Cloning and expression of mycobacterial genes for diagnosis and therapy.

The first part of the thesis describes the characterization of 19 kDa antigen from M. bovis BCG. The gene was expressed in E. coli and was detected using the monoclonal antibody CMA134.1. It was shown to be inducible in E. coli by performing Western blot analysis of induced and non-induced lysogens. The complete nucleotide sequence of the gene was determined and shown to be identical to that of 19 kDa gene from M. tuberculosis. This gene has the potential to be used as a diagnostic tool for the early detection of bovine tuberculosis. The second part of the thesis describes an attempt to clone, characterize and sequence the two genes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS) from M. tuberculosis. Polymerase chain reaction technique was used to amplify the coding DNA sequences and the amplified DNA product of the reaction was used as the probe to screen the genomic library of M. tuberculosis. Putative genes for dihydrofolate reductase and thymidylate synthase were isolated from the genomic library. Both of these putative genes failed to complement the E. coli deficient strains of DHFR and TS genes. The complete nucleotide sequence of both these genes was determined. CLUSTAL analysis of both sequences showed only low homology with published Dihydrofolate reductase and Thymidylate synthase sequences

High concordance in grading reticulin fibrosis and cellularity in patients with myeloproliferative neoplasms

The myeloproliferative neoplasms primary myelofibrosis, polycythemia vera, and, rarely, essential thrombocythemia are characterized by variable degrees of bone marrow fibrosis, either at presentation or upon progression. The increasing use of emerging therapies that may alter disease biology and morphology demands accurate and reproducible assessment of fibrosis grade. To assess concordance of hematopoietic cellularity and fibrosis grading, three hematopathologists independently evaluated a total of 728 bone marrow biopsies from 261 patients with myeloproliferative neoplasms on three clinical trials using fedratinib (SAR302503), a JAK2 inhibitor, including 249 taken at baseline and 479 on therapy. Concordance between the pathologists was evaluated by Pearson correlation coefficient (cellularity) and unweighted kappa statistic (fibrosis grade). There was high correlation of cellularity assessment (r = 0.92) and fibrosis grading (kappa = 0.83) between the three pathologists. Concordance with World Health Organization (WHO) grade 3 samples was higher compared with grades 0, 1, and 2. Concordance of fibrosis grading in pretreatment samples was superior to that of post-treatment samples (kappa = 0.83 and 0.79, respectively, P = 0.023). Our analysis suggests that the updated 2008 WHO reticulin fibrosis grading system is highly reproducible, even in patients undergoing JAK2 inhibitor therapy. This system is practically applicable to establish baseline fibrosis grade as well as changes in fibrosis in subsequent samples on therapy

Safety and Efficacy of Fedratinib in Patients With Primary or Secondary Myelofibrosis: A Randomized Clinical Trial

Myelofibrosis (MF) is a BCR-ABL-negative myeloproliferative neoplasm characterized by anemia, splenomegaly, debilitating constitutional symptoms, and shortened survival. Fedratinib, a JAK2-selective inhibitor, previously demonstrated clinically beneficial activity in patients with MF in early-phase trials