Modern History of Hansen's Disease in Korea.

Modern history of Hansen's Disease (HD) in Korea begins with nationwide use of the chemotherapeutic agent Diamino Diphenyl Sulphone for the patients in 1955. Definition of the case was different from time to time. Based on World Health Organization (WHO) criteria, Ministry of Health and Welfare (MOHW) reported 4,393 registered patients and same number 4,393 as new cases in 1977. This is the turning point they accepted patient reporting system of WHO, but total number of registered and managed as leprosy patients was 28,029 in 1977, which means the people who needs HD service from government at that time. The number of new cases decreased from 4,393 in 1977, 39 in 1996 to 4 in 2017. Regarding to new cases, it takes 40 years to accomplish from thousands level to below 10. Now we have 166 active cases (registered patients) and reported them as patients to the WHO. Korea Civil Assistance Command invited Dr. RG Cochrane who visited Korea for six weeks to make blue print for eradication of HD in Korea. With his advice and MOHW set HD project and plan for manpower to solve HD problems in 1955. Dr. Joon Lew and his colleagues founded Korean Leprosy Prevention Association in 1947 to combat leprosy, enlighten the public, and solve social problems caused by HD. The Korean Leprosy Prevention Association led by him changed its name to the Korean Leprosy Association in 1956, and grew into the current Korean Hansen Welfare Association. This organization is now playing a leading role in the eradication and management of HD in Korea.

Epigallocatechin-3-gallate attenuates the AIM2-induced secretion of IL-1ß in human epidermal keratinocytes.

The pro-inflammatory cytokine interleukin-1ß (IL-1ß) plays a central role in the pathogenesis of psoriasis. Keratinocytes are a major source of IL-1ß and express absent in melanoma 2 (AIM2). AIM2 recognizes a double-stranded DNA and initiates the IL-1ß-processing of inflammasome. The AIM2 inflammasome is a cytosolic multiprotein complex composed of AIM2, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Epigallocatechin-3-Gallate (EGCG), a major polyphenolic component of green tea, has anti-inflammatory properties. In the current study, we investigated the issue of whether or how EGCG suppresses AIM2 inflammasome in human epidermal keratinocytes, neonatal (HEKn). Treatment with EGCG, before or after IFN-γ priming, attenuated poly(dA:dT)-induced IL-1ß secretion in HEKn cells. Pre-treatment with EGCG reduced the level of IFN-γ-induced priming signal via the down-regulation of pro-IL-1ß and pro-capspase-1 in HEKn cells. Furthermore, treatment with EGCG attenuated poly(dA:dT)-induced ASC oligomerization and caspase-1 activation in IFN-γ-primed HEKn cells. These results suggest that EGCG attenuates AIM2-induced IL-1ß secretion by suppressing both IFN-γ-mediated priming and poly(dA:dT)-induced ASC oligomerization of inflammasomes in human epidermal keratinocytes.

Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

BACKGROUND: Endoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells. METHODS: RAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR. RESULTS: Palmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα. CONCLUSIONS: The findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

Expression of Nucleotide-oligomerization Domain (NOD) and Related Genes in Mouse Tissues Infected with Mycobacterium leprae.

The nucleotide-oligomerization domain (NOD) is an important molecule involved in host defense against bacterial infection. To study the role of NODs in the host response to Mycobacterium leprae, we measured the mRNA levels of NODs and related genes in infected mouse tissues. The mRNA expression of NOD1, NOD2, caspase-1 and ASC was increased in mouse footpads. Whereas NOD2 expression in macrophages was increased at 2 and 24 h post-infection with M. leprae, there was no expression of NOD1 at these time points. An increase in caspase-1 expression was observed at 2 h and continued at 24 h. However, the expression of ASC was increased only at the early time point. The expression of caspase-1 is regulated by NOD2-dependent pathway in established HEK 293. Our results suggest NOD2, rather than NOD1, may be associated with the host response to M. leprae and that caspase-1 activation is essential for the host response.

Palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB, p38, and ERK in human dermal fibroblasts.

It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function.

Quantification of MSCs involved in wound healing: use of SIS to transfer MSCs to wound site and quantification of MSCs involved in skin wound healing.

Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1 × 10(6) cells were seeded onto the centre of the SIS. MSC-seeded SIS complexes were injected onto full-thickness skin wounds made on the dorsum of nude mice. Tracing of MSC-seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 2-3 × 10(5) from day 1 to day 10. The expression of skin regeneration-related growth factors was confirmed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration-related growth factors that enable the recovery of the skin. An optimal number of about 3 × 10(5) MSCs injected into the site was found to adapt themselves to the skin wound-healing process effectively.

Palmitate induces RIP1-dependent necrosis in RAW 264.7 cells.

