Gue-Tae Chae

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)‐γ, interleukin (IL)‐10, IL‐12 and tumour necrosis factor (TNF)‐α 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‐γ, and TNF‐α 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.

Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae.

Summary To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae‐specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129‐bp fragment of the RLEP repetitive sequence or a 360‐bp fragment of the 18‐kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18‐kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty‐one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18‐kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

DNA-PCR and RT-PCR for the 18-kDa gene of Mycobacterium leprae to assess the efficacy of multi-drug therapy for leprosy.

DNA-PCR and reverse transcription (RT)-PCR for the 18-kDa protein of Mycobacterium leprae were used to examine the efficacy of multi-drug therapy (MDT) in leprosy. MDT was administered for 0-24 months. Fourteen (63.6%) of 22 patients showed positive PCR results after treatment for 12 months and the positive results decreased to 30% after 24 months of MDT. These results did not correlate with the bacterial index (BI) or the IgM antibody titre for the phenolic glycolipid (PGL)-1. One-dimensional densitometric analysis of agarose gels from PCR from the longitudinal study showed a gradual reduction of the 360-bp band after 12-24 months of MDT. RT-PCR for mRNA of the 18-kDa protein successfully tracked bacterial RNA changes in the biopsies and confirmed a decrease in the RNA of M. leprae in patients after MDT for 12 months. Thus, DNA- and RT-PCR for the 18-kDa protein of M. leprae are effective in assessing the efficacy of MDT for leprosy.

Vitamin D Receptor Gene TaqI, BsmI and FokI Polymorphisms in Korean Patients with Tuberculosis

Background The active metabolite (1, 25-dihydroxycholecalciferol) of vitamin D (25-hydroxycholecalciferol) leads to activation of macrophages and deficiency of vitamin D seems to be involved in the risk of tuberculosis. The effects of vitamin D are exerted by interaction with the vitamin D receptor (VDR) and may be influenced by polymorphism in the VDR gene. In this study, variation in the VDR gene was investigated in Korean population with tuberculosis. Methods We typed three VDR polymorphisms of restriction endonuclease sites for TaqI, BsmI and FokI in 155 patients with tuberculosis and 105 healthy volunteers. Results The frequencies of FokI genotypes determined from TB patients were 29.13% for FF, 56.31% for Ff, and 14.56% for ff. We observed 1.4-fold increased prevalence of the Ff genotype in TB patients compared with normal healthy groups (p=0.0857). However, there was no significant association between the genotype groups, TB patient and normal control, for FokI polymorphism. There was also no significant association between VDR gene and tuberculosis in another polymorphism (BsmI and TaqI). Conclusion Three polymorphisms (TaqI, BsmI and FokI) in the VDR gene do not appear to be responsible for host susceptibility to human tuberculosis in 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.

The Role of Intracellular Receptor NODs for Cytokine Production by Macrophages Infected with Mycobacterium leprae

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1β and TNF-α was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-κB activation and cytokine expression. Treatment with M. leprae significantly increased NF-κB activation and expression of TNF-α and IL-1β in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.

Identification of catalase-like activity from Mycobacterium leprae and the relationship between catalase and isonicotinic acid hydrazide (INH).

As Mycobacterium leprae proliferate inside macrophages, it has been speculated that catalase encoded by katG may protect the bacilli from deleterious effects of peroxide generated from the macrophage and may also play a crucial role in the survival of M. leprae in vivo. However, unlike that of M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implicating that isoniazid, which is activated to a potent tuberculocidal agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. These results raise a question as to how M. leprae avoids H202-mediated killing inside macrophages. To understand the survival of M. leprae in macrophages, the present study attempted to detect catalase-like activity in M. leprae. Catalase-like activity was found in M. leprae cell lysate by the diaminobenzidine (DAB) staining method with non-denaturing polyacrylamide gel electrophoresis. An ammonium sulphate precipitation study revealed that the catalase-like activity was precipitable with 80% ammonium sulphate. The effect of isoniazid (INH) on M. leprae growth was also tested by RT-PCR and radiorespirometric assay to examine catalase-like activity in M. leprae, because INH was activated by catalase. It was found that the viability of M. leprae was decreased at a concentration of 20 microg/ml by radiorespirometric assay and it was inhibited at higher concentrations as determined by RT-PCR. These data suggest that a catalase-like activity other than that encoded by katG is present in M. leprae.

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.

The adipogenic effect of palmitate in mouse bone marrow-derived mesenchymal stem cells

In the present study, we examined the effect of palmitate on adipocyte differentiation in mouse bone marrow-derived mesenchymal stem cells (MSCs). Obesity is associated with increased adipocyte differentiation of preadipocytes or MSCs. Free fatty acids (FFAs) have been reported to have an influence on adipogenesis. Thus, the elevated level of FFAs in obese individuals may set off a vicious cycle of events leading to further increased adipogenesis. Palmitate is one of the most abundant FFAs in plasma. The results in this study showed that treatment with palmitate for 3 days, followed by maintenance with insulin for 4 days (palmitate 3d/insulin 4d), induced lipid accumulation and adipocyte protein 2 (aP2) expression in bone marrow-derived MSCs. Our results also showed that treatment with palmitate 3d/insulin 4d induced expressions of adipogenic transcription factors, namely CCAAT enhancer-binding protein (C/EBP) β, C/EBPα, and PPARΓ in bone marrow-derived MSCs. In comparison of palmitate with stearate, the levels of lipid accumulation, aP2, and adipogenic transcription factors in cells treated with palmitate 3d/insulin 4d were generally higher than those of cells treated with same concentration of stearate 3d/insulin 4d. Taken together, our results suggest that palmitate is a potent adipogenic FFA that triggers adipocyte differentiation of bone marrow-derived MSCs.

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice.

RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2) expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-gamma genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.