Case Series of Mycobacterium Abscessus Infections Associated with a Trigger Point Injection and Epidural Block at a Rural Clinic / 한국역학회지

OBJECTIVES: The aim of this report is to investigate Mycobacterium abscessus infections at a rural clinic and carry out a surveillance program to determine the extent and source of these infections. METHODS: The authors conducted an active surveillance investigation of 36 patients who had visited the clinic since 1 July 2008. Clinical specimens were collected from the patients and an envirnmental investigation. Pulsed-field gel elctrophoresis (PFGE) was performed for comparing with M. abscessus isolates from the patients. RESULTS: Six specimens were obtained from the 6 patients respectively and 22 environmental samples were obtained. M. abscessus was isolated from the wounds of two patients, and various nosocomial pathogens, but not M. abscessus, were isolated from the surrounding environment. Two strains of M. abscessus from patients were identical as a result of PFGE. CONCLUSION: Infection control education including proper hand hygiene should be emphasized for physicians performing invasive procedures. There also needs to be more attention for invasive procedures management, including trigger point injection and epidural block in rural clinics.

Cytokine Profiles of Macrophages by Mycobacterium abscessus Conditional Morphotype Variants and Comparison of Their Extracellular-Protein Expressions

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Previously, it was documented that a spontaneously formed rough variant of M. abscessus causes persistent and invasive infection in mice, while a smooth isogenic variant does not. However, little is known for immune responses elicited by M. abscessus variants artificially induced by culture conditions and their culture filtrate antigens. Thus, morphological variants of M. abscessus type strain (ATCC19977T) were generated by an acidic and low oxygen culture conditions. Overall comparison between the variant and its original smooth strain showed that the rough variant was less virulent than original smooth strain in murine bone-marrow derived macrophage. To understand the basis for the difference, the protein expression pattern in the culture filtrates of each strain was analyzed by 1-dimensional electrophoresis. Generally, the protein expressions were more influenced by pH conditions than oxygen pressures. Interestingly, several proteins, mainly lower than 30 kDa molecular weight, were uniquely expressed in normal culture conditions. In contrast, several high molecular weight proteins (>55 kDa) were induced by acidic and low oxygen culture conditions. These findings not only provide new insights of association between morphological change and the virulence, but may also be useful in the design of immunological diagnosis and vaccines for M. abscessus infection.

Analysis of RET Gene Point Mutation in a Family with Multiple Endocrine Neoplasia Type 2A / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Multiple Endocrine Neoplasia type 2A (MEN 2A) is a syndrome that encompasses medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism. Since MEN 2A is inherited as autosomal dominant, early detection and treatment is crucial. A genetic analysis of RET proto-oncogene of the family members of an index patient diagnosed as MEN 2A is reported. SUBJECTS AND METHOD: A patient diagnosed as MEN 2A and his 13 family members across two generations were studied. Initially, DNA was extracted from the peripheral blood leukocyte of family members and PCR amplification of exons 10, 11, 13, 14, 15, and 16 was performed, followed by investigation of point mutation on the RET proto-oncogene using a DNA sequence analyzer. Cervical ultrasonography was carried out in the 3 nephews who were revealed to have RET proto-oncogene point mutation. RESULTS: Point mutations of TGC (cys) to TGG (Trp) at codon 634 of exon 11 at RET proto-oncogene was detected by using automatic DNA sequence analyzing method in the index patient. The same point mutation was identified in 7 of the 13 family members. Cervical ultrasonography revealed bilateral thyroid nodules in all 3 nephews who had point mutations of RET proto-oncogene. CONCLUSION: With the genetic analysis of RET proto-oncogene, limitations of the conventional calcitonin stimulation test may be overcome, and a more complete approach can be achieved through early diagnosis by carrying out this screening test for point mutations in family members of the patient with MEN 2A.

