Biomedical subjects

W A Day

Publications and source records attributed to W A Day.

At least 19 recordsLinked to original sources

PubMed2006

Anthrax vaccines: Pasteur to the present.

Anthrax has been a major cause of death in grazing animals and an occasional cause of death in humans for thousands of years. Since the late 1800s there has been an exceptional international history of anthrax vaccine development. Due to animal vaccinations, the rate of infection has dropped dramatically. Anthrax vaccines have progressed from uncharacterized whole-cell vaccines in 1881, to pXO2-negative spores in the 1930s, to culture filtrates absorbed to aluminum hydroxide in 1970, and likely to recombinant protective antigen in the near future. Each of these refinements has increased safety without significant loss of efficacy. The threat of genetically engineered, antibiotic and vaccine resistant strains of Bacillus anthracis is fueling hypothesis-driven research and global techniques--including genomics, proteomics and transposon site hybridization--to facilitate the discovery of novel vaccine targets. This review highlights historical achievements and new developments in anthrax vaccine research.

Animals↗

PubMed2001

Cloning and characterization of a Campylobacter jejuni iron-uptake operon.

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C).

Bacterial Outer Membrane Proteins↗

PubMed2001

Shigella flexneri LuxS quorum-sensing system modulates virB expression but is not essential for virulence.

Quorum-sensing systems regulate the expression of virulence factors in a wide variety of plant and animal pathogens, including members of the Enterobacteriaceae. Studies of Shigella virulence gene expression have demonstrated that maximal expression of genes encoding the type III secretion system and its substrates and maximal activity of this virulence organelle occur at high cell density. In these studies, we demonstrate that the expression of ipa, mxi, and spa invasion operons is maximal in stationary-phase bacteria and that conditioned media derived from stationary-phase cultures enhance the expression of these loci. In contrast, expression of virB, a transcription factor essential for the expression of invasion loci, peaks in late log phase; accordingly, virB expression is enhanced by a signal(s) present in conditioned media derived from late-log-phase cultures. Autoinducer 2 (AI-2), a quorum signaling molecule active in late log phase, was synthesized by Shigella species and enteroinvasive Escherichia coli and shown to be responsible for the observed peak of virB expression. However, AI-2 does not influence invasion operon expression and is not required for Shigella virulence, as mutants deficient in AI-2 synthesis are fully virulent. The implications of these findings with regard to both virB and invasion operon expression and the evolution of circuitries governing virulence gene expression are discussed.

Animals↗

PubMed2001

Pathoadaptive mutations that enhance virulence: genetic organization of the cadA regions of Shigella spp.

Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in Shigella species that have evolved from LDC-expressing Escherichia coli. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of Shigella enterotoxins and that the gene encoding LDC, cadA, was abolished by large chromosomal deletions in each Shigella species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of the cad region were sequenced from the four Shigella species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral E. coli chromosome displaced the cadA locus to form distinct genetic linkages that are unique to each Shigella species. Hybridization studies, using an E. coli K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the Shigella genomes. None of these novel gene arrangements were observed in representatives of all E. coli phylogenies. Collectively, these observations indicate that inactivation of the cadA antivirulence gene occurred independently in each Shigella species. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal E. coli, strong pressures in host tissues selected Shigella clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.

Adaptation, Biological↗

PubMed2000

Role of catalase in Campylobacter jejuni intracellular survival.

The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.

Animals↗

PubMed1997

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.

Campylobacter jejuni↗

PubMed1995

Characterization of lipoproteins containing a truncated form of apolipoprotein B, apolipoprotein B32.

We have characterized the lipoproteins isolated from the plasma of a human subject who was heterozygous for a mutation yielding a truncated apolipoprotein B (apo-B32). The apo-B32 lipoproteins were isolated from the plasma high density lipoprotein (HDL) fraction by heparin-Sepharose chromatography. Although the chemical composition of the apo-B32-containing lipoproteins was similar to that of normal HDL, the mean diameter of the apo-B32 lipoproteins was larger than typical apo-AI-containing HDL particles. On agarose gels, the apo-B32 lipoproteins had pre-beta mobility similar to that of normal very low density lipoproteins. Analysis of the purified apo-B32 lipoproteins by SDS-polyacrylamide gel electrophoresis revealed the presence of both apo-AI and apo-E. In order to further analyze the properties of apo-B32, we developed an apo-B32 expression vector and generated stable rat hepatoma cell lines expressing apo-B32. In these cell lines, the apo-B32 protein was secreted in a d > 1.21 g/ml lipoprotein. Oleic acid supplementation of the cell-culture media had no measurable affect on the density distribution of the apo-B32 lipoproteins that were secreted by the cells.

Animals↗

PubMed1989

Histology of port wine stains after copper vapour laser treatment.

We report histological changes in four patients with port wine stains treated with 578 nm yellow light from a high power copper vapour laser. Histology showed that selective damage occurred to the ectatic blood-vessels in the dermis, without haemorrhage and damage to non-vascular structures, and without scarring. The initial damage to the overlying epidermis was not permanent, and the damaged ectatic vessels returned to normal size or were completely necrosed and replaced by collagen.

Adolescent↗

PubMed1988

Ultrastructural localization of Tamm-Horsfall protein in human kidney using immunogold electron microscopy.

The ultrastructural localization of Tamm-Horsfall protein (THP) was studied in paraformaldehyde-fixed human renal biopsies. Pre-embedding and post-embedding immunogold labelling techniques were developed utilizing a monoclonal antibody specific for human urinary THP. With the pre-embedding technique, membrane contrast was enhanced by osmification thus allowing precise localization of gold particles. Reasonable tissue penetration of antibodies was achieved without compromising ultrastructural detail. The hydrophilic resin LR White was used for post-embedding labelling to ensure maximum penetration of antibodies. However, sections had only mild osmification and consequently localization of label was less certain. Both labelling techniques gave similar results. THP was found to be associated with two renal cell types. Epithelial cells lining the thick ascending limb of Henle's loop had gold label closely associated with the whole cell plasmalemma, with some of these cells having an apparently random distribution of label throughout the cytoplasm. Only the luminal plasmalemma of epithelial cells lining distal convoluted tubules were found to be labelled. Basolateral membranes and the cytoplasm of these cells were negative. The use of a monoclonal antibody of defined specificity combined with the two immunolabelling procedures represents a precise reliable method for studying ultrastructural localization of THP in the human kidney.

Antibodies, Monoclonal↗

PubMed1988

Nicotine decreases the porosity of the rat liver sieve: a possible mechanism for hypercholesterolaemia.

Nicotine was fed to rats for 6 weeks, as a weight adjusted dose equivalent to that of a human being smoking 50 to 100 cigarettes per day. Those rats fed nicotine developed hypercholesterolaemia. Scanning electron microscopy showed the porosity of the hepatic sinusoidal endothelium of nicotine fed animals was about 40% that of control animals. The decline in porosity was found to be due to a reduction in diameter rather than number of fenestrae. We believe that this decreased hepatic sinusoidal porosity may alter cholesterol homeostasis by increasing the circulation time of chylomicron remnants too large to pass through the fenestrae. This phenomenon may be an aetiological factor in the known correlation between cigarette smoking, atherosclerosis, and coronary heart disease in humans.

Animals↗

PubMed1986

The liver sinusoidal cells. Their role in disorders of the liver, lipoprotein metabolism and atherogenesis.

A review of the morphological and metabolic interactions between hepatocytes, the various sinusoidal cells and sinusoidal blood is presented. This field of investigation is rapidly expanding, with widespread implications in the regulation of physiological processes and diseases in the liver and other organs. Although most of the review is of already published work, some areas incorporate personal speculations of the probable role of sinusoidal cells in disease processes.

Alcoholism↗

PubMed1986

Ultrastructural studies of the portal transport of fat in chickens.

Dietary fat is transported in the chicken by portomicrons; these large lipoproteins enter the portal blood of the small intestinal villi. We have shown by electron microscopy that avian portomicrons resemble mammalian chylomicrons in size, but their mode of transport differs. Portomicrons enter the intestinal blood vessels through endothelial intracytoplasmic vesicles, whereas chylomicrons enter the intestinal lymphatics through gaps between endothelial cells. We have also shown that the sinusoidal endothelium of the chicken liver, like that of the mammal, is fenestrated. Because the fenestrae are relatively few in number, the endothelium is less porous in the chicken than in the rat. We postulate that this prevents the hepatocytes from being swamped by dietary fat, but makes the chicken susceptible to diet-induced atherosclerosis.

Animals↗

PubMed1985

An ultrastructural study of adenocarcinoma of the small intestine in sheep.

