Identification of lymphoma-associated antigens using SEREX.

Serological analysis of recombinant complementary deoxyribonucleic acid (cDNA) expression libraries (SEREX) is a powerful approach to identify immunogenic cancer-associated proteins using antibodies that are naturally present in the serum of cancer patients. This technique involves the screening of a relevant cDNA expression library with patient serum that has been cleaned to remove any antibodies that may recognize bacterial and/or viral proteins. Once antigens have been identified and their reactivity has been confirmed with a second round of screening, the gene encoding the protein can be sequenced and identified. Antigens can then be screened with a panel of allogenic sera from other patients and control individuals. This identifies disease-specific antigens, which may be useful diagnostic markers or, alternatively, targets for immunotherapy. This chapter describes the SEREX methodology in full.

Serologic detection of diffuse large B-cell lymphoma-associated antigens.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of all adult non-Hodgkin's lymphomas, yet the understanding of its underlying genetic abnormalities remains poor. Our present study used the serological analysis of recombinant cDNA expression libraries (SEREX) technique to identify DLBCL-associated antigens. SEREX screening of testis libraries has previously identified cancer-testis antigens (CTAs) that may act as disease-specific targets for immunotherapy. Screening a testis cDNA expression library with serum from a DLBCL patient identified a total of 94 positive clones, representing 28 distinct antigens. Two of these antigens were novel, 8 were previously uncharacterised, and the remainder were proteins of known function. Screening of the antigens with sera from DLBCL (n = 10), acute myeloid leukaemia (AML, n = 10) and chronic myeloid leukaemia (CML, n = 10) patients, alongside normal healthy donor controls (n = 20), revealed that 7 of the antigens were recognised by DLBCL sera but not normal donor sera, whilst 2 of these antigens were also recognised by leukaemic sera. Some of the genes identified here were already known to be transcribed in DLBCL. The mRNA expression of the majority of the remaining antigens was confirmed in DLBCL cell lines using reverse-transcriptase PCR (RT-PCR). Our study identified a number of DLBCL associated antigens that may be suitable as prognostic/diagnostic markers and/or for the immunotherapy of haematologic malignancies.

Phenotypic and functional changes in glial cells as a function of age.

Both in vivo and in vitro investigations point to an important role for the immune system in the development of age-related neurodegeneration. Microglia isolated from aged female F344 rats, 18-20 months, show a higher percentage of cells with an ameboid morphology indicative of activation, whereas, astrocytes had a quiescent morphology. The ability of astrocytes and microglia to attenuate toxin-induced neuronal injury was examined. Post-natal day 1-3 pup cells optimally rescued neurons from Abeta-induced toxicity, whereas mixed glial cells from 18-20 month old rats were unable to rescue neurons from Abeta-induced toxicity. Our results suggested the appearance of a neurotoxic co-factor, therefore we investigated the basal level of nitric oxide and pro-inflammatory cytokines to determine if altered levels of immune mediators play a role in the toxicity. Mitogen-stimulated nitric oxide production increased 10 fold with age of donor, whereas, only the pup cells expressed an increase in TNF-alpha production. Basal levels of pro-inflammatory cytokines, as measured by RNA protection assays, increased with age. In particular, IL-1beta was increased 2 fold between adult and aged glial cells. The elevated cytokine expression may contribute to enhanced susceptibility to neurodegenerative diseases.