OBJECTIVE: The kinase receptor-interacting protein (RIP) 1, a serine/threonine protein kinase, is a key signaling molecule for necrosis. The possible involvement of RIP1 in palmitate-induced macrophage death and its underlying molecular mechanism was investigated in this study. METHODS: Cell viability was measured by an MTT reduction assay. The type of cell death was determined by staining with annexin V, propidium iodide (PI) and the APOPercentage dye, and by examining cell morphology using transmission electron microscopy. The down-regulation of RIP1 was performed by siRNA transfection. Intracellular reactive oxygen species (ROS) were measured by staining with H(2)DCF-DA. RESULTS: Palmitate largely induced necrosis in RAW 264.7 cells, whereas C2-ceramide induced apoptosis. Palmitate-induced necrosis was inhibited by Necrostatin-1, an inhibitor of RIP1, and by RIP1 siRNA transfection, whereas ordinary cell death was not inhibited by z-VAD-fmk. In addition, the presence of palmitate caused a significant increase in intracellular ROS levels compared to control cells. Pre-treatment with Tempol, a cell permeable ROS scavenger, and MnTBAP, an inhibitor of mitochondrial oxidative stress, protected cells from palmitate-induced cell death. Furthermore, the down-regulation of RIP1 by siRNA transfection significantly decreased palmitate-induced ROS generation compared to control cells. CONCLUSION: The findings reported herein indicate that palmitate induces necrotic cell death via RIP1-dependent ROS generation in RAW 264.7 cells. These findings may provide a new mechanism that explains the link between elevated levels of free fatty acids (FFAs) and macrophage death.

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.

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-1ß 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-1ß 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.

Hydrogen peroxide induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.

A novel mechanism for H2O2-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H2O2-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H2O2 treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H2O2-treated cells. Collectively, these findings demonstrate that H2O2 induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.

Comparison of two mice strains, A/J and C57BL/6, in caspase-1 activity and IL-1beta secretion of macrophage to Mycobacterium leprae infection.

A/J mice were found to have amino acid differences in Naip5, one of the NOD-like receptors (NLRs) involved in the cytosolic recognition of pathogen-associated molecular patterns and one of the adaptor proteins for caspase-1 activation. This defect was associated with a susceptibility to Legionella infection, suggesting an important role for Naip5 in the immune response also to other intracellular pathogens, such as Mycobacterium leprae. In this study, the immune responses of macrophages from A/J mice against M. leprae were compared to those of macrophages from C57BL/6 mice. Infection with M. leprae induced high levels of TNF-alpha production and NF-kappaB activation in A/J and C57BL/6 macrophages. Caspase-1 activation and IL-1beta secretion were also induced in both macrophages. However, macrophages from A/J mice exhibited reduced caspase-1 activation and IL-1beta secretion compared to C57BL/6 macrophages. These results suggest that NLR family proteins may have a role in the innate immune response to M. leprae.

Leprotic cervical spondylodiscitis.

Leprosy is a chronic infectious disease caused by the Mycobacterium leprae that leads to leprotic neuropathy involving the peripheral nerve and several characteristic skin lesions. Skeletal involvement can occur in peripheral joints, such as the wrist and the ankle. However, there is no report of an axial leprotic lesion involving the spine or paraspinal soft tissue. The authors report the first case of a leprotic cervical lesion involving the axial skeletal system. A 48-year-old male presented with neck pain and severe pain in the right suprascapular area and left arm. Preoperative MRI of the cervical spine revealed signal changes in the prevertebral soft tissue at the level of the C3, 4, 5 vertebral bodies. There were a lower signal intensity on T1-weighted image and high signal intensity on T2WI of the bone marrow at the level of the C5 and C6 vertebral bodies, and a C5/6 segmental ossification of the posterior longitudinal ligament. There were herniated cervical disc on the left C5/6 with C6 root and the right side of C6/7 with a C7 root compression. He was previously diagnosed with leprosy when he was 14 years old and received treatment intermittently over the course of 7 years. But patient did not disclose his past history. Surgical intervention was conducted using an anterior cervical approach. An incision was made in the anterior longitudinal ligament at C5/6, and a pinkish gray friable gelatinous material was observed on the C5/6 disc and on the anterior lower one-third surface of the C5 vertebral body. Specimens were obtained and subjected to pathological evaluation and microbiological culture. After C5/6 and C6/7 discectomies, nerve root decompression and autologous iliac bone grafting were performed at the C5/6 and C6/7 levels. The C5-6-7 vertebrae were fixed with an Atlantis cervical locking plate and a screw system. The pathological report indicated chronic inflammation with heavy plasma cell infiltration on the specimen. We sent the specimens to the Institute of Hansen's Disease, and polymerase chain reaction for leprosy tested positive. After surgery, his pain disappeared and he was given a prescription for antileprotic drugs. The authors describe the first case of leprotic cervical spondylodiscitis that was operatively treated in a 48-year-old patient with known leprosy history since his 14 years old.

Mycobacterial infections in coal workers' pneumoconiosis patients in South Korea.

Coal workers' pneumoconiosis (CWP) is the most common occupational disease in South Korea and is an important factor in the development of infections with Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM). In the current study, we identified mycobacterial species that cause pulmonary infections in CWP patients, using rpoB DNA-PCR-restriction analysis. Among the 129 CWP patients studied, 35 (27.1%) were diagnosed as having mycobacterial infections. Among these, the proportion of NTM infections (21/35, 60.0%) was higher than that for MTB infections (14/35, 40.0%). Of the 21 NTM strains, the most common was M. intracellulare (6/21, 28.6%), followed by M. avium (5/21, 23.8%). We also compared the characteristics of CWP patients between the MTB and NTM infection groups. A higher proportion of CWP patients with NTM infections compared with those with MTB infections had a history of having been involved in rock work (38.1% vs 21.4%), and had complicated CWP (85.7% vs 35.7%) and a past history of TB treatment (61.9% vs 50.0%). We also discovered 3 MTB mutants that are resistant to first-line anti-TB drugs, in CWP patients. These results demonstrate the features of pulmonary mycobacterial infections with a predominance of NTM in CWP patients in South Korea.

Hydrogen peroxide induces autophagic cell death in C6 glioma cells via BNIP3-mediated suppression of the mTOR pathway.

Oxidative stress by exposure to H(2)O(2) induces various types of cell death depending on cell type and conditions. We report herein on a study of the mechanisms underlying H(2)O(2)-induced cell death in C6 glioma cells. The findings show that H(2)O(2) triggers a caspase-independent autophagic cell death in these cells. The findings also show that H(2)O(2) induces the dephosphorylation of the mammalian target of rapamycin (mTOR) at Ser 2481 and the p70 ribosomal protein S6 kinase (p70S6K) at Thr389 in a Bcl-2/E1B 19kDa interacting protein 3 (BNIP3)-dependent manner. BNIP3 has the capacity to inhibit mTOR activity and mTOR inhibition plays a role in autophagic induction. This suggests that BNIP3 may mediate H(2)O(2)-induced autophagic cell death through the suppression of mTOR. The findings show that the down-regulation of BNIP3 by BNIP3 siRNA prevents C6 cells from undergoing H(2)O(2)-induced autophagic cell death. Collectively, these results suggest that H(2)O(2) induces autophagic cell death in C6 cells via the BNIP3-mediated suppression of the mTOR pathway.

Rifampicin Inhibits the LPS-induced Expression of Toll-like Receptor 2 via the Suppression of NF-kappaB DNA-binding Activity in RAW 264.7 Cells.

Rifampicin is a macrocyclic antibiotic which is used extensively for treatment against Mycobacterium tuberculosis and other mycobacterial infections. Recently, a number of studies have focused on the immune-regulatory effects of rifampicin. Therefore, we hypothesized that rifampicin may influence the TLR2 expression in LPS-activated RAW 264.7 cells. In this study, we determined that rifampicin suppresses LPS-induced TLR2 mRNA expression. The down-regulation of TLR2 expression coincided with decreased production of TNF-alpha. Since NF-kappaB is a major transcription factor that regulates genes for TLR2 and TNF-alpha, we examined the effect of rifampicin on the LPS-induced NF-kappaB activation. Rifampicin inhibited NF-kappaB DNA-binding activity in LPS-activated RAW 264.7 cells, while it did not affect IKKalpha/beta activity. However, rifampicin slightly inhibited the nuclear translocation of NF-kappaB p65. In addition, rifampicin increased physical interaction between pregnane X receptor, a receptor for rifampicin, and NF-kappaB p65, suggesting pregnane X receptor interferes with NF-kappaB binding to DNA. Taken together, our results demonstrate that rifampicin inhibits LPS-induced TLR2 expression, at least in part, via the suppression of NF-kappaB DNA-binding activity in RAW 264.7 cells. Thus, the present results suggest that the rifampicin-mediated inhibition of TLR2 via the suppression of NF-kappaB DNA-binding activity may be a novel mechanism of the immune-suppressive effects of rifampicin.

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.

Effect of rifampicin to inhibit rapamycin-induced autophagy via the suppression of protein phosphatase 2A activity.

Recently, a number of studies have focused on the secondary effects of rifampicin. In the present study, we assessed whether rifampicin influences the rapamycin-induced autophagy of RAW 264.7 cells. Here, we demonstrate that the rapamycin-induced autophagy is dependent on protein phosphatase (PP) 2A activity and rifampicin inhibits the activity of PP2A by reducing expressions of PP2A subunits A and C. In addition, rifampicin slightly, but significantly, inhibited the rapamycin-induced dephosphorylation of p70 ribosomal protein S6 kinase (p70S6K) at Thr421/Ser424, which are regulated dually by both rapamycin and PP2A, but not at the rapamycin dephosphorylation site located at Thr389. These results show that rifampicin inhibits rapamycin-induced autophagy, at least in part, via the suppression of PP2A activity.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells.

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.