Debra J. Bevitt

Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin-producing polyketide synthase from Saccharopolyspora erythraea

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6‐deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3′ regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin‐producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N‐terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co‐purified with DEBS 2 and DEBS 3 and had an N‐terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.

New monoclonal antibodies to oestrogen and progesterone receptors effective for paraffin section immunohistochemistry.

Assessment of oestrogen and progesterone receptors (ER and PgR) in breast cancer is widely used for the prediction of response to endocrine therapy and as a prognostic marker. Cytosolic assays have been replaced in many centres by immunochemical techniques, which have many advantages including applicability to small samples, simplicity, and cost‐effectiveness. This study describes the generation and characterisation of two novel murine monoclonal antibodies recognizing ER and PgR, designated NCL‐ER‐6F11 and NCL‐PGR respectively, which are effective in heat‐treated formalin‐fixed, paraffin‐embedded tissue. The antibodies have been characterized by Western blotting and by immunohistochemistry on normal and pathological breast and other tissues. NCL‐ER‐6F11 has been shown to compare favourably with a currently available ER antibody. These antibodies may prove of value in the assessment of hormone receptor status in human breast cancer.

Expression of ADAMTS metalloproteinases in the retinal pigment epithelium derived cell line ARPE-19: Transcriptional regulation by TNFα

ADAMTS (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type I motifs) are multidomain proteins with demonstrated metalloproteinase functionality and have potential roles in embryonic development, angiogenesis and cartilage degradation. We present here investigations of ADAMTS expression in an ocular cell type, ARPE-19, with a view to implicating them in retinal matrix turnover. Expression analysis was undertaken using a combination of reverse transcription polymerase chain reaction (RT-PCR) and Northern blotting experiments, which together detected the expression of mRNAs for several ADAMTS proteins, all of which have active site motifs characteristic of matrix metalloproteases (MMPs). These included ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS5, ADAMTS6, ADAMTS7 and ADAMTS9. The expression of mRNA isoforms for ADAMTS7 and ADAMTS9 were also detected. Following stimulation with TNFalpha, ADAMTS1, ADAMTS6 and both ADAMTS9 transcripts expressed in ARPE-19 cells showed a potent upregulation. The expression of ADAMTS genes in ARPE-19 cells and the transcriptional stimulation of some family members by TNFalpha may implicate them in inflammatory eye disease and the compromise of retinal matrix structure, which is evident in age-related macular degeneration (ARMD) and other retinal pathologies.

Comparison of cytotoxic T‐lymphocyte responses to hepatitis C virus core protein in uninfected and infected individuals

Cytotoxic T lymphocytes have been implicated in the control of hepatitis C virus (HCV) infection. Recognition by cytotoxic T lymphocytes of epitopes within HCV core protein has been defined previously by in vitro stimulation with synthetic peptides. The aim of this study has been to examine cytotoxic T‐lymphocyte responses generated against peptides produced naturally following intracellular processing of viral protein. Antigen‐specific cytotoxic T‐lymphocyte lines were generated from both HCV uninfected and infected individuals by culturing CD8+ T cells with autologous dendritic cells loaded intracytoplasmically with recombinant HCV core protein. Analysis of the epitopes recognized by core protein‐specific cytotoxic T lymphocytes used synthetic peptides that were selected based on their predicted binding to HLA‐A*0201 molecules. Core protein‐specific cytotoxic T lymphocytes derived from HCV uninfected and infected individuals were able to lyse autologous target cells pulsed with each of 5 predicted epitopes. Generation of HCV‐specific cytotoxic T lymphocytes using dendritic cells as antigen presenting cells provides a method of comparing the potential repertoire of cytotoxic T‐lymphocyte responses to the responses that occur in chronically infected individuals. No evidence of a qualitatively different response by patient cytotoxic T lymphocytes was apparent which might explain persistence of the virus. J. Med. Virol. 58:239–246, 1999.

Hepatitis C virus density heterogeneity and viral titre in acute and chronic infection: a comparison of immunodeficient and immunocompetent patients

BACKGROUND Heterogeneities in the buoyant density of hepatitis C virus RNA have been reported in different groups of patients, and have been attributed to differential binding of viral particles to beta-lipoproteins and IgG, and the presence of hepatitis C virus nucleocapsids in circulation. It may be that hepatitis C virus density heterogeneity correlates with the severity of liver disease, hepatitis C virus RNA titre, and the immunocompetence of the patient. METHODS AND RESULTS We have analysed five immunodeficient patients (one with hypogammaglobulinaemia and selective IgA deficiency, one with X-linked agammaglobulinaemia, three with common variable immunodeficiency) who have been acutely infected with the same batch of intravenous immunoglobulin contaminated with hepatitis C virus (genotype 1a). The course of hepatitis C virus infection in these patients was compared to one immunocompetent patient who presented with acute hepatitis C virus and progressed to chronic disease, and seven immunocompetent patients with chronic hepatitis C. Serum samples were analysed by differential flotation ultracentrifugation in NaCl solution (density 1.063 g/ml). The high and low density fractions were tested for the presence of RNA by RT-PCR. Serum samples were also quantified for hepatitis C virus RNA (Amplicor HCV Monitor kit, Roche Diagnostic Systems). Three quarters of the acutely infected patients analysed presented with low density hepatitis C virus. Low density hepatitis C virus was absent in most chronic infections but persisted in two patients with common variable immunodeficiency. High density hepatitis C virus was detected in the chronic phase in all acutely infected patients in whom the disease persisted, and was present in all samples from PCR-positive patients with chronic infection. Immunodeficient patients had significantly higher hepatitis C virus RNA titres on presentation than immunocompetent patients, but there was no correlation between titre and clinical course of infection. CONCLUSIONS Heterogeneities in the buoyant density of hepatitis C virus RNA have been identified in the patient groups studied. Low density hepatitis C virus is detected more often in acute infection and high density hepatitis C virus is detected more often in chronic infection. Despite acute infection via the same route of infection with the same hepatitis C virus strain, the five immunodeficient patients studied all followed a different clinical course.

Intervening Early: Attendance and Performance Monitoring as a Trigger for First Year Support in the Biosciences.

Abstract A centralised system monitoring attendance and performance among first year students in Biomedical Sciences has been established at Newcastle University. Early signs of absence and poor performance trigger immediate intervention by academic staff, with the aim of providing support for students at risk of failure or withdrawal. Difficulties associated with monitoring attendance in large lecture classes are avoided by monitoring attendance only at ‘high stakes’ classes, namely practicals and seminars. Level of attendance at non-lecture classes was a predictor of academic achievement and the early intervention strategy was associated with improvements in attendance. Student perceptions of attendance monitoring were evaluated and found to be positive. Meeting with absent and underperforming students at the earliest possible opportunity has proved an effective way of promoting dialogue between staff and students who are experiencing difficulties.

Immunohistochemical assessment of hepatitis C virus antigen in cholestatic hepatitis after liver transplantation

