros, P. endodontalis, and P. gingivalis in 71, 60, and 52% of carious samples, respectively. Correlation matrices of the real-time PCR data revealed significant positive associations between M. micros and P. endodontalis detection and inflammatory degeneration of pulpal tissues. These anaerobes have been strongly implicated in endodontic infections that occur as sequelae to carious pulpitis. Accordingly, the data suggest that the presence of high levels of these bacteria in carious lesions may be indicative of irreversible pulpal pathology.

Bacteria, Anaerobic↗

PubMedDate not supplied

Binding of Porphyromonas gingivalis gingipains to human CD4(+) T cells preferentially down-regulates surface CD2 and CD4 with little affect on co-stimulatory molecule expression.

The role of Porphyromonas gingivalis cysteine proteinases (gingipains) in the evasion of host cell-mediated immunity has not been fully determined. In this study, modulation by gingipains of accessory and co-stimulatory molecule expression on human CD4(+) T cells was evaluated. Arg-gingipain rather than Lys-gingipain binds to resting CD4(+) T cells in the presence of serum. The constitutive expression of CD28 on T cells was slightly up-regulated following challenge with gingipains, whereas CD45 and CD3 were not affected. Binding of anti-CD2 and anti-CD4 monoclonal antibodies (mAbs) was reduced after challenge of T cells with gingipains, but restored to 50 and 100%, respectively, of control levels, after 48h of incubation in medium depleted of gingipains. The induced expression, by anti-CD3 mAb, of CTLA-4, CD25, and CD40 ligand (CD40L) was decreased following incubation of T cells with gingipains which also led to decreased response to anti-CD3 and anti-CD28 mAbs as shown by reduction of interleukin-2 (IL-2) production. Cumulatively, these results indicate that activated gingipains attach to T cells and preferentially cleave CD2 and CD4 molecules, with potential to impair T cell responses at periodontal sites.

Adhesins, Bacterial↗