Genetic Variation/Evolution and Differential Host Responses Resulting from In-Patient Adaptation of Mycobacterium avium.

Members of the Mycobacterium avium complex (MAC) are characterized as nontuberculosis mycobacteria and are pathogenic mainly in immunocompromised individuals. MAC strains show a wide genetic variability, and there is growing evidence suggesting that genetic differences may contribute to a varied immune response that may impact the infection outcome. The current study aimed to characterize the genomic changes within M.avium isolates collected from single patients over time and test the host immune responses to these clinical isolates. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on 40 MAC isolates isolated from 15 patients at the Department of Medical Microbiology at St. Olavs Hospital in Trondheim, Norway. Isolates from patients (patients 4, 9, and 13) for whom more than two isolates were available were selected for further analysis. These isolates exhibited extensive sequence variation in the form of single-nucleotide polymorphisms (SNPs), suggesting that M. avium accumulates mutations at higher rates during persistent infections than other mycobacteria. Infection of murine macrophages and mice with sequential isolates from patients showed a tendency toward increased persistence and the downregulation of inflammatory cytokines by host-adapted M. avium strains. The study revealed the rapid genetic evolution of M. avium in chronically infected patients, accompanied by changes in the virulence properties of the sequential mycobacterial isolates.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT-PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cysteine-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.