The ultrastructural appearance at primary and metastatic sites of ten ovine small intestinal adenocarcinomas was that of scirrhous tubular adenocarcinoma. Polygonal undifferentiated tumor cells had desmosomes, folded nuclei, and moderate numbers of mitochondria but few other organelles. More differentiated cells were columnar with apical microvilli and basal nuclei. They contained granular endoplasmic reticulum, Golgi and secretory granules. Microvillus-lined intracytoplasmic lumina (5-10 micron diameter), fibrous filaments (10 nm diameter, up to 1.4 micron length) and tubular paracrystalline arrays (hexagonal symmetry, 37-nm periodicity) in lumina and secretory granules were seen in some tumor cells in all ten sheep.

Adenocarcinoma↗

PubMed1985

Neutrophils adherent to a nonphagocytosable surface (glomerular basement membrane) produce oxidants only at the site of attachment.

Adherence of neutrophils to glomerular basement membrane containing immunoglobulin G aggregates was accompanied by a marked increase in oxygen uptake (eightfold). Very little of the O2 consumed was recovered as superoxide, measured by cytochrome c reduction, or as H2O2, measured with horseradish peroxidase and scopoletin. When neutrophils were incubated with the basement membrane preparation in the presence of cerium chloride to detect H2O2, electron micrographs showed cerium perhydroxide deposits in the contact area between the cells and the basement membrane, but not on the remainder of the cell surface. The results imply that superoxide is produced only where the plasma membrane is in contact with the basement membrane matrix, and that it mostly breaks down to H2O2 or undergoes other reactions at this site. The longer lifetime of H2O2 compared with that of superoxide allows some of the H2O2 produced to be detected in the medium. The results also suggest that the area of contact between the neutrophil and surfaces such as basement membrane is inaccessible to proteins in the medium, eg, cytochrome c. Circulating scavengers such as superoxide dismutase or catalase, or proteolytic inhibitors, may therefore be unable to control events occurring at this site.

Basement Membrane↗

PubMed1983

Hepatic sinusoidal endothelium in sheep: an ultrastructural reinvestigation.

Following perfusion fixation of livers from fetal, neonatal, and adult sheep, the ultrastructure of the fenestrated sinusoidal endothelium was examined. In contrast to an earlier report which utilized immersion fixation, we found the fenestrae to be unoccluded by diaphragms or basal lamina, thus offering only a discontinuous barrier between the blood and the space of Disse and hepatocytes. This observation was confirmed by perfusing the liver with marker particles. The fenestrae appear, therefore, to have a sieving function, regulating the metabolism of particulate matter such as lipoproteins in sheep in a similar manner to that which has been shown to occur in rats.

Animals↗

PubMed1983

Hepatic sinusoidal endothelium in goats.

The ultrastructure of the hepatic sinusoids of the goat was examined. Contrary to current belief concerning this and other ruminant species, we found the sinusoids to be lined by a smooth, continuous layer of endothelium, broken only by the presence of numerous, unoccluded fenestrae which provide a direct path of communication between the sinusoidal lumen and the space of Disse. Therefore, we suggest that a sieving function similar to that which has been shown to occur in rats may be attributed to the sinusoidal endothelium of the goat.

Animals↗

PubMed1981

Acute promyelocytic leukemia: cytogenetics and bone-marrow culture.

Six patients were diagnosed as having acute promyelocytic leukemia (APL) according to FAB criteria. One patient conformed to the M3 variant. Informative cytogenetic results (G-banding) on five of the patients showed that three of them, including the M3 variant, had the 15;17 translocation in bone-marrow or blood cells. Cells with the translocation were accompanied by cells with a normal karyotype in all patients and no other chromosomal abnormality was present. This first report of the 15;17 translocation from the South Pacific region is relevant to the uneven geographical distribution of APL patients with the translocation. Five of the six patients including the M3 variant, showed a distinctive pattern of cell growth in agar culture characterized by a profusion of small, uniform clusters containing 6-20 cells with the appearance of promyelocytes. The remaining patient had a pattern of cell growth more typical of M2 acute leukemia. This cell growth pattern may be useful in diagnosing and monitoring the course of APL.

Aged↗

PubMed1981

Mouse glomerular culture.

A method is described for the culture of isolated intact mouse glomeruli. Cultures showed adherent glomeruli after 2 days and from 2-4 days a monolayer of cells began to develop around the points of adherence. The predominant cell (Type I) was 80-200 micrometer in diameter with cytoplasmic extensions, later becoming polyhedral and often multinucleated. Type II spindle cells, 70-100 micrometer in length, were less prominent in the earlier stages and later formed sheaves between Type I cells. Small phagocytic Type III cells, 10-20 micrometer in diameter, were not prominent in cultures from normal kidneys and tended to form small clusters on top of the Type I cell monolayer.

Animals↗