Background: Patients with common variable immunodeficiency may exhibit rapidly progressive hepatitis when infected with hepatitis C virus (HCV), leading to cirrhosis and liver failure. Liver transplantation in these patients may result in a cholestatic form of HCV reinfection with exceptionally high virus loads. Aims: To report an immunohistochemical investigation of the pretransplant and post-transplant liver of one such patient. Methods/Results: On immunohistochemical staining of frozen sections with anti-HCV core monoclonal antibody or fluorescein labelled human polyclonal anti-HCV IgG, no HCV antigens were demonstrated in the native cirrhotic liver removed at transplant, despite a viral load of 106.4 genomes/g. The transplanted liver, collected six weeks post-transplant, exhibited cholestatic recurrent hepatitis, had an HCV virus load of 1010 genomes/g of liver, and revealed HCV antigen in the cytoplasm of most hepatocytes, with a pronounced periportal distribution. No virus antigen was demonstrable in other cell types. The core antigen was also detected in paraffin wax embedded, formaldehyde fixed tissue of this liver after high temperature antigen retrieval, but not in the native cirrhotic liver or a selection of HCV positive livers collected pretransplant from immunocompetent patients. Attempts to delineate the distribution of E1, NS3, and NS4 antigens were unsuccessful because monoclonal antibodies to these antigens produced “false positive” staining of foci of hepatocytes in the post-transplant livers of HCV seronegative patients with cholestasis. Conclusion: This case provided an opportunity to study the natural development of HCV during acute infection in the absence of an immune response, and may help to elucidate the pathogenesis of HCV recurrence in liver allografts.

Expression of mutant and wild-type TIMP3 in primary gingival fibroblasts from Sorsby's fundus dystrophy patients

Gingival fibroblast cell lines were derived from Sorsbys fundus dystrophy (SFD) patients carrying the S181C TIMP3 and the E139X TIMP3 mutations. These cell lines were grown in culture to study expression of the wild-type and mutant tissue inhibitor of metalloproteinase 3 (TIMP3) alleles from a normal diploid cell type. Firstly, patient cells were found to co-express the wild-type and mutant TIMP3 alleles, S181C TIMP3 or E139X TIMP3, at the mRNA level using restriction fragment length polymorphism (RFLP) analysis. A SpeI RFLP for E139X TIMP3 is described. Low levels of endogenous TIMP3 protein expression were elevated using the natural polysaccharide calcium pentosan polysulfate (CaPPs) in combination with the cytokine IL-1alpha. Immunoblotting detected protein expression from both wild-type and mutant alleles, S181C TIMP3 or E139X TIMP3. S181C TIMP3 from these cells was found to dimerise and retain MMP2 inhibitory activity. To facilitate studies of the E139X TIMP3 protein, the allele was expressed using HighFive insect cells. In this cell type, the E139X TIMP3 was synthesised as a mixture of monomer and dimer. Both monomeric and dimeric E139X TIMP3 protein retained MMP2 inhibitory activity in gelatin zymography. Expression of mutant E139X or S181C TIMP3 protein from a normal diploid patient-derived fibroblast cell had no effect on either MMP2 or MMP9 expression or activation whilst transcribed from their normal promoter context.

Comparison of human respiratory syncytial virus A2 and 8/60 fusion glycoprotein gene sequences and mapping of sub-group specific antibody epitopes.

The fusion glycoprotein, F, of human respiratory syncytial virus is a principal target of neutralising antibodies and an important protective immunogen. Among sub‐group A strains of the virus the F gene is highly conserved. A comparison of F gene sequences of two sub‐group B strains, 8/60 and 18537, indicates that the gene also is conserved within this sub‐group. However, both limited sequence variability and antigenic variation occurs between F genes from different virus sub‐groups. Such variability may be important in the failure of natural‐ and vaccine‐induced immunity and it is thus important to identify the variable epitopes. Three anti‐F MAbs exhibiting sub‐group specific neutralisation and binding to recombinant F glycoprotein were studied. Comparison of A2 and 8/60 F gene sequences revealed 64 predicted varient amino acids. In order to map the variant amino acids responsible for sub‐group specific binding, three sets of chimaeric genes, in which different domains of A2 and 8/60 F were exchanged, were created and expressed. Sub‐group specificity mapped to the N‐terminal region of F1 for two MAbs (RS2B8 and RS348) and to the C‐terminal region for the third. By using site‐directed mutagenesis, sub‐group specific binding of MAbs RS2B8 and RS348 was attributed to a predicted loop region between residues 200 and 216. This loop carried four residues variant between the sub‐groups. Change of at least two was necessary to abrogate MAb binding. J. Med. Virol. 63:168–177, 2001.