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Biomedical subjects

S Hughes

Publications and source records attributed to S Hughes.

At least 91 records · Page 5Linked to original sources
PubMed2000

Roles of endothelial cell migration and apoptosis in vascular remodeling during development of the central nervous system.

OBJECTIVE: To examine the roles of apoptosis, macrophages, and endothelial cell migration in vascular remodeling during development of the central nervous system. METHODS: The terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique was combined with Griffonia simplicifolia isolectin B4 histochemistry to detect apoptotic endothelial cells in retinal whole-mount preparations derived from rats at various stages of postnatal development as well as from rat pups exposed to hyperoxia. Macrophages were detected by immunohistochemistry with the monoclonal antibody ED1, and proliferating endothelial cells were identified by incorporation of bromodeoxyuridine. RESULTS: Only small numbers of TUNEL-positive endothelial cells were detected during normal development of the retinal vasculature, with the apoptotic cell density in the inner plexus peaking during the first postnatal week and decreasing markedly during the second week, at a time when vessel retraction was widespread. Neither apoptotic endothelial cells nor macrophages were apparent at sites of initiation of vessel retraction. TUNEL-positive endothelial cells were observed in vessels destined to remain. Hyperoxia induced excessive vessel withdrawal, resulting in the generation of isolated vascular segments containing apoptotic endothelial cells. A topographical analysis showed low numbers of proliferating endothelial cells at sites of angiogenesis whereas vascular proliferation was increased in the adjacent inner plexus. CONCLUSIONS: Endothelial cell apoptosis and macrophages do not initiate vessel retraction, but rather contribute to the removal of redundant cells throughout the vasculature. We suggest that vessel retraction is mediated by endothelial cell migration and that endothelial cells derived from retracting vascular segments are redeployed in the formation of new vessels. Only when retraction results in compromised circulation and redeployment is not possible, does endothelial cell apoptosis occur.

Animals↗
PubMed1999

In vitro motility speed of slow myosin extracted from single soleus fibres from young and old rats.

1. Isolated soleus muscle fibres from aged rats contract more slowly than those from young rats. To determine whether this effect is due to a difference between the myosin molecules, we measured the rate at which actin filaments are driven over a myosin coated surface in the presence of ATP by using a novel in vitro motility assay where myosin is extracted from single muscle fibre segments. 2. Motility was dependent on the myosin density on the coverslip. In regions of high myosin density, actin motility was orientated parallel and anti-parallel to the direction of flow during myosin adhesion to the coverslip. In contrast, in regions of lower myosin density, actin motility was more random. The speed was about 20 % higher in the high density regions (P < 0.001). Further, the speed of filaments in the high density region, moving away or towards the fibre was less variable (P < 0.05) than that of more randomly moving filaments in the low density region. 3. The speed with myosin from slow soleus fibres of young adult rats (3-6 months old; v = 1.43 +/- 0.23 microm s-1; mean +/- s.d.) was faster (P < 0.001) than with myosin from aged rats (20-24 months old; v = 1.27 +/- 0.23 microm s-1). 4. No difference in myosin isoforms between young adult and aged fibres could be detected using electrophoretic and immunocytochemical techniques. Fibres of both ages expressed the beta/slow myosin heavy chain (MyHC) isoform and slow isoforms of essential and regulatory myosin light chains (MyLCs). 5. It is concluded that an age-related alteration in myosin contributes to the slowing of the maximum shortening velocity (V0) observed in soleus muscle fibres expressing the beta/slow MyHC isoform.

Actins↗
PubMed1999

Differential sensitivity of distinct Chlamydia trachomatis isolates to IFN-gamma-mediated inhibition.

Resistance to the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis has been mapped to MHC class II-restricted, IL-12-dependent CD4+ T cells that secrete a type 1 profile of proinflammatory cytokines, which includes IFN-gamma and TNF-alpha. The relative contribution of IFN-gamma is controversial, however, due to variation in results presented by different laboratories. To determine whether C. trachomatis strain differences contributed to this apparent conflict, the relative resistance of IFN-gamma-deficient mice to murine and human strains of C. trachomatis was compared. All human serovars were much more sensitive to the direct inhibitory actions of IFN-gamma than the MoPn strain. Furthermore, genital clearance of human serovar D in the C57BL/6 mouse was mediated by class II-independent mechanisms that probably involved local production of IFN-gamma by cells of the innate immune system. TNF-alpha also contributed indirectly to host resistance against all strains tested. The differential susceptibility of distinct C. trachomatis strains to effector cytokines such as IFN-gamma could not have been predicted by interstrain biologic variation or by the profile of cytokines stimulated during infection. These findings indicate that strain variation should be considered in situations where related isolates of a given parasite produce conflicting data in models of infection and immunity. They also suggest that stimulation of mucosal IFN-gamma activity is a relevant goal for a human chlamydial vaccine.

Animals↗
PubMed1999

The chicken 9E3/CEF4 CXC chemokine is the avian orthologue of IL8 and maps to chicken chromosome 4 syntenic with genes flanking the mammalian chemokine cluster.

The gene encoding the chicken chemokine 9E3/CEF4 was cloned, sequenced, and mapped; 9E3/CEF4 was the first nonmammalian cytokine cDNA to be cloned and has significant amino acid identity with both human IL8 and human GROalpha. These results show that this cytokine is chicken IL8 and not GROalpha. The exon:intron structure of chicken IL8 corresponds almost exactly to that of human IL8 and differs from those of other known mammalian CXC chemokine genes. Analysis of the predicted amino acid sequence suggests that overall protein structure is conserved between human and chicken IL8, but that the receptor binding sites are not. Genetic distance analysis also suggests that this gene encodes chicken IL8. A number of potential regulatory sequences similar to those found in human IL8 have been identified in the promoter. These include (5'-3') a hepatocyte NF-1 binding site, an NF-kappaB binding site, and a TATAAA box. The human AP-1 binding site and CCAT box are poorly conserved in the promoter of the chicken gene, but there are other potential AP-1 binding sites and a potential CCAT box. The human IRF-1 and octamer binding sites seem to be absent. However, the chicken gene promoter contains a GATA motif not present in the promoter of human IL8. Sequence comparisons also identify conserved regions in the promoter that may function as transcription factor binding sites as yet undescribed in the human IL8 promoter. Promoter sequence polymorphisms have been identified in chicken lines C and 61, but neither lie in any of the regulatory regions mentioned above. Chicken IL8 contains nine repeats of the "instability" motif ATTTA in the 3' untranslated region (UTR) in exon 4. A multiple restriction single-stranded conformational polymorphism was identified which enabled chicken IL8 to be genetically mapped to Chromosome (Chr) 4, linked to SPP1 and ALB1, and thus showing conserved synteny with mouse Chr 5 and human Chr 4. This is the first nonmammalian chemokine gene to be genetically mapped.

Amino Acid Sequence↗
PubMed1999

Peritrophins of adult dipteran ectoparasites and their evaluation as vaccine antigens.

Several peritrophins of larvae of Lucilia cuprina (sheep blowfly) have demonstrated potential as vaccine antigens, and some have been characterised and cloned. These proteins are tightly associated with the peritrophic matrix, a chitinous tube or sac lining the lumen of the gut of most insects. The peritrophins require strong denaturants for their removal from peritrophic matrix. We now report the preliminary characterisation of peritrophins of the adult stage of L. cuprina and Haematobia irritans exigua (buffalo fly). Similar SDS-PAGE profiles were obtained for proteins extracted in SDS or urea from isolated adult peritrophic matrices of both species. Radioiodination of urea-extracted peritrophins improved sensitivity, indicating numerous proteins of 15-75 kDa. Direct radioiodination of L. cuprina peritrophic matrix preferentially labelled high molecular weight complexes and proteins of 80-90 kDa. Two-dimensional gel analyses of a urea extract of adult L. cuprina peritrophic matrix revealed that most proteins were moderately acidic. Antibodies produced against SDS-extracted peritrophins, or against sonicated peritrophic matrices of these two flies were crossreactive. The sera also appeared to recognise SDS-extracted components of Triton X-100 treated and washed adult peritrophic matrix of the mosquito, Aedes vigilax (Skause). This profile altered as the peritrophic matrix matured. In concordance with extracts from the adult L. cuprina and H.i. exigua peritrophic matrices, proteins in the 50-75 kDa region were immunodominant. The vaccine potential of the peritrophins of these Diptera were examined following vaccination of cattle and rabbits with adult H.i. exigua or L. cuprina peritrophins. When the adult life stages of H.i. exigua or two mosquitoes, A. vigilax and A. aegypti (Linnaeus), were fed on the sera or blood of vaccinated hosts, there were no detrimental effects to any life cycle stages of these Diptera.

Aedes↗
PubMed1999

The effect of age on in vitro motility speed of slow myosin extracted from single rat soleus fibres.

The effect of age on the motor protein myosin was examined in a novel in vitro motility assay. Myosin was extracted from soleus fibres of young (3-6 month) and old (20-24 month) rats. All fibres expressed the type I myosin heavy chain (MyHC) and the slow isoforms of the myosin light chains (MyLCs). In vitro motility speed was significantly (P < 0.001) faster in the young adult (1.43 +/- 0.23 microm s-1) than in the aged group (1.27 +/- 0.23 microm s-1). The result indicates that the age-related decrease in contractile speed observed in slow fibres may be the effect of a change in the properties of myosin with age.

Aging↗
PubMed1999

The effect of doxycycline treatment on the development of protective immunity in a murine model of chlamydial genital infection.

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) worldwide. Antibiotics are effective in treating infection; however, reinfection is common. This observation has led to the conclusion that infection fails to elicit a protective antichlamydial immune response. It was postulated that high reinfection rates might be due to early eradication of organisms from genital tissue after antibiotic intervention, which could negatively influence the development of naturally acquired protective immunity. This hypothesis was tested by use of a murine model of female genital infection. The findings show that doxycycline intervention of infection, although very effective in eradicating chlamydiae from genital tissue and preventing upper genital tract disease, significantly inhibits the development of protective immunity. If antibiotic intervention of human chlamydial genital infection has a similar effect on protective immunity, it could have important implications in the understanding of immunity to infection and future public health efforts to control chlamydial STD.

Animals↗
PubMed1999

Warm-blooded isochore structure in Nile crocodile and turtle.

The genomes of warm-blooded vertebrates are characterized by a strong heterogeneity in base composition, with GC-rich and GC-poor isochores. The GC content of sequences, especially in third codon positions, is highly correlated with that of the isochore they are embedded in. In amphibian and fish genomes, GC-rich isochores are nearly absent. Thus, it has been proposed that the GC increase in a part of mammalian and avian genomes represents an adaptation to homeothermy. To test this selective hypothesis, we sequenced marker protein genes in two cold-blooded vertebrates, the Nile crocodile Crocodylus niloticus (10 genes) and the red-eared slider Trachemys scripta elegans (6 genes). The analysis of base composition in third codon position of this original data set shows that the Nile crocodile and the turtle also exhibit GC-rich isochores, which rules out the homeothermy hypothesis. Instead, we propose that the GC increase results from a mutational bias that took place earlier than the adaptation to homeothermy in birds and before the turtle/crocodile divergence. Surprisingly, the isochore structure appears very similar between the red-eared slider and the Nile crocodile than between the chicken and the Nile crocodile. This point questions the phylogenetic position of turtles as a basal lineage of extant reptiles. We also observed a regular molecular clock in the Archosauria, which enables us, by using a more extended data set, to confirm Kumar and Hedges's dating of the bird-crocodile split.

Alligators and Crocodiles↗
PubMed1999

Clearance of Chlamydia trachomatis from the murine genital mucosa does not require perforin-mediated cytolysis or Fas-mediated apoptosis.

The molecular mechanisms of resistance to genital infection with the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis are unknown. A role for major histocompatibility complex class II-restricted, interleukin-12-dependent CD4(+) T cells has been established, but the functional activity of these cells does not depend on secretion of gamma interferon. Here we examined the potential contribution of T-cell-mediated cytotoxicity and apoptosis to mucosal clearance of MoPn by using mice deficient in the molecular mediators of target cell lysis. Animals lacking perforin, Fas, Fas ligand, or both perforin and Fas ligand were infected genitally with C. trachomatis MoPn and monitored for expression of immunity to chlamydial antigens and clearance of MoPn from the genital mucosa. In each case, the profile of spleen cytokine production, the magnitude of the host antibody response, and the kinetics of chlamydial clearance were similar to those of genetically intact controls. Compensatory overproduction of tumor necrosis factor alpha, an alternate mediator of apoptosis in certain cell types, did not appear to account for the ability of mutant mice to resolve Chlamydia infections. These results fail to support CD4(+) T-cell-mediated apoptosis or CD8(+) T-cell-mediated cytotoxicity as being critical to the clearance of C. trachomatis MoPn urogenital infections.

Animals↗
PubMed1999

Chlamydial colonization of multiple mucosae following infection by any mucosal route.

Chlamydia trachomatis inoculated by any mucosal route colonized multiple murine mucosae and, in most cases, the spleen, liver, and kidneys. Cell-to-cell transmission, systemic dissemination, and autoinoculation of infectious fluids may have contributed to chlamydial spread. Intermucosal trafficking of protective T cells cannot be accurately evaluated by using live chlamydial challenges.

Animals↗
PubMed1999

A collaborative approach to management of chlamydial infection among teenagers seeking contraceptive care in a community setting.

OBJECTIVES: To develop and assess a coordinated model of care for effective management of genital chlamydial infection in young women, identified through a selective screening programme in a community based teenage health clinic. METHODS: Selective screening for genital Chlamydia trachomatis was undertaken among young women aged 13-19 years who were having a routine cervical smear test, being referred for termination of pregnancy, or who reported behavioural risk factors, for, and/or symptoms of, genital infection. Collaboration among family planning, genitourinary medicine (GUM), and public health staff was used to enhance management of infected individuals, with particular focus on partner notification. RESULTS: 94 young women had confirmed genital chlamydial infection, representing 11% of those tested. All index patients received appropriate antibiotic therapy and follow up; 93 (99%) of these were counselled by a health adviser, of whom 62 (66%) were able to provide sufficient details for partner notification, resulting in treatment of male partners associated with 51 (82%) of these young women. Younger age (< or = 16 years) was significantly associated with delay in attending for treatment. CONCLUSIONS: Effective management of genital chlamydial infection is achievable in settings outside GUM clinics using a collaborative approach which incorporates cross referencing between community based services and GUM clinics.

Adolescent↗
PubMed1999

Case studies in osteoporosis: a problem based learning intervention for family physicians.

OBJECTIVE: To develop and evaluate a practice based small group (PBSG) learning intervention on osteoporosis for primary care physicians. METHODS: A needs assessment on osteoporosis was performed and objectives for a continuing medical education (CME) program developed by an interdisciplinary advisory committee. Nine clinical cases were developed for evaluation by CME participants with a trained facilitator and content expert using the PBSG format. The effect of the CME intervention was evaluated using a pre and post-test consisting of objective structured clinical examination stations and standardized patients. RESULTS: Fifty-four family physicians participated in 4 pilot PBSG learning sessions. The program format, content, and participant satisfaction was highly rated (> 3.35:4.0). Participants expected the program to have a significant effect on the practices (3:40:4.0). Ninety-eight percent of participants improved their pretest scores, with a mean increase of 13% (range 1-36%). CONCLUSION: Based on our experience, we advocate the use of PBSG learning interventions as an effective and acceptable method of providing CME by rheumatologists for their family physician colleagues. This format appears to be associated with a significant effect on knowledge, skills, and behavior as assessed by our study.

Aged↗