B cell activation by cytomegalovirus.

Human cytomegalovirus is shown to be a nonspecific polyclonal B cell activator. The B cell response is independent of virus replication and requires little, if any, T cell help.

Patient-focused outcomes following detection in a hospital-based screening programme for C282Y haemochromatosis.

BACKGROUND: Haemochromatosis is a common genetic disease in populations of a northern European origin. However, there is uncertainty as to whether it is a condition that should be screened for. AIMS: To determine the proportion of persons, in a public hospital setting, who were homozygous for the C282Y mutation for hereditary haemochromatosis and the proportion of these persons who would benefit from therapeutic phlebotomy. METHODS: All persons who had blood submitted for pathology testing, had total iron-binding capacity and iron measured and transferrin saturation calculated, and where this result exceeded 40%, genotyping for the C282Y mutation was carried out. RESULTS: Of 18,779 patients screened, 887 (5.4%) were found to have transferrin saturation greater than 40%. Thirty-five of these were homozygous for the C282Y mutation. Fourteen were previously known to be affected and six of these were non-compliant with venesection. Venesection was commenced in 5 of the 21 newly diagnosed subjects. CONCLUSIONS: The proportion of detected subjects who commenced venesection was significant. Results suggest that clinical penetrance is higher in Australia than other countries and that even in the environment of a large tertiary teaching hospital, phenotypic screening identifies cases of hereditary haemochromatosis, which are likely to benefit from treatment.

Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma.

Loss of the Epstein-Barr virus (EBV) genome from Akata Burkitt lymphoma (BL) cells is coincident with a loss of malignant phenotype, despite the fact that Akata and other EBV-positive BL cells express a restricted set of EBV gene products (type I latency) that are not known to overtly affect cell growth. Here we demonstrate that reestablishment of type I latency in EBV-negative Akata cells restores tumorigenicity and that tumorigenic potential correlates with an increased resistance to apoptosis under growth-limiting conditions. The antiapoptotic effect of EBV was associated with a higher level of Bcl-2 expression and an EBV-dependent decrease in steady-state levels of c-MYC protein. Although the EBV EBNA-1 protein is expressed in all EBV-associated tumors and is reported to have oncogenic potential, enforced expression of EBNA-1 alone in EBV-negative Akata cells failed to restore tumorigenicity or EBV-dependent down-regulation of c-MYC. These data provide direct evidence that EBV contributes to the tumorigenic potential of Burkitt lymphoma and suggest a novel model whereby a restricted latency program of EBV promotes B-cell survival, and thus virus persistence within an immune host, by selectively targeting the expression of c-MYC.

Diverse familial malignant tumors and Epstein-Barr virus.

Continued monitoring of a family for new malignant tumors has revealed diverse immunological and neoplastic disorders during a 15-year period. In 1966, the proband developed lymphoma. In 1975, his antibody titers to Epstein-Barr virus (EBV) became elevated, and again, he developed a malignant lymphoma. He also had borderline hypo-immunoglobulin A, died of glioblastoma multiforme in 1977, and at autopsy, had adenomatous colonic polyps. His eldest brother has normal immunoglobulin levels, but developed immune thrombocytopenia in 1973 and had elevated EBV antibody titers in 1980. Another brother had hypo-immunoglobulin A, thymoma in 1965, and adenomas and adenocarcinoma of the colon. Two other brothers succumbed to glioblastoma in 1968 and 1969. The father of the proband had bronchiectasis in 1952, hypo-immunoglobulin M documented in 1972, and elevated EBV antibody titers 5 years preceding development of a malignant lymphoma. The latter contained 10 EBV genome equivalents/cell by EBV viral DNA/DNA reassociation kinetics analysis. The proband's grandmother had died of an immunoglobulin G-secreting myeloma in 1977, and his grandfather had borderline low immunoglobulin M, elevated EBV antibody titers, and hypopharyngeal carcinoma in 1980. Predisposition to oncogenesis in this family was probably inherited.

Requirements for immunoglobulin synthesis in leukocyte cultures exposed to human cytomegalovirus.

T cell-depleted leukocytes from normal healthy donors lacking antibody to cytomegalovirus (CMV) were induced to secrete immunoglobulin (Ig) by exposure to inactivated CMV. The responses to two virus strains were compared. One strain had been reported to require specific T cell help to induce Ig synthesis, and the other had been reported to induce Ig synthesis in the presence of only very few cells. Both viruses induced T cell-depleted leukocytes from one group of seronegative donors to secrete Ig. However, both viruses failed to induce leukocytes from a second group of donors to secrete Ig unless B cell growth factor was added as a source of T cell help. These findings suggest that certain individuals are hyperresponsive to CMV and raise the possibility that this group may be most likely to develop mononucleosis after primary infection with CMV.

Cytotoxic flow-cytometric crossmatches (flow-tox): a comparison with conventional cytotoxicity crossmatch techniques.

Detection and avoidance of donor-reactive antibodies in the sera of potential organ transplant recipients is key to a successful transplant outcome. Techniques of antibody detection that use flow cytometry are more sensitive than those that rely upon a visual determination of cytotoxicity. However, as conventionally performed, flow-cytometric crossmatches do not distinguish between cytotoxic (complement fixing) and noncytotoxic antibodies because both types of antibodies can bind to a cell and be detected by laser-activated fluorochrome photon emission. In 1989 we described two techniques for detecting cytotoxic antibodies using flow-cytometric techniques [1]. In 1990, we expanded the application of these new techniques that we called flow cytotoxicity assays or "Flow-Tox" [2]. Flow-Tox crossmatches demonstrate an increase in both sensitivity and specificity over conventional cytotoxicity crossmatches.

Using green fluorescent protein to study intracellular signalling.

Subcellular compartmentalisation of signalling molecules is an important phenomenon not only in defining how a signalling pathway is activated but also in influencing the desired physiological output of that pathway (e.g. cell growth or differentiation, regulation of metabolism, cytoskeletal changes etc.). Biochemical analyses of protein and lipid compartmentalisation by, for example, subcellular fractionation presents many technical difficulties. However, this aspect of cell signalling research has seen a major revolution thanks to the cloning and availability of a variety of mutant green fluorescent protein derivatives with distinct molecular properties. Mutants with increased brightness, altered excitation and emission maxima, altered stability and differential sensitivity to pH, are now in widespread use for following the trafficking and function of proteins in living cells and for monitoring the intracellular environment. In this review we focus on some of the recent developments in the use of green fluorescent proteins for studying intracellular signalling pathways often with special reference to the actions of insulin. We also discuss the future utility of these proteins to analyse protein--protein interactions in signalling pathways using fluorescence resonance energy transfer.

Review and evaluation of MRI nonuniformity corrections for brain tumor response measurements.

Current MRI nonuniformity correction techniques are reviewed and investigated. Many approaches are used to remedy this artifact, but it is not clear which method is the most appropriate in a given situation, as the applications have been with different MRI coils and different clinical applications. In this work four widely used nonuniformity correction techniques are investigated in order to assess the effect on tumor response measurements (change in tumor volume over time): a phantom correction method, an image smoothing technique, homomorphic filtering, and surface fitting approach. Six brain tumor cases with baseline and follow-up MRIs after treatment with varying degrees of difficulty of segmentation were analyzed without and with each of the nonuniformity corrections. Different methods give significantly different correction images, indicating that rf nonuniformity correction is not yet well understood. No improvement in tumor segmentation or in tumor growth/shrinkage assessment was achieved using any of the evaluated corrections.

Molecular mechanisms of insulin-stimulated glucose uptake in adipocytes.

The stimulation of muscle and adipose tissue glucose metabolism, which is ultimately responsible for bringing about post-absorptive blood glucose clearance, is the primary clinically relevant action of insulin. Insulin acts on many steps of glucose metabolism, but one of the most important effects is its ability to increase the rate of cellular glucose transport. This results from the translocation of the insulin-responsive transporter isoform, GLUT4, from intra-cellular vesicular storage sites to the plasma membrane. In adipocytes, a substantial amount of cellular GLUT4 is located in a specific highly insulin-responsive storage pool, termed GLUT4 Storage Vesicles (GSVs). GLUT4 can also translocate to the plasma membrane from the recycling endosomal pool which also additionally contains the GLUT1 isoform of glucose transporter and the transferrin receptor. In this article we review the molecular mechanism by which insulin stimulates GLUT4 translocation in adipose cells, including the nature of the signaling pathways involved and the role of the cytoskeleton.

Method for image equalization of ROI fluoroscopic images using mask localization, selection and subtraction.

In region of interest (ROI) fluoroscopy, a filter is used to drastically reduce the X-ray dose to the patient peripheral to an ROI, and mask subtraction is used to equalize the displayed image brightness. Methods are described for an optimized search to locate the ROI and select or construct a mask image using a descriptor look up table (DLUT) based on ROI size, relative ROI/periphery brightness and ROI shape and orientation. After mask subtraction, the brightness values of the periphery are comparable to those of the ROI providing for equalized display. This method was tested successfully on 50 actual fluoroscopic images of two anthromorphic phantoms. The methodology developed for real-time applications was successfully simulated in PC code and full real-time implementation is underway.

Proteins specified by bovine herpesvirus-2.

Polypeptides synthesized in bovine testes cells infected with bovine herpesvirus type 2 were labeled with [35S]methionine and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thirty-eight virus-induced proteins, ranging in molecular weight from 32,000 to 149,000, were detectable by analysis of whole cell lysates between postinfection hours 2 and 36. A similar number was immunoprecipitated by rabbit antiserum to bovine herpesvirus type 2. Twelve proteins incorporated [3H]glucosamine. On the basis of their temporal characteristics and their pattern of synthesis in the presence of cycloheximide and dactinomycin, 3 proteins, including at least one that was nondetectable in the absence of drugs, were classified as alpha proteins, 4 as beta proteins, 1 as a beta/gamma protein, and 27 as gamma proteins. Host cell protein synthesis was not reduced substantially until postinfection hours 22 to 28.

Head and neck tumour immunology: basic concepts and new clinical implications.

An understanding of the immune system and its modes of action is fundamental to understanding the causes, natural history, management and treatment of many diseases. As such, a grasp of the principles of immunology is essential for every physician.This paper represents a succinct overview of the immune system, discussing the major components in turn, in respect of structure, function and integrated organisation, in relation to head and neck cancer.

Epstein-Barr virus shed in saliva is high in B-cell-tropic glycoprotein gp42.

Epstein-Barr virus is an orally transmitted human herpesvirus that infects epithelial cells and establishes latency in memory B lymphocytes. Movement of virus between the two cell types is facilitated by changes in amounts of an envelope glycoprotein, gp42, which are effected by interaction of gp42 with HLA class II in a B cell. Here we used the differential ability of virus to bind to CD21-positive B cells and CD21-negative epithelial cells, which is also influenced by levels of gp42, to determine that the majority of virus shed in saliva is derived from an HLA class II-negative cell.

Alcohol and iron: one glass of red or more?

The consumption of excessive amounts of alcohol affects human iron homeostasis and an association of iron overload and heavy alcohol consumption has been recognized for many years. Both major proteins of iron metabolism, ferritin and transferrin, are affected by alcohol. Increased hepatic iron levels are seen in a high proportion of alcoholic subjects, sometimes causing confusion in diagnosis between alcoholic liver disease and iron-overload disease. The pattern of deposition of this iron in alcoholics, however, differs from that seen in the iron-overload disease haemochromatosis. Excessive alcohol consumption causes transferrin to become carbohydrate deficient, which allows it to be used as an efficient biochemical marker of alcohol abuse. It is concluded that alcohol consumption in moderation is unlikely to have deleterious health consequences.

Haemochromatosis: understanding the mechanism of disease and implications for diagnosis and patient management following the recent cloning of novel genes involved in iron metabolism.

Haemochromatosis, a common recessive genetic disorder in people of Northern European descent, is an iron storage disorder characterized by excessive hepatic iron accumulation resulting from disruption of the regulation of intestinal iron absorption. The identification of novel genes involved in the control of iron absorption from the diet has allowed improved understanding of iron metabolism in health and disease. In particular, the identification of the haemochromatosis gene (HFE) and more recently the transferrin receptor 2 gene (TfR2) together with the specific mutations in these genes which result in hepatic iron overload, has enhanced our understanding of the pathophysiology of haemochromatosis. However, because of the wide variation in phenotypic expression of the disease, there now exists a considerable challenge to diagnosis and patient management.