HOX gene analysis in the osteogenic differentiation of human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the capacity to differentiate into osteoblasts during osteogenesis. Several studies attempted to identify osteogenesis-related genes in hMSCs. Although HOX genes are known to play a pivotal role in skeletogenesis, their function in the osteogenesis of hMSCs has not yet been investigated in detail. Our aim was to characterize the expression of 37 HOX genes by multiplex RT-PCR to identify the ones most probably involved in osteogenic differentiation. The results showed that the expression patterns of four HOX genes were altered during this process. In particular, the expression levels of HOXC13 and HOXD13 were dramatically changed. Real-time PCR and Western blot analysis were performed in order to further analyze the expression of HOXC13 and HOXD13. The qRT-PCR results showed that transcription of HOXC13 was up-regulated by up to forty times, whereas that of HOXD13 was down-regulated by approximately five times after osteogenic differentiation. The Western blot results for the HOXC13 and HOXD13 proteins also corresponded well with the real-time PCR result. These findings suggest that HOXC13 and HOXD13 might be involved in the osteogenic differentiation of hMSCs.

Genotypic analysis of Mycobacterium leprae isolates from Japan and other Asian countries reveals a global transmission pattern of leprosy.

The genotype of single-nucleotide polymorphism type 3, CTC, at positions 14676, 164275, and 2935685, along with four copies of 6 bp repeats in the rpoT gene, was predominant for isolates originating in the Japanese mainland. Type 1, CGA, type 2, CTA, and type 3 were detected from Korea, Indonesia, and Myanmar. No isolates with four copies of 6 bp were detected from Myanmar, Okinawa, and Japanese Brazilian patients. Type 4, TTC, with three copies of 6 bp, was detected only from Japanese Brazilians. The results indicate that infection occurred in Brazil and the disease developed later in Japan.

Differential production of interleukin-10 and interleukin-12 in mononuclear cells from leprosy patients with a Toll-like receptor 2 mutation

Toll-like receptor 2 (TLR2) is a key mediator of the immune response to mycobacterial infections, and mutations in TLR2 have been shown to confer susceptibility to infection with mycobacteria. This study investigated the profiles of cytokines, such as interferon (IFN)-gamma, interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-alpha in response to Mycobacterium leprae in peripheral blood mononuclear cells (PBMC) with the TLR2 mutation Arg677Trp, a recently reported polymorphism that is associated with lepromatous leprosy. In leprosy patients with the TLR2 mutation, production of IL-2, IL-12, IFN-gamma, and TNF-alpha by M. leprae-stimulated PBMC were significantly decreased compared with that in groups with wild-type TLR2. However, the cells from patients with the TLR2 mutation showed significantly increased production of IL-10. There was no significant difference in IL-4 production between the mutant and wild-type during stimulation. Thus, these results suggest that the TLR2 signal pathway plays a critical role in the alteration of cytokine profiles in PBMC from leprosy patients and the TLR2 mutation Arg677Trp provides a mechanism for the poor cellular immune response associated with lepromatous leprosy.

Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea

Interleukin-12 receptor beta 1 ( IL12RB1), interleukin-12 receptor beta 2 ( IL12RB2), and interferon gamma receptor 1 ( IFNGR1) perform important roles in the host defense against intracellular pathogens such as Mycobacteria. Several mutations within their genes have been confirmed as associated with increased susceptibility to mycobacterial infection. However, the association between mutations of the IL12RB1, IL12RB2, and IFNGR1 encoding genes and lepromatous leprosy has not been studied. This study screened for polymorphisms within IL12RB1, IL12RB2, and IFNGR1 encoding genes in the Korean populations using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) DNA sequencing assay, and an association study was performed using the missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), and 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14M), and 1443 T/C (L467P) for the IFNGR1 encoding genes. There were no differences in the genotype and allele frequencies of IL12RB1 and IFNGR1 genes between 93 lepromatous leprosy patients and 94 control subjects. In conclusion, missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14 M), and 1443 T/C (L467P) of the IFNGR1 encoding genes have no association with the susceptibility to lepromatous leprosy in the Korean population.

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.

A polymorphism in the toll-like receptor 2 is associated with IL-12 production from monocyte in lepromatous leprosy

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections, and the mutations in the TLR2 have been shown to confer the susceptibility to infection with mycobacteria. We previously reported the detection of TLR2 Arg677Trp mutation in lepromatous leprosy. Here, the events triggered by TLR2 in response to cell lysate of Mycobacterium leprae(MLL), the causative agent of leprosy, were investigated. Upon stimulation with MLL, monocytes produced TNF-alpha and Interleukin-12 (IL-12), which play a role in the innate immune response to infection. Anti-TLR2 mAb blocked greater than 50 per cent of the MLL-induced production of IL-12. We also performed the functional study on TLR2 by measurement of IL-12 production in serum and monocytes from leprosy patients with TLR2 mutation (Arg677Trp). The monocytes obtained from patients with the TLR2 mutation, in comparison to the wild-type TLR2, is significantly less responsive to MLL. It was also confirmed that patients with TLR2 mutation showed significantly lower serum levels of IL-12, in comparing with TLR2 wild-type. Our results reveal that innate immune response of monocytes against M. lepraeis mediated by TLR2, and suggest that the mutation in the intracellular domain of TLR2 gene is associated with IL-12 production in lepromatous leprosy.

Mycobacterium leprae typing by genomic diversity and global distribution of genotypes

The genetic diversity and related global distribution of 51 Mycobacterium leprae isolates were studied. Isolates were obtained from leprosy patients from 12 geographically distinct regions of the world and two were obtained from nonhuman sources. Polymerase chain reaction (PCR) followed by DNA sequencing was performed targeting the rpoT gene of M. leprae. Isolates were classified into two groups based on the number of tandem repeats composed of 6 base pairs in the rpoT gene. Isolates from Japan (except Okinawa) and Korea belonged to one group, while those from Southeast Asian countries, Brazil, Haiti and Okinawa in Japan belonged to a second genotype. M. leprae obtained from two nonhuman sources (an armadillo and a mangabey monkey) revealed the latter genotype. These results demonstrate the genetic diversity of M. leprae and the related genotype-specific distribution in the world.