An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with ¹²⁵I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.     

Collagen-induced arthritis in TNF receptor-1-deficient mice: TNF receptor-2 can modulate arthritis in the absence of TNF receptor-1

TNF is a potent proinflammatory cytokine important for the development of arthritis in human and animals. We have investigated the roles of TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in collagen-induced arthritis (CIA) by inducing CIA in mice genetically deficient in TNFR1. TNFR1-/- mice developed arthritis with similar incidence and severity as TNFR1+/- littermates, indicating that TNFR1 is redundant for the development of CIA. Anti-type II collagen (CII) antibody levels and T cell responses to CII did not differ between TNFR1-/- mice and controls. Neutralization of TNF with soluble TNF binding protein suppressed the development of arthritis in TNFR1+/- mice but not in TNFR1-/- mice, indicating that TNFR2 cannot substitute for TNFR1 for the proinflammatory function. To further investigate the functions of TNFR2, TNFR1-/- mice were injected with murine TNF-alpha at different stages during the course of CIA. Repeated TNF-alpha injection during the early induction phase enhanced the development of arthritis, but inhibited arthritis when administered during the late progression phase. These results show that the engagement of TNFR2 by TNF is involved in the development of CIA in the absence of TNFR1 and that opposing signals can be transduced by TNFR2.     

Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method

A commercial line blot using recombinant antigens was compared with a commercial ELISA and 'in-house' IFA (reference test). Two panels were evaluated: Panel A was selected to distinguish between primary infections (89), past infections (20) and seronegatives (8) in immunocompetent individuals. In panel B, patients with a high number of reactivations were included: immunosuppressed patients (37), lymphoma (19), nasopharyngeal carcinoma (10), chronic fatigue syndrome (14). Blood donors (43) and cross-reactive sera (29) were added as controls. Line blot and IFA were concordant in 94% of primary infections, 100% of seronegatives and 100% of past infections, similar to ELISA. Results differed significantly with regard to reactivations. When compared with IFA, the incidence of reactivations was overestimated by the blot, 24 and 58% in blood donors and cross-reactive sera, respectively. ELISA showed a similar problems with 21 and 34% indeterminate results, respectively. The line blot is easy to carry out, has a good concordance with the reference IFA for primary infections, and is, therefore, a sufficient choice for distinguishing primary infection from seronegative and past infection. EBV reactivation assessment will require other methods such as EBV viral load.     

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8⁺ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.     

Research on cancer diagnosis in Malaysia: current status

Cancer is a major morbidity and mortality concern in Malaysia. Based on National Cancer Registry data, the Malaysian population is estimated to bear a cancer burden of about 40,000 new cases per year, and a cumulative lifetime risk of about 1:4. Cancer research in Malaysia has to consider needs relevant to our population, and resources constraints. Hence, funding bodies prioritise cancers of high prevalence, unique to our community and posing specific clinical problems. Cancer diagnosis is crucial to cancer management. While cancer diagnosis research largely aims at improvements in diagnostic information towards more appropriate therapy, it also impacts upon policy development and other areas of cancer management. The scope of cancer diagnosis upon which this paper is based, and their possible impact on other R&D areas, has been broadly categorized into: (1) identification of aetiological agents and their linkages to the development of precancer and cancer (impact on policy development, cancer prevention and treatment), (2) cancer biology and pathogenesis (impact on cancer prevention, treatment strategies and product development), (3) improvements in accuracy, sensitivity and specificity in cancer detection, monitoring and classification (impact on technology development) and (4) prognostic and predictive parameters (impact on treatment strategies). This paper is based on data collected by the Working Group on Cancer Diagnosis Research for the First National Conference on Cancer Research Coordination in April 2004. Data was collated from the databases of Institutions/Universities where the authors are employed, the Ministry of Science, Technology and Innovation (MOSTI) and targeted survey feedback from key cancer researchers. Under the 7th Malaysia Plan, 76 cancer projects were funded through the Intensified Research in Priority Areas (IRPA) scheme of MOSTI, amounting to almost RM15 million of grant money. 47(61.8%) of these projects were substantially in cancer diagnosis, accounting for 65.6% (RM 9.7 million) of cancer project funds. The 8th Malaysia Plan saw a change in research strategy. The IRPA agency fielded several top-down projects which encouraged a multicentre and multidisciplinary approach. This resulted in larger funding per project i.e. RM32 million for 49 projects. There was also a surge of interest in drug development and natural products. Because of this shift in direction, cancer diagnosis projects constituted only 51% of IRPA-funded cancer projects. Nonetheless funding for cancer diagnosis research has exceeded that of the 7th Malaysia Plan, being RM12.5 million by March 2004. The majority of such research is carried out at the Universities, engaging a large number of young scientists and postgraduate students (51 MSc and 21 PhD). A lot of research findings presented at scientific meetings have not yet been published and there is a glaring shortage of patents and commercialization of research findings (such as creation of test kits). Because diagnosis is very much a part of clinical practice, many researchers felt satisfied and confident that their work will be translated into practice and will significantly improve diagnostic services in Malaysia. National guidelines and consensus development on at least three malignancies i.e. breast cancer, oral cancer and lymphoma, have substantial basis in local R&D work. Problems encountered in research included (1) insufficient funding to realize research objectives, (2) lack of local expertise (most research assistants are inexperienced BSc graduates with no or minimal research experience), (3) inadequate technical support from vendors during equipment failure, (4) inexperienced Institutional development units to assist in product development, (5) lack of venture capital for commercialization of findings, and (6) inadequate incentives to undertake research. Researchers pointed out that plans to promote research should include the establishment of (1) regional and national cancer tissue banks, (2) a National Cancer Research Institute, (3) a dedicated cancer research fund, (4) a registry of cancer researchers, (5) national research coordinators, (6) improved coverage by the National Cancer Registry, (7) more international collaboration, (8) a better career structure for researchers, (9) improved Institutional support for product realization, and (10) better recognition for cancer researchers.