This study evaluated bacterial etiology and antibiotic susceptibility in patients diagnosed with community-acquired perforated appendicitis over a 12-year-period. We retrospectively reviewed records of adult patients diagnosed with perforated appendicitis at an 800-bed teaching hospital between January 2000 and December 2011. In total, 415 culture-positive perforated appendicitis cases were analyzed. Escherichia coli was the most common pathogen (277/415, 66.7%), followed by Streptococcus species (61/415, 14.7%). The susceptibility of E. coli to ampicillin, piperacillin/tazobactam, ceftriaxone, cefepime, amikacin, gentamicin, and imipenem was 35.1%, 97.1%, 97.0%, 98.2%, 98.9%, 81.8%, and 100%, respectively. The overall susceptibility of E. coli to quinolones (ciprofloxacin or levofloxacin) was 78.7%. During the study period, univariate logistic regression analysis showed a significant decrease in E. coli susceptibility to quinolones (OR = 0.91, 95% CI 0.84–0.99, P = 0.040). We therefore do not recommend quinolones as empirical therapy for community-acquired perforated appendicitis.
Upregulated ERK1/2 activity is often correlated with AKT activation during prostate cancer (PCa) progression, yet their functional relation needs elucidation. Using androgen-deprived LNCaP cells, in which ERK1/2 activation occurs in strong correlation with AKT activation, we found that AKT-mediated B-Raf regulation is necessary for ERK1/2 activation.
Specifically, in response to androgen deprivation, AKT upregulated B-Raf phosphorylation at Ser445 without affecting A-Raf or C-Raf-1. This effect of AKT was abolished by Arg25 to Ala mutation or truncating (Δ4-129) the pleckstrin homology domain of AKT, indicating that the canonical AKT regulation is important for this signaling. Intriguingly, although a constitutively active AKT containing N-terminal myristoylation signal could sufficiently upregulate B-Raf phosphorylation at Ser445 in LNCaP cells, subsequent MEK/ERK activation still required hormone deprivation. In contrast, AKT activity was sufficient to induce not only B-Raf phosphorylation but also MEK/ERK activation in the hormone refractory LNCaP variant, C4-2. These data indicate that androgen depletion may induce MEK/ERK activation through a synergy between AKT-dependent and -independent mechanisms and that the latter may become deregulated in association with castration resistance. In support, consistent AKT-mediated BRaf regulation was also detected in a panel of PCa lines derived from the cPten-/- L mice before and after castration. Our results also demonstrate that AKT regulates androgen receptor levels partly via the Raf/MEK/ERK pathway. This study reveals a novel crosstalk between ERK1/2 and AKT in PCa cells.
Prostate cancer; androgen withdrawal; AKT; B-Raf; ERK1/2
In Korea recently, nontuberculous mycobacteria (NTM) have been more frequently isolated in respiratory specimens, while Mycobacterium tuberculosis (MTB) isolations have decreased. The major NTM lung disease species in Korea are M. intracellulare, M. avium, and M. abscessus, whereas M. kansasii is a rare species. This retrospective study was performed to determine if there are region-specific characteristics of lung disease-causing NTM species in Ulsan, a highly industrialized city in Korea.
Between January 2010 and July 2013, the results of all acid-fast bacilli (AFB) cultures of respiratory specimens performed at Ulsan University Hospital (Ulsan, Korea) were collected. NTM were identified and regional differences of NTM species were compared.
AFB cultures were performed on 33,567 respiratory specimens, obtained from 10,208 patients, during the study period. Further, 10% of the specimens (3,287/33,567) were AFB culture-positive [MTB, 2,288/3,287 (70%); NTM 999/3,287 (30%)]. The proportion of NTM isolations gradually increased between 2010 and 2013, at 25% and 38%, respectively. The most common NTM species was M. intracellulare (356/999, 36%), followed by M. kansasii (295/999, 30%), M. avium (161/999, 16%), M. abscessus (117/999, 12%) and M. fortuitum (39/999, 4%). This trend was maintained throughout the study period.
In Ulsan, NTM isolation from respiratory specimens is increasing, consistent with previous studies performed in Korea. The distribution of respiratory NTM species, however, differed from previous studies that were performed in other regions of Korea: M. kansasii was the second most common NTM species in Ulsan. In Ulsan, there is a regional difference in the NTM species isolated.
