Biomedical subjects
Publications and source records attributed to P N Nelson.
From dengue to herpes and influenza to AIDS, the phytoestrogens that are present in many fruits and vegetables have been shown to exert anti-viral properties. Here we review the various different anti-viral mechanisms employed by phytoestrogens.
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OBJECTIVE: To examine whether human endogenous retrovirus K10 is associated with autoimmune rheumatic disease. DESIGN: A novel multiplex reverse transcription polymerase chain reaction (RT-PCR) system was developed to investigate HERV-K10 mRNA expression in patients with rheumatoid arthritis. METHODS: 40 patients with rheumatoid arthritis, 17 with osteoarthritis, and 27 healthy individuals were recruited and total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and analysed using multiplex RT-PCR for the level of HERV-K10 gag mRNA expression. Southern blot and DNA sequencing confirmed the authenticity of the PCR products. RESULTS: Using the histidyl tRNA synthetase (HtRNAS) gene as a housekeeping gene in the optimised multiplex RT-PCR, a significantly higher level of HERV-K10 gag mRNA expression was found in rheumatoid arthritis than in osteoarthritis (p = 0.01) or in the healthy controls (p = 0.02). CONCLUSION: There is enhanced mRNA expression of the HERV-K10 gag region in rheumatoid arthritis compared with osteoarthritis or healthy controls. This could contribute to the pathogenesis of rheumatoid arthritis.
The characterization of monoclonal antibodies (MAbs) with regard to reactivity and specificity is important for the successful application as a molecular probe and/or diagnostic reagent. Furthermore, it is recognized that some monoclonal reagents perform well in some assay systems but not others. In this study, the reactivity profiles of two anti-myosin MAbs (H1 and DH2, raised against human cardiac myosin) were evaluated in enzyme-linked immunosorbent assay (ELISA), slot-blotting, and immunocytochemistry. Both antibodies performed well in slot-blotting against myosin heavy chain preparations from cardiac and skeletal muscle and from non-human sources. In general, MAb H1 demonstrated strong to moderate reactivity in all assay systems, whilst MAb DH2 faired poorly in ELISA. MAb H1 also showed reactivity to synthetic peptides of myosin, one of which possessed a motif (ERRDA, single amino acid code) that was found in other human and nonhuman myosin protein sequences that could explain its cross-reactive profile. Intriguingly, this motif was found on viral and other pathogenic agents associated with myocarditis. Hence, it is speculated that this region could give some credence to the mechanism of molecular mimicry associated with some cardiac diseases. Overall, MAb H1 may serve as a useful probe of myosin structure.
Human endogenous retrovirus HERV-K like-sequences have been implicated in certain cancers. We developed a novel multiplex RT-PCR system for HERV-K that yielded a 533 bp product together with a smaller sized product (319 bp) of the house keeping gene, histidyl tRNA synthetase (HtRNAS). The latter spanned an intron that also served to validate target cDNA. PCR amplicons of HERV-K and HtRNAS were visualised using a gel documentation system and the pixel intensity used to derive semi-quantitative levels of viral expression. Our data showed that HERV-K10 was significantly elevated in MCF-7 cells treated with estrogen. Interestingly, HERV-K expression was higher in MCF-7 cells selected with adriamycin. RT-PCR combined with Southern blotting also detected HERV-K from breast cancer tissue using laser capture microscopy. This study highlights the presence of HERV-K in the breast cancer cell lines MCF-7 and MCF-7 ADR and confirms HERV-K10 transcripts in the cell line T47D. We believe this study to be a novel approach in determining levels of HERV-K expression and for detecting this virus in cancer cell lines and tissues.
Human endogenous retroviruses (HERVs) are a significant component of a wider family of retroelements that constitute part of the human genome. These viruses, perhaps representative of previous exogenous retroviral infection, have been integrated and passed through successive generations within the germ line. The retention of HERVs and isolated elements, such as long-terminal repeats, could have the potential to harm. In this review we describe HERVs within the context of the family of known transposable elements and survey these viruses in terms of superantigens and molecular mimics. It is entirely possible that these mechanisms provide the potential for undesired immune responses.
Recombinant antibodies are important tools for biomedical research and are increasingly being used as clinical diagnostic/therapeutic reagents. In this article, a background to humanized antibodies is given, together with details of the generation of antibody fragments--for example, single chain Fv fragments. Phage antibody fragments are fast becoming popular and can be generated by simple established methods of affinity enrichment from libraries derived from immune cells. Phage display methodology can also be used for the affinity enrichment of existing antibody fragments to provide a reagent with a higher affinity. Here, phage antibodies are demystified to provide a greater understanding of the potential of these reagents and to engage clinicians and biomedical scientists alike to think about potential applications in pathology and clinical settings.
Human endogenous retroviruses (HERVs) are a family of viruses within our genome with similarities to present day exogenous retroviruses. HERVs have been inherited by successive generations and it is possible that some have conferred biological benefits. However, several HERVs have been implicated in certain cancers and autoimmune diseases. This article demystifies these retroviruses by providing an insight into HERVs, their means of classification, and a synopsis of HERVs implicated in cancer and autoimmunity. Furthermore, the biological roles of HERVs are explored.
We have recently described the novel autoantigen plasminogen activator inhibitor (PAI-1) in systemic lupus erythematosus (SLE). The aim of this study was to determine the prevalence and clinical significance of anti-PAI-1 autoantibodies in patients with SLE. Autoantibodies to recombinant PAI-1 were measured in retrospective sera of 48 lupus patients by immunoassay in order to assess their clinical significance. This showed that 71% of sera from 48 lupus patients had significantly elevated anti-PAI-1 autoantibodies as compared with normal control subjects (P < 0.0001). There was a weak but significant (P < 0.043) correlation with anti-dsDNA autoantibodies. In longitudinal studies, autoantibodies against PAI-1 correlated with clinical parameters measured by the BILAG disease activity index including global clinical score. Our study demonstrates the high frequency of novel autoantibodies to PAI-1 in patients with lupus. The serial clinical correlations with anti-PAI-1 autoantibodies also support the hypothesis that these autoantibodies may play a pathogenic role in lupus.
The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.
Acid phosphatases (APs) are a family of enzymes that are widespread in nature, and can be found in many animal and plant species. Mystery surrounds the precise functional role of these molecular facilitators, despite much research. Yet, paradoxically, human APs have had considerable impact as tools of clinical investigation and intervention. One particular example is tartrate resistant acid phosphatase, which is detected in the serum in raised amounts accompanying pathological bone resorption. This article seeks to explore the identity and diversity of APs, and to demonstrate the relation between APs, human disease, and clinical diagnosis.
The generation of monoclonal antibodies (MAbs) specific for quail neural crest may provide valuable tools for studying the differentiation of embryonic precursor cells. Unfortunately, relatively few antibodies are available because of the difficulty in obtaining sufficient cells for in vivo immunization strategies. We have overcome this problem by using intrasplenic immunization with formaldehyde-fixed cells harvested from neural crest cultures. In addition, booster injections of cultured whole-embryo cells were administered intraperitoneally. Following two fusions, a total of 18 hybridomas were generated with antibody reactivity to the cytoplasm of neural crest cells. Furthermore, 32 were reactive against both somite (a noncrest mesodermal control) and crest cultures, whilst 15 were not reactive. Out of those hybridomas reactive with neural crest, six designated 160D, 164D, OE, 12E, 120E and 124E were further characterized. Interestingly MAb supernatants OE, 12E, 120E, and 124E exhibited reactivity against some but not all neural crest cells suggesting that they might recognise subpopulations. Immunoglobulin isotyping of supernatants revealed that 4 (160D, 164D, OE, and 120E) were IgM and 2 (12E and 124E) were IgG(2b). On assessing their reactivity against human tissue sections, all six hybridoma supernatants cross-reacted with neuroendocrine cells within appendix, colon and rectum. These MAbs could provide novel reagents for the understanding of neural crest development.
Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping and molecular modelling, monoclonal antibodies enable the antigenic profiling and visualisation of macromolecular surfaces. In addition, monoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents. Furthermore, the continuous culture of hybridoma cells that produce these antibodies offers the potential of an unlimited supply of reagent. In essence, when compared with the rather limited supply of polyclonal antibody reagents, the feature of a continuous supply enables the standardisation of both the reagent and the assay technique. Clearly, polyclonal and monoclonal antibodies have their advantages and disadvantages in terms of generation, cost, and overall applications. Ultimately, monoclonal antibodies are only produced when necessary because their production is time consuming and frustrating, although greatly rewarding (at least most of the time!). This is especially apparent when a monoclonal antibody can be applied successfully in a routine pathology laboratory or can aid in the clinical diagnosis and treatment of patients. In this article, the generation and application of monoclonal antibodies are demystified to enable greater understanding and hopefully formulate novel ideas for clinicians and scientists alike.
BACKGROUND: In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix. AIM: To test whether the expression of MMP-9 in Hodgkin's disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin's disease cell line, L428. METHODS: MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin's disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkin's disease cell line, L428. RESULTS: MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkin's disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkin's disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin's disease cell line. Induction of LMP-1 expression in the Hodgkin's disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography. CONCLUSIONS: These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkin's disease tumours and by the Hodgkin's disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.
The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.
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We have analyzed paraffin sections from 190 patients with histologically confirmed Hodgkin's disease (HD) for the presence of Epstein-Barr virus (EBV) using in situ hybridization to detect the EBV-encoded Epstein-Barr virus early RNAs (EBERs) and immunohistochemistry to identify latent membrane protein-1 (LMP1) expression. EBV was present in the tumor cells in 51 HD cases (27%) and was mainly confined to the mixed cellularity and nodular sclerosis subtypes. There was no difference between EBV-positive and EBV-negative HD patients with regard to age, clinical stage, presentation, and the number of alternating chemotherapy cycles of ChIVPP and PABIOE received. The complete remission rate after study chemotherapy was 80% in EBV-positive patients versus 69% in EBV-negative patients (P =.05). The 2-year failure-free survival rate was significantly better for EBV-positive patients when compared with the EBV-negative HD group (P =.02). Although 2-year and 5-year overall survival rates were better for EBV-positive HD patients, the differences were not statistically significant (P =.18 and P =.40, respectively). In conclusion, the results confirm the favorable prognostic value of EBV in the tumor cells of HD patients and suggest important differences in response to chemotherapy between EBV-positive and EBV-negative patients.
Since its initial description over twenty years ago the PCR has become one of the most valuable and flexible tools available to biomedical research. Subsequently, refinements and modifications to the basic approach, many of which have been described in this review, have enabled the application of the PCR to many areas of diagnostic medicine and have ensured its rapid acceptance as a routine test in many pathology disciplines. The growing importance of molecular approaches to the diagnosis of disease, particularly in histopathology, will continue to secure an ever expanding role for the PCR in diagnostic pathology.