PubMed HealthSearch

Biomedical subjects

D J Holland

Publications and source records attributed to D J Holland.

14 recordsLinked to original sources

Anterograde transport of herpes simplex virus type 1 in cultured, dissociated human and rat dorsal root ganglion neurons.

The mechanism of anterograde transport of herpes simplex virus was studied in cultured dissociated human and rat dorsal root ganglion neurons. The neurons were infected with HSV-1 to examine the distribution of capsid (VP5), tegument (VP16), and glycoproteins (gC and gB) at 2, 6, 10, 13, 17, and 24 h postinfection (p.i.) with or without nocodazole (a microtubule depolymerizer) or brefeldin A (a Golgi inhibitor). Retrogradely transported VP5 was detected in the cytoplasm of the cell body up to the nuclear membrane at 2 h p.i. It was first detected de novo in the nucleus and cytoplasm at 10 h p.i., the axon hillock at 13 h p.i., and the axon at 15 to 17 h p.i. gC and gB were first detected de novo in the cytoplasm and the axon hillock at 10 h p.i. and then in the axon at 13 h p.i., which was always earlier than the detection of VP5. De novo-synthesized VP16 was first detected in the cytoplasm at 10 to 13 h p.i. and in the axon at 16 to 17 h p.i. Nocodazole inhibited the transport of all antigens, VP5, VP16, and gC or gB. The kinetics of inhibition of VP5 and gC could be dissociated. Brefeldin A inhibited the transport of gC or gB and VP16 but not VP5 into axons. Transmission immunoelectron microscopy confirmed that there were unenveloped nucleocapsids in the axon with or without brefeldin A. These findings demonstrate that glycoproteins and capsids, associated with tegument proteins, are transported by different pathways with slightly differing kinetics from the nucleus to the axon. Furthermore, axonal anterograde transport of the nucleocapsid can proceed despite the loss of most VP16.

Animals

Anterograde transport of herpes simplex virus proteins in axons of peripheral human fetal neurons: an immunoelectron microscopy study.

Herpes simplex virus (HSV) reactivates from latency in the neurons of dorsal root ganglia (DRG) and is subsequently transported anterogradely along the axon to be shed at the skin or mucosa. Although we have previously shown that only unenveloped nucleocapsids are present in axons during anterograde transport, the mode of transport of tegument proteins and glycoproteins is not known. We used a two-chamber culture model with human fetal DRG cultivated in an inner chamber, allowing axons to grow out and penetrate an agarose barrier and interact with autologous epidermal cells in the outer chamber. After HSV infection of the DRG, anterograde transport of viral components could be examined in the axons in the outer chamber at different time points by electron and immunoelectron microscopy (IEM). In the axons, unenveloped nucleocapsids or focal collections of gold immunolabel for nucleocapsid (VP5) and/or tegument (VP16) were detected. VP5 and VP16 usually colocalized in both scanning and transmission IEM. In contrast, immunolabel for glycoproteins gB, gC, and gD was diffusely distributed in axons and was rarely associated with VP5 or VP16. In longitudinal sections of axons, immunolabel for glycoprotein was arrayed along the membranes of axonal vesicles. These findings provide evidence that in DRG axons, virus nucleocapsids coated with tegument proteins are transported separately from glycoproteins and suggest that final assembly of enveloped virus occurs at the axon terminus.

Axons

The axonal transmission of herpes simplex virus to epidermal cells: a novel use of the freeze substitution technique applied to explant cultures retained on cover slips.

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a technical challenge. We developed a technique to study the axonal transport of the Herpes simplex virus from dorsal root ganglia sensory neurones to epidermal cells. Autologous explants of human foetal dorsal root ganglia and skin were cultured on plastic cover slips. Axon fascicles grew from the ganglia to the epidermal cells and the ganglia were inoculated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from paraformaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, and embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the resin block, which retained the tissue. The resin block was placed in fresh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block. After trimming, serial sections were easily obtained and successfully immunolabelled for viral proteins. This is a convenient technique for immunolabelling tissue grown on cover slips in which the preservation of the ultrastructural interactions between different cells is important. It should be adaptable to a number of cell-culture applications and has a number of advantages over other techniques.

Axons

Emerging viruses.

An emerging virus is a term applied to a newly discovered virus, one that is increasing in incidence or with the potential to increase in incidence. Many viruses fit into this definition. HIV is the clearest example of a previously unknown virus that has now produced one of the largest pandemics in history. Recent advances have occurred in the identification and understanding of new hantaviruses in the Americas, causing an acute respiratory disease. The possible causal role of human herpesvirus 8 in Kaposi's sarcoma has gained support, whereas that of a newly discovered flavivirus in causing hepatitis has not been confirmed. A major advance has been evidence showing that the bovine spongiform encephalopathy agent is almost certainly the cause of a new variant of Creutzfeldt-Jakob disease. Although new viruses are discovered almost yearly (e.g., Australian bat lyssavirus), other "older" viruses (e.g., dengue) are reemerging, infecting millions of people every year with significant mortality.

Animals

A comparison of chemical dipsticks read visually or by photometry in the routine screening of urine specimens in the clinical microbiology laboratory.

Two different commercially available urine dipsticks were evaluated to determine their usefulness as a screening test for the detection of non-infected urine specimens. The reactions of both dipstick strips were read visually and, in addition, one was read by semi-automated reflectance photometry. 2928 consecutive routine urine specimens received by the microbiology laboratory during normal working hours underwent testing by the conventional method of microscopy and culture and the results were compared with those obtained by dipstick testing. There were poor correlations between microscopy for red and white blood cells and dipstick results for blood and leucocyte esterase respectively. The dipsticks had a low positive predictive value (PPV) in identifying infected urines. In contrast, dipsticks had a very high negative predictive value (NPV) of 98.6-99.5% and correctly identified a significant proportion (about 1/3) of non-infected urines as reported by the conventional method. Introduction of dipstick testing of urine specimens in our hospital has led to significant time saving as dipstick-negative urines are not processed further (except for specific clinical indications) and there has also been a significant decrease (25%) in the number of specimens submitted to the laboratory. Further efficiencies may be obtainable with increased automation.

Adolescent

Mycobacterium neoaurum infection of a Hickman catheter in an immunosuppressed patient.

Mycobacterium neoaurum is a rapidly growing mycobacterium that has only once before been reported as a cause of bacteremia in an immunocompromised patient. We report on a patient who developed bacteremia after an allogeneic bone marrow transplantation and from whose blood (taken via a Hickman catheter) M. neoaurum was isolated on three separate occasions. There was a clinical response to therapy with ticarcillin/clavulanate and tobramycin, and the organism appeared to be susceptible to the former in vitro. The Hickman catheter was removed because of persistence of the organism in cultures of blood taken from the catheter. M. neoaurum may be a rare cause of infections in immunocompromised patients and should not be dismissed as an environmental contaminant.

Adolescent

Antimicrobial therapy and prevention of spontaneous bacterial peritonitis.

Spontaneous bacterial peritonitis is a frequent and serious infection in cirrhotic patients with ascites. A high index of suspicion is required for early diagnosis and rapid institution of treatment. The common micro-organisms involved in SBP are the aerobic Gram-negative bacilli and Gram-positive cocci that inhabit the intestine. Empiric antibiotic therapy active against these organisms should be instituted as soon as possible to improve survival. Third generation cephalosporins are very effective and safe as the initial empiric antibiotic regimen. Alternatives include beta-lactam-clavulanic acid combinations and other broad-spectrum antibiotics, although cost benefit considerations are important in selection. If cultures and susceptibility tests allow, antibiotic therapy should be altered to provide optimum narrow-spectrum and cost-effective treatment. Recent evidence suggests that (at least in the case of cefotaxime), 5-day treatment is equally effective as 10-day treatment. Except in patients awaiting liver transplantation, antibiotic prophylaxis of SBP is not recommended at present, as the few trials performed have not been able to demonstrate superior results for survival, hospital admissions or cost-effectiveness, over prompt diagnosis and therapy of individual episodes of SBP.

Anti-Bacterial Agents