Nontuberculous mycobacteria (NTM); M. kansasii; Ulsan
We evaluated high-performance liquid chromatography (HPLC) for
species identification of mycobacteria from various clinical specimens in an
urban hospital in South Korea between January 2005 and December 2009.
In the study period 24,774 cultures were completed, yielding the 3215
clinical isolates cultivated for mycobacteria and positive cultures that had
mycolic acid investigated by HPLC. For species identification, we compared
HPLC patterns of clinical isolates with 33 standard
There were 3 different HPLC groups with single, double, and
triple-cluster patterns representing 9, 20, and 4 mycobacterial species,
respectively. Species identification rates of HPLC for Mycobacterium
tuberculosis and nontuberculous mycobacteria (NTM) were found
to be 100% and 95.6%, respectively. Among mycobacterial
isolates, 12.1% were NTM-positive. There were 20 different NTM
species with frequencies of 0.3%~15.5%.
The HPLC method was highly sensitive identifying NTM isolated from
mycobacteria; Mycobacterium tuberculosis; nontuberculous mycobacteria; HPLC
The discovery of potent BRAF inhibitors has revolutionized therapy for BRAF-mutant melanoma, yet NRAS-mutant melanoma remains without effective therapy. Since direct pharmacological inhibition of RAS has thus far been unsuccessful, we explored system biology approaches to identify synergistic drug combination(s) that can mimic direct RAS inhibition. Here, leveraging an inducible mouse model of NRAS-mutant melanoma, we show that pharmacological MEK inhibition activates apoptosis but fails to trigger cell cycle arrest, in contrast to complete NRAS extinction in vivo by genetic means. Network modeling pinpointed CDK4 as a key driver of this differential phenotype. Accordingly, combined pharmacological inhibition of MEK and CDK4 in vivo led to significant synergy in therapeutic efficacy. Taken together, our data suggest a gradient model of oncogenic NRAS signaling to the canonical MAPK cascade, where the output is gated, resulting in de-coupling of discrete downstream biological phenotypes in the setting of incomplete inhibition. Such a gated signaling model provides a novel framework to identify non-obvious co-extinction target(s) for combined pharmacological inhibition in NRAS-mutant melanomas.
We used HPLC and AdvanSure real-time PCR (LG Life Sciences, Korea) to retrospectively analyze non-tuberculous mycobacteria (NTM) in 133 clinical specimens. The specimens were culture-positive for NTM and the HPLC method identified 130 strains of mycobacteria from the cultures (97.7%) at the species level. Among the isolates, 48 Mycobacterium. kansasii (36.1%), 39 M. intracellulare (29.3%), 17 M. avium (12.8%), 16 M. abscessus (12.0%), 6 M. fortuitum (4.5%), 2 M. szulgai (1.5%), 2 M. gordonae (1.5%), and 3 unclassified NTM strains (2.3%) were identified. The real-time PCR assay identified 60 NTM-positive specimens (45.1%), 65 negative specimens (48.9%), and 8 M. tuberculosis (TB)-positive specimens (6.0%). The real-time PCR assay is advantageous because of its rapid identification of NTM. However, in our study, the real-time PCR assay showed relatively low sensitivity (45.1%) when using direct specimens including sputum and bronchoalveolar lavage (BAL) fluid. HPLC is useful as it discriminates NTM at the species level, although it is time-consuming and requires specific equipment and technical expertise. A combination of both methods will be helpful for the rapid and accurate identification of mycobacteria in clinical laboratories.
Non-tuberculous mycobacteria; Real-time PCR; HPLC
The environmental factors that contribute to the development of autoimmune diseases are largely unknown. Endemic pemphigus foliaceus in humans, known as Fogo Selvagem (FS) in Brazil, is mediated by pathogenic IgG4 autoantibodies against desmoglein1 (Dsg1). Clusters of FS overlap with those of leishmaniasis, a disease transmitted by sand fly (Lutzomyia longipalpis) bites. In this study we show that salivary antigens from the sand fly, and specifically the LJM11 salivary protein, are recognized by FS antibodies. Anti-Dsg1 monoclonal autoantibodies derived from FS patients also cross-react with LJM11. Mice immunized with LJM11 generate anti-Dsg1 antibodies. Thus, insect bites may deliver salivary antigens that initiate a cross-reactive IgG4 antibody response in genetically susceptible individuals and lead to subsequent FS. Our findings establish a clear relationship between an environmental, non-infectious antigen and the development of potentially pathogenic autoantibodies in an autoimmune disease.
Tumor-initiating stem-like cells (TICs) are resistant to chemotherapy and associated with hepatocellular carcinoma (HCC) caused by HCV and/or alcohol-related chronic liver injury. Using HCV Tg mouse models and patients with HCC, we isolated CD133+ TICs and identified the pluripotency marker NANOG as a direct target of TLR4, which drives the tumor-initiating activity of TICs. These TLR4/NANOG–dependent TICs were defective in the TGF-β tumor suppressor pathway. Functional oncogene screening of a TIC cDNA library identified Yap1 and Igf2bp3 as NANOG-dependent genes that inactivate TGF-β signaling. Mechanistically, we determined that YAP1 mediates cytoplasmic retention of phosphorylated SMAD3 and suppresses SMAD3 phosphorylation/activation by the IGF2BP3/AKT/mTOR pathway. Silencing of both YAP1 and IGF2BP3 restored TGF-β signaling, inhibited pluripotency genes and tumorigenesis, and abrogated chemoresistance of TICs. Mice with defective TGF-β signaling (Spnb2+/– mice) exhibited enhanced liver TLR4 expression and developed HCC in a TLR4-dependent manner. Taken together, these results suggest that the activated TLR4/NANOG oncogenic pathway is linked to suppression of cytostatic TGF-β signaling and could potentially serve as a therapeutic target for HCV-related HCC.
Our objective was to evaluate the relationship between intrauterine exposure to cadmium and the presence of atopic dermatitis in infants 6 months of age, adjusted for covariates including exposure to other heavy metals. The present research is a component of the Mothers' and Children's Environmental Health (MOCEH) study, a multi-center birth cohort project conducted in Korea. Study subjects were restricted to pregnant women in whom cadmium and lead levels were measured at delivery and whose infants were assessed for the presence of atopic disease at 6 months of age. The odds ratio (OR) for the presence of atopic dermatitis in 6-month-old infants whose cord blood had elevated cadmium levels, after adjustment for other covariates, was 2.350 (95% CI, 1.126-4.906). The OR for the presence of atopic dermatitis in infants whose cord blood had elevated lead levels was not significant. In the present study, the cord blood cadmium level was significantly associated with the presence of atopic dermatitis in 6-month-old infants; this was not true of the cord blood lead level. To the best of our knowledge, this is the first prospective study to show a relationship between prenatal exposure to cadmium and atopic dermatitis in infancy.
Atopy; Cadmium; Lead; Cord; Dermatitis, Atopic
Cancer stem cells (Tumor-initiating stem-like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)-infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4-NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto-oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine-treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG-dependent genes that inhibit TGF-β signaling in TISCs. Tlr4 expression is higher in TISCs compared to CD133−/CD49f+ cells, and DNA hypomethylation, histone acetylation and de-methylation drive Tlr4 induction through NOTCH signaling or Hif-1α. Taken together, Tlr4 is induced by hypoxia and NOTCH signaling and is a universal proto-oncogene responsible for the genesis of TLR4-NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC.
Alcohol; HCV; HCC; Tumor-initiating stem-like cells; Obesity
A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Nontuberculous mycobacteria; Mycobacterium; Mycobacterium riyadhense; Mycobacterium simulans
Despite its initial positive response to hormone ablation therapy, prostate cancers invariably recur in more aggressive, treatment resistant forms. The lack of our understanding of underlying genetic alterations for the transition from androgen-dependent (AD) to ADI prostate cancer growth hampers our ability to develop target-driven therapeutic strategies for the efficient treatment of ADI prostate cancer.
By screening a library of activated human kinases, we have identified TPL2, encoding a serine/threonine kinase, as driving ADI prostate cancer growth. TPL2 activation by over-expressing either wild-type or a constitutively activated form of TPL2 induced ADI growth, whereas the suppression of TPL2 expression and its kinase activity in ADI prostate cancer cells inhibited cell proliferation under androgen-depleted conditions. Most importantly, TPL2 is upregulated in ADI prostate cancers of both the Pten deletion mouse model and the clinical prostate cancer specimens.
Together these data suggest that TPL2 kinase plays a critical role in the promotion of ADI prostate cancer progression. Furthermore, the suppression of TPL2 diminishes ADI prostate cancer growth and a high frequency of TPL2 overexpression in human ADI prostate cancer samples validates TPL2 as a target for the treatment of this deadly disease.
The Beijing family of Mycobacterium tuberculosis has been emerging in the world. However, there are few nationwide data of genotypic distribution in Korea. This study aimed to identify the genotypic diversity of clinical isolates of M. tuberculosis and to demonstrate the population of Beijing family in Korea. We collected 96 clinical M. tuberculosis isolates from 11 university hospitals nationwide in Korea from 2008 to 2009. We observed 24 clusters in IS6110-RFLP analysis and 19 patterns in spoligotyping. Seventy-five isolates were confirmed to be Beijing family. Two isolates of the K strain and 12 isolates of the K family strain were also found. We found that drug resistance phenotypes were more strongly associated with Beijing family than non-Beijing family (P=0.003). This study gives an overview of the distribution of genotypes of M. tuberculosis in Korea. These findings indicate that we have to pay more attention to control of M. tuberculosis strains associated with the Beijing family.
Mycobacterium tuberculosis; IS6110-RFLP; Spoligotyping; Beijing Family; K Strain
Autophagy is an active homeostatic degradation process for the removal or turnover of cytoplasmic components wherein the LC3 ubiquitin-like protein undergoes an Atg7 E1-like enzyme/Atg3 E2-like enzyme-mediated conjugation process to induce autophagosome biogenesis1–4. Besides its cytoprotecive role, autophagy acts on cell death when it is abnormally upregulated. Thus, the autophagy pathway requires tight regulation to ensure that this degradative process is well balanced. Two death effector domains (DED1/2) containing cellular FLICE-like inhibitor protein (cFLIP) and viral FLIP (vFLIP) of Kaposi’s sarcoma-associated herpesvirus (KSHV), Herpesvirus saimiri (HVS), and Molluscum contagiosum virus (MCV) protect cells from apoptosis mediated by death receptors5,6. Here, we report that cellular and viral FLIPs suppress autophagy by preventing Atg3 from binding and processing LC3. Consequently, FLIP expression effectively represses cell death with autophagy, as induced by rapamycin, an mTor inhibitor and an effective anti-tumour drug against KSHV-induced Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL)7,8. Remarkably, either a DED1 α2-helix ten amino-acid (α2) peptide or a DED2 α4-helix twelve amino-acid (α4) peptide of FLIP is individually sufficient for binding FLIP itself and Atg3, with the peptide interactions effectively suppressing Atg3–FLIP interaction without affecting Atg3-LC3 interaction, resulting in robust cell death with autophagy. Our study thus identifies a checkpoint of the autophagy pathway where cellular and viral FLIPs limit the Atg3-mediated step of LC3 conjugation to regulate autophagosome biogenesis. Furthermore, the FLIP-derived short peptides induce growth suppression and cell death with autophagy, representing biologically active molecules for potential anti-cancer therapies.
Oligonucleotide chips targeting the bacterial internal transcribed spacer region (ITS) of the 16S-23S rRNA gene, which contains genus- and species-specific regions, were developed and evaluated. Forty-three sequences were designed consisting of 1 universal, 3 Gram stain-specific, 9 genus-specific, and 30 species-specific probes. The specificity of the probes was confirmed using bacterial type strains including 54 of 52 species belonging to 18 genera. The performance of the probes was evaluated using 825 consecutive samples that were positive by blood culture in broth medium. Among the 825 clinical specimens, 708 (85.8%) were identified correctly by the oligonucleotide chip. Most (536 isolates, or 75.7%) were identified as staphylococci, Escherichia coli, or Klebsiella pneumoniae. Thirty-seven isolates (4.5%) did not bind to the corresponding specific probes. Most of these also were staphylococci, E. coli, or K. pneumoniae and accounted for 6.3% of total number of the species. Sixty-two specimens (7.5%) did not bind the genus- or species-specific probes because of lack of corresponding specific probes. Among them, Acinetobacter baumannii was