Differential Roles of Toll-like Receptor (TLR) 2 and 4 between PPD- and 38-kDa-induced Proinflammatory Cytokine Productions in Human Monocytes

In this study, we investigated the role of toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) pathways involved in the tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 expression after stimulation with purified protein derivatives (PPD) or native 38-kDa protein antigen (Ag) of Mycobacterium tuberculosis H37Rv in human primary monocytes. Both PPD and 38-kDa Ag significantly induced TNF-alpha and IL-6 in human primary monocytes. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] are rapidly phosphorylated in human monocytes stimulated with the PPD or 38-kDa Ag. Both p38 and ERK 1/2 activation are essential for PPD- or 38-kDa-induced TNF-alpha and IL-6 production. The inhibition of TLR2 and TLR4 by specific antibodies significantly abrogated the 38-kDa-induced secretion of TNF-alpha and IL-6, whereas blockade of TLR2, but not TLR4, was responsible for the PPD-induced TNF-alpha and IL-6 production in human monocytes. Collectively, these data suggest that the PPD and 38-kDa Ag differentially interact with TLR2 and TLR4, which in turn mediate an essential role for the early inflammatory immune responses during human tuberculosis.

Identification of Proteins Induced at Hypoxic and Low pH Conditions in Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis likely reside within a granuloma as a dormant state. An area of necrosis forms at the center of lung granulomas. Within this area, the bacteria are deprived of nutrients and exposed to harsh conditions, including low pH and anoxia. The response of M. tuberculosis to low pH and low oxygen conditions was investigated in both cellular and extracellular proteins by two-dimensional polyacrylamide gel electrophoresis analysis and MALDITOF. Several proteins intensively expressed under low pH and/or hypoxic conditions were found. In the culture filtrate, PhoS1 (Rv0934) and ScoB (Rv2503c) were found in significant amounts under both the low oxygen and acidic stress conditions. These results indeed extend our understanding of acidic response as well as hypoxic in M. tuberculosis and provide an important insight into physiology of the latent bacilli.

The Phospholipase-Protein Kinase C-MEK-ERK Pathway is Essential in Mycobacteria-induced CCL3 and CCL4 Expression in Human Monocytes

BACKGROUND: Little information is available on the identification and characterization of the upstream regulators of the signal transduction cascades for Mycobacterium tuberculosis (M. tbc)-induced ERK 1/2 activation and chemokine expression. We investigated the signaling mechanisms involved in expression of CCL3/MIP-1 and CCL4/MIP-1 in human primary monocytes infected with M. tbc. METHODS: MAP kinase phosphorylation was determined using western blot analysis with specific primary antibodies (ERK 1/2, and phospho-ERK1/2), and the upstream signaling pathways were further investigated using specific inhibitors. RESULTS: An avirulent strain, M. tbc H37Ra, induced greater and more sustained ERK 1/2 phosphorylation, and higher CCL3 and CCL4 production, than did M. tbc H37Rv. Specific inhibitors for mitogen-activated protein kinase (MAPK) kinase (MEK; U0126 and PD98059) significantly inhibited the expression of CCL3 and CCL4 in human monocytes. Mycobacteria-mediated expression of CCL3 and CCL4 was not inhibited by the Ras inhibitor manumycin A or the Raf-1 inhibitor GW 5074. On the other hand, phospholipase C (PLC) inhibitor (U73122) and protein kinase C (PKC)- specific inhibitors (GO6976 and Ro31-8220) significantly reduced M. tbc-induced activation of ERK 1/2 and chemokine synthesis. CONCLUSION: These results are the first to demonstrate that the PLC-PKC-MEK-ERK, not the Ras-Raf-MEK-ERK, pathway is the major signaling pathway inducing M. tbc-mediated CCL3 and CCL4 expression in human primary monocytes.

Mycobacteria-induced Interleukin-12 Expression by Human Monocyte-derived Macrophages Is Negatively Regulated by Phosphatidylinositol 3-kinase and ERK 1/2 Pathways

