Interleukin-7 (IL-7) and IL-7 splice variants affect differentiation of human neural progenitor cells.

Alternative splicing of pre-mRNA increases proteomic diversity, a crucial mechanism in defining tissue identity. We demonstrate differentially spliced interleukin (IL)-7 in distinct anatomic areas in the adult, in developing human brains and in normal human neuronal progenitor (NHNP) cells. IL-7c (c, the canonical form spanning all six exons) or its variants IL-7 delta 5, delta 4 or delta 4/5 were cloned and expressed as recombinant proteins. IL-7 and splice variants were able to shift the differentiation of NHNP cells as compared with the diluent control (P<0.01) defined by anti-beta (III)-tubulin and glial fibrillary acidic protein expression, with different degrees (IL-7c>delta 4/5>IL-7 delta 5); IL-7 delta 4 exhibited a significantly weaker potency. Differentiation was confirmed by transcriptome analysis of IL-7c-stimulated neural NHNP cells, resulting in 58 differentially expressed genes; some of these are involved in neural differentiation, for example, the developmentally regulated transcription factor krüppel-like factor 12, musashi 2, a translational regulator of cell fate or the sonic hedgehog receptor patch 1. This suggests that IL-7 influences neural development at a molecular level by participating in human brain architecture through glia cell formation: a paradigm that alternative splicing in cytokines, for example, for IL-7, has a physiological role in human organ development and progenitor cell differentiation.

Expression analysis and functional activity of interleukin-7 splice variants.

Alternative splicing results in multiple protein isoforms derived from a single gene. The magnitude of this process ranges from a complete loss of function to gain of new function. We examined, as a paradigm, alternative splicing of the non-redundant human cytokine, interleukin-7 (IL-7). We show that extensive IL-7 splicing in human tissues of different histology, including MTB+ granuloma lesions, transformed tissue and tumor cell lines. IL-7 splice variants were expressed as recombinant proteins. A differentially spliced IL-7 isoform, lacking exon 5, leads to STAT-5 phosphorylation in CD4+ and CD8+ T cells, promotes thymocyte maturation and T-cell survival. Human tumor lesions show aberrant IL-7 isoform expression, as compared with the autologous, non-transformed tissue. Alternatively spliced cytokines, such as IL-7, represent candidates for diagnostics and therapeutic interventions.

Isolation and characterization of an immunogenic fragment of heat shock protein 60 from Trichophyton mentagrophytes.

Heat shock protein 60 (hsp60) were isolated from several fungal, protozoal and many bacterial pathogens and successfully used for protective vaccination in some infection models. This work focuses on the isolation of recombinant hsp60 from the dermatophyte, Trichophyton mentagrophytes as a potentially protective antigen in trichophytosis. With the help of a previously tested set of degenerated primers, it was used reverse transcriptase polymerase chain reaction (RT-PCR) for isolation of partial cDNA of the hsp60 T. mentagrophytes (labelled hsp60-TM814), which was cloned into cloning vector. The sequencing of hsp60-TM814 cDNA and global alignment confirmed homology of the hsp60-TM814 with other members of the hsp60 family. Hsp60-TM814 cDNA corresponds to the region encoding the immunoprotective fragment of the hsp60 from Histoplasma capsulatum, used successfully in mouse model of histoplasmosis. A recombinant fragment (r-hsp60-TM664), 220 amino acids in length, was prepared in a prokaryote expression system, and its identity confirmed by mass spectroscopy. High immunogenicity of r-hsp60-TM664 was proven after subcutaneous immunization of mice. Immunized mouse sera recognized r-hsp60-TM664 on Western blots as well as hsp60 from mouse liver lysate and lysate of Candida albicans.