Reactivation of the Epstein-Barr Virus Leading to Acute Liver Failure in a Patient Living with HIV.

We report a case of a 46-year-old female living with HIV since 2010 who was originally from Malawi and had settled in the UK in 2001. She was admitted to our hospital with confusion and quickly noted to have a decreased Glasgow Coma Scale of 10/15. Her biochemical parameters showed the presence of elevated liver function tests (LFTs), clotting abnormalities, and her ammonia were found to be >400 mmol/L with a severe metabolic acidosis (pH = 7.05). She was treated for HIV with combined antiretroviral therapy, namely tenofovir disoproxil fumarate, emtricitabine (FTC) and cobicistat boosted atazanavir 2 years previously and had normal LFTs at that time. Her HIV-1 viral load was 1400 copies/ml on admission after recently having an undetectable viral load 2 months previously, and her CD4 count was 480. Her relevant past medical history included insulin-dependent diabetes mellitus. Her other medications included insulin, ramipril, sertraline, amitriptyline, and zopiclone. Toxicology and viral hepatitis screen were negative. Epstein Barr virus (EBV) serology showed evidence of previous exposure, but she was found to have a very high EBV viral load of 55,000 copies/ml, which given her serology, was very likely to be a reactivation of EBV infection rather than a primary EBV infection. In the intensive care unit, the patient deteriorated and died very quickly. The postmortem examination showed extensive hepatic necrosis with collapse. To our knowledge, this is the first case report to show an association between EBV reactivation and fulminant hepatic failure in an individual living with HIV.

Regulation of nerve growth and patterning by cell surface protein disulphide isomerase.

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.

Targeting chondroitinase ABC to axons enhances the ability of chondroitinase to promote neurite outgrowth and sprouting.

BACKGROUND: There is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of ß-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the ß-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth. CONCLUSIONS / SIGNIFICANCE: The novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson's disease.

Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

BACKGROUND: There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. METHODOLOGY/PRINCIPAL FINDINGS: To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of ß-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. CONCLUSION/SIGNIFICANCE: Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

HIV - lessons from a late diagnosis.

Late HIV diagnosis is the most important predictor of HIV-related morbidity and mortality in the UK and often results from missed testing opportunities during earlier contact with health services. The HPA now recommends routine HIV testing be commissioned as a priority for all general medical admissions in high prevalence areas, such as Milton Keynes. We present the case of a patient admitted to our Medical Admissions Unit (MAU) managed initially for presumed septic complications of metastatic disease who was later found to have terminal HIV disease. In keeping with UK-wide experience which we review, a local audit following this case found MAU HIV test coverage increased after routine testing but not after staff education alone, and resulted in implementation of routine HIV testing in our MAU.

Use of steroids for management of varicella pneumonia.

Varicella pneumonia (VP) is a critical complication of varicella infection and still carries significant morbidity and mortality, often requiring intensive care unit admission. Current accepted treatment is with intravenous aciclovir and organ support, if required. We report two cases of VP with acute respiratory failure, successfully treated with intravenous steroids in addition to aciclovir. Further research into the benefits of steroid therapy in VP is warranted.

Fusion of FNA-cytology and gene-expression data using Dempster-Shafer Theory of evidence to predict breast cancer tumors.

Decision-in decision-out fusion architecture can be used to fuse the outputs of multiple classifiers from different diagnostic sources. In this paper, Dempster-Shafer Theory (DST) has been used to fuse classification results of breast cancer data from two different sources: gene-expression patterns in peripheral blood cells and Fine-Needle Aspirate Cytology (FNAc) data. Classification of individual sources is done by Support Vector Machine (SVM) with linear, polynomial and Radial Base Function (RBF) kernels. Out put belief of classifiers of both data sources are combined to arrive at one final decision. Dynamic uncertainty assessment is based on class differentiation of the breast cancer. Experimental results have shown that the new proposed breast cancer data fusion methodology have outperformed single classification models.

A herpesvirus vector can transduce axotomized brain neurons.

If gene therapy is to be used to promote axon regeneration after spinal cord injury, a suitable vector for transgene delivery must be obtained. Replication-defective herpes simplex virus (HSV) vectors are promising candidates. We have examined whether they can express a LacZ transgene in injured neurons of adult rat brain. We transected the medial forebrain bundle, injected replication-defective HSV/LacZ vectors close to the lesion site, and looked for transgene expression at 2-14 days after the lesion. The vectors carried the LacZ transgene controlled either by the cytomegalovirus immediate-early promoter (vector CS5) or the HSV latency-associated promoter (vector CS1). CS5 transfected many cells near the lesion at 2 days, but did not give persistent expression at 5 days. CS1, in contrast, labeled many neurons in midbrain regions remote from the injection site at 5 days, and much of this expression remained at 12-14 days. The neurons of most interest were in the substantia nigra pars compacta and parabrachial nuclei, which were axotomized by the lesion. Vector-driven beta-galactosidase expression was detected in neurons in both regions. These were confirmed as axotomized by double immunofluorescence for c-Jun. By 12-14 days, many substantia nigra neurons had disappeared but some transduced neurons remained; there was no net loss of transduced neurons from the parabrachial nuclei. These results show that an HSV vector is capable of transducing axotomized cells in the central nervous system and producing transgene expression in them for at least 2 weeks after injection.