Both interleukin (IL)-12, an important cytokine skewing the immune response towards a Th1 cytokine profiles, and tumor necrosis factor (TNF)-alpha, are thought to be critical factors in defenses against mycobacteria. In this study, we evaluated the roles of phosphatidylinositol 3-kinase (PI 3-K), and extracellular signal-regulated kinase (ERK) 1/2 pathways in the expression of IL-12 in human monocyte-derived macrophages (MDMs) after stimulation with Mycobacterium tuberculosis H37Rv (M. tbc) or the Triton X-114 solublized proteins (TSP) of M. tbc. Both M. tbc and TSP rapidly phosphorylated ERK 1/2, and Akt in human MDMs. Inhibition of PI 3-K-Akt pathway by specific inhibitors (LY294002 and wortmannin) dramatically increased M. tbc- or TSP-induced IL-12 p40 and p35 mRNA and IL-12 production. In addition, blockade of ERK 1/2 pathway by specific inhibitors (PD98059 and U0126) significantly increased the mRNA levels and cytokine production in M. tbc- or TSP-treated MDMs. On the contrary, M. tbc- or TSP-induced TNF-a production was significantly depressed in human MDMs by pretreatment with inhibitors of PI 3-K or ERK pathways. The M. tbc or TSP stimulation decreased ERK 1/2 phosphorylation by 70% in the presence of wortmannin or LY294002, suggesting that some cross-talk between the PI 3-K-Akt and mitogen-activated protein kinase kinase (MEK)-ERK pathways may be operating in human monocytes during mycobacterial infection. PI 3-K activity is partially required for the M. tbc- or TSP-induced ERK 1/2 phosphorylation. Collectively, these data suggest that the PI 3-K and ERK 1/2 pathways play a central role in the negative regulation of IL-12, but not TNF-a, production by M. tbc.

Transduction of yeast cytosine deaminase mediated by HIV-1 Tat basic domain into tumor cells induces chemosensitivity to 5-fluorocytosine

Enzyme/prodrug approach is one of the actively developing areas for cancer therapy. In an effort to develop more effective enzyme/prodrug systems, cell-permeable cytosine deaminase was produced by fusing yeast cytosine deaminase (yCD) in frame with RKKRRQRRR domain of HIV-1 Tat which is an efficient delivery peptide of the foreign proteins into cells. The purified Tat-yCD fusion protein expressed in Escherichia coli was readily transduced into mammalian cells in a time- and dose-dependent manner. A significant level of the transduced Tat-yCD protein was recovered in the cell and was stable for 24 h as indicated by both results of the enzymatic assay of 5-fluorocytosine (5-FC) conversion to 5-fluorouracil (5-FU) and Western blot analysis. The cells transduced with Tat-yCD become highly sensitive to the cytotoxicity of 5-FC, while cells treated with yCD are unaffected by 5-FC. In addition, a strong bystander effect was observed with conditioned media from cells transduced with Tat-yCD added to non-transduced cells. Tat-yCD fusion protein demonstrated here for its ability to transduce into cells and convert nontoxic prodrug 5-FC to the toxic antimetabolite 5-FU, may be a useful approach for cancer therapy.

Role of Mitogen-Activated Protein Kinase Pathways in the Production of TNF-alpha, IL-10, and MCP-1 by Mycobacterium tuberculosis H37Rv-Infected Human Blood Monocytes

Clinical manifestations of tuberculosis are closely associated with the initial responses of macrophages to mycobacteria. In this study, we investigated the signal transduction pathways for the secretion of cytokines and chemokines [tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1)] in human blood monocytes infected with Mycobacterium tuberculosis H37Rv. M. tuberculosis H37Rv infection induced the secretion of significant amounts of TNF-alpha, IL-10, IL-8, and MCP-1 from human blood monocytes. Analysis of mitogen-activated protein kinase (MAPK) activation [extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase] showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MEK-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF-alpha production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human blood monocytes infected with M. tuberculosis H37Rv. However, IL-8 secretion was regulated neither by ERK1/2 nor p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-alpha. IL-10, and MCP-1 by human blood monocytes during M. tuberculosis H37Rv infection.

Human glutamate dehydrogenase is immunologically distinct from other mammalian orthologues

Five monoclonal antibodies (mAbs) that recognize human glutamate dehydrogenase (GDH) have been selected and designated as monoclonal antibodies hGDH60-6, hGDH60-8, hGDH63-10, hGDH63-11, and hGDH91-14. A total of five mAbs recognizing different epitopes of the enzyme were obtained, two of which inhibited human GDH activity. When total proteins of human homogenate separated by SDS- PAGE, were probed with mAbs, a single reactive protein band of 55 kDa, which co-migrated with purified recombinant human GDH was detected. When the purified GDH was incubated with each of the mAbs, its enzyme activity was inhibited by up to 58%. Epitope mapping analysis identified, two subgroups of mAbs recognizing different peptide fragments. Using the individual anti-GDH antibodies as probes, the cross reactivities of brain GDH obtained from human and other animal brain tissues were investigated. For the human and animal tissues tested, immunoreactive bands on Western blots appeared to have the same molecular mass of 55 kDa when hGHD60-6, hGHD60-8, or hGHD91-14 mAbs were used as probes. However, the anti-human GDH mAbs immunoreactive to bands on Western blots reacted differently on the immunoblots of the other animal brains tested, i.e., the two monoclonal antibodies hGDH63-10 and hGDH63-11 only produced positive results for human. These results suggest that human brain GDH is immunologically distinct from those of other mammalian brains. Thorough characterization of these anti-human GDH mAbs could provide potentially valuable tool as immunodiagnostic reagents for the detection, identification and characterization of the various neurological diseases related to the GDH enzyme.

Expression and Characterization of Human Immunodeficiency Virus Type 1 Envelope Mutant Glycoproteins by Using Baculovirus Expression System

Considerable effort has been directed at understanding the structure and function of HIV-1 envelope glycoproteins. It has been difficult to characterize HIV-1 envelope glycoproteins due to the limited availability of these proteins from virus particles or infected cells. To facilitate the structural and functional analysis of HIV-1 envelope glycoproteins, recombinant baculoviruses were generated to express wild type or mutant HIV-1 envelope glycoproteins. The gp160 precursor protein as well as the gp120 glycoprotein were detected in the cell infected with recombinant BacENVw.t containing wild type HIV-1 envelope gene. In the insect cells infected with recombinant BacENVc with mutations at the cleavage site of gp160, a precursor form of envelope glycoprotein was produced, but not secreted into the culture medium. However, the insect cells infected with recombinant BacENVc/t containing both mutations at the cleavage site and membrane spanning region produced mutant envelope glycoproteins that were efficiently secreted into the culture medium in the form of precursor. Therefore the recombinant HIV-1 envelope glycoproteins produced in this system would be useful as immunogens in the development of a vaccine against AIDS.

A Case of Primary Malignant Melanoma in Parotid Gland / 대한이비인후과학회지

Reports on primary malignant melanoma arising from parotid gland are extremely rare. We report a case of malignant melanoma which was presented in the parotid gland with no other primary lesions detectable. The main clinical presentation was a progressively enlarging, asymptomatic mass in the parotid gland. The 29-year-old patient underwent a total parotidectomy and right modified radical neck dissection type I. The patient was subsequently treated by postoperative high-dose radiotherapy. The most common symptom of primary malignant melanoma in the parotid gland is a progressively enlarging, asymptomatic, firm, and fixed mass. Radical excision is the treatment of choice. The role of radiotherapy, chemotherapy and immunotherapy remains unclear. Although rare, primary malignant melanoma should be considered in the differential diagnosis of parotid gland tumor. We report the case with a review of the literature.

Expression of MAGE 3 Gene Product in Squamous Cell Carcinomas of the Head and Neck / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: MAGE 3 gene may constitute a potential target for cancer immunotherapy since it is expressed in a variety of cancers but not in normal tissues except the testis. In this study, expression and intracellular location of MAGE 3 gene products were investigated using squamous cell carcinomas of the head and neck. MATERIALS AND METHODS: MAGE 3 protein expression was screened in 40 squamous cell carcinomas, 2 tumor lines, 20 benign diseases, and 20 normal tissues of the head and neck. Immunohistochemical staining with anti-MAGE 3 mAb 57B was conducted from fresh frozen specimens. A correlation between MAGE 3 expression and clinicopathological parameters was also evaluated. RESULTS: MAGE 3 gene product was detected in squamous cell carcinomas (18/40, 45%) and in tumor lines (2/2, 100%), but not in benign diseases and normal tissues. MAGE 3 gene product was identified as a cytoplasmic protein of cancer cells without staining in normal epithelia and stromal tissues coexisting adjacent to cancer cells. No significant correlation between MAGE 3 expression and clinicopathological parameters including tumor cell differentiation, age, gender, primary site, tumor stage, and metastasis was drawn. CONCLUSIONS: MAGE 3 antigen could represent a potential target for immunotherapy in head and neck squamous cell carcinomas.