Identification of Mycobacterium tuberculosis vaccine candidates using human CD4+ T-cells expression cloning.

To identify Mycobacterium tuberculosis (Mtb) antigens as candidates for a subunit vaccine against tuberculosis (TB), we have employed a CD4+ T-cell expression screening method. Mtb-specific CD4+ T-cell lines from nine healthy PPD positive donors were stimulated with different antigenic substrates including autologous dendritic cells (DC) infected with Mtb, or cultured with culture filtrate proteins (CFP), and purified protein derivative of Mtb (PPD). These lines were used to screen a genomic Mtb library expressed in Escherichia coli and processed and presented by autologous DC.

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein.

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32(C)-Mtb39-Mtb32(N). Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-gamma responses directed against the first two components of the polyprotein and a strong CD8(+) T cell response directed exclusively against Mtb32(C).

P704P, P712P, and P775P: A genomic cluster of prostate-specific genes.

Background: Discovery of prostate cancer- and tissue-specific genes will lead to an increased understanding of the molecular events associated with the malignant transformation and tumorigenesis of prostate cells. Such understanding will likely result in the development of promising new markers for screening, diagnosis, and prognosis, as well as potential therapeutic approaches for combating this disease.

Methods: A PCR-based subtraction method was combined with a high-throughput microarray screening approach to identify prostate tissue- and/or cancer-specific genes.

Expression cloning.

Expression cloning involves the selection of specific polypeptides, generated from a cDNA or genomic DNA library, based on certain characteristics of the expressed proteins, such as antibody or ligand binding, recognition by T-cells, function, or complementation of cell defects. Here we describe the detailed construction of a genomic, random shear lambda expression library, adsorption of anti Escherichia coli antibody from antiserum, the screening of an expression library with specific antisera, and the cloning of genes with potential use in the diagnosis of infectious disease. This approach has been used successfully by our laboratory for the discovery of antigenic components of diagnostics and vaccines for several infectious agents including: Mycobacterium tuberculosis, Anaplasma phagocytophila (formerly Ehrlichia spp.

Serum antibodies to lipophilin B detected in late stage breast cancer patients.

Lipophilin B mRNA is overexpressed in approximately 70% of breast tumors and shows a high degree of correlation with the mRNA expression profile of mammaglobin. This is further supported by the recent finding that, like other members of the secretoglobulin-uteroglobin family, mammaglobin and lipophilin B form a heteroduplex. The studies described show that there are pre-existing antibodies to lipophilin B peptide in the sera of breast cancer patients with different stages and grade of tumor and that this response is different from that seen to recombinant mammaglobin and native mammaglobin-lipophilin B complex.

Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera.

The ability to identify prostate tumor or prostate tissue specific genes that are expressed at high levels and use their protein products as targets could greatly aid in the diagnosis and treatment of prostate cancer. Using a polymerase chain reaction (PCR)-based subtraction technique, we have recovered the recently described KLK4 (prostase) gene from human prostate cDNA. In this study, KLK4 gene expression in human prostate tumors was further characterized using cDNA quantitative PCR and immunohistochemistry, demonstrating that the gene is specifically expressed at both the mRNA and protein levels in normal human prostate tissue, and in both primary and metastatic prostate tumor samples.

Application of a multigene reverse transcription-PCR assay for detection of mammaglobin and complementary transcribed genes in breast cancer lymph nodes.

Background: Mammaglobin mRNA expression is found in 70-80% of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P, and gamma-aminobutyrate type A receptor pi subunit (GABApi) complement the expression of mammaglobin. Collectively the expression profile of these four genes could be used as a diagnostic and prognostic indicator for breast cancer.

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis.

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis.