A rhodanine agent active against non-replicating intracellular Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Antibiotic therapy targeting chronic mycobacterial disease is often ineffective due to problems with the emergence of drug resistance and non-replicating persistent intracellular antibiotic resistant phenotypes. Strategies which include agents able to enhance host cell killing mechanisms could represent an alternative to conventional methods with the potential for host clearance if active against dormant phenotypes. Investigations of agents with potential activity against non-replicating mycobacteria however are restricted due to a need for assays that can assess bacterial viability without having to culture. RESULTS: This study describes the development and use of a pre16S ribosomal gene RNA/DNA ratio viability assay which is independent of the need for culture, supported by a novel thin layer accelerated mycobacterial colony forming method for determining viability and culturability of MAP in intracellular environments. We describe the use of these tools to demonstrate intracellular killing activity of a novel rhodanine agent (D157070) against the intracellular pathogen Mycobacterium avium subspecies paratuberculosis (MAP) and show that the culturability of MAP decreases relative to its viability on intracellular entry suggesting the induction of a non-culturable phenotype. We further demonstrate that D157070, although having no direct activity against the culturability of extracellular MAP, can bind to cultured MAP cells and has significant influence on the MAP transcriptome, particularly with respect of delta(L )associated genes. D157070 is shown to be taken up by bovine and human cells and able to enhance host cell killing, as measured by significant decreases in both culturability and viability of intracellular MAP. CONCLUSIONS: This work suggests that pre16srRNA gene ratios represent a viable method for studying MAP viability. In addition, the rhodanine agent D157070 tested is non-toxic and enhances cell killing activity against both growing and latent MAP phenotypes

Suppression of anchorage-independent growth after gene transfection.

A novel procedure for isolating anchorage-dependent cells has been developed. It involves negative selection of cells growing in suspension followed by clonal replica screening for anchorage-dependent growth. Cells which have regained anchorage-dependent growth have been isolated from a library of the Chinese hamster ovary cell line, CHO-K1, transfected with pSV2neo and human genomic DNA. One anchorage-dependent clone, 1042AC, has been studied in detail. Anchorage-dependent growth of 1042AC is stable when cultured as adherent monolayers, but revertants appear rapidly when cultured in suspension. Suppression is unlikely to be due to loss or mutation of hamster genes conferring anchorage-independent growth as hybrids between 1042AC and CHO-K1 have the suppressed phenotype of 1042AC. Furthermore, a population of cells obtained from the hybrid by selecting for revertants to anchorage-independent growth showed selective loss of the transgenome derived from 1042AC. The growth suppression was not due to transfection of the human Krev-1 gene, which has previously been shown to restore anchorage-dependent growth, nor was there any evidence of alteration in the endogenous hamster Krev-1 gene. However, evidence for a human gene being responsible for the suppressed phenotype has not been obtained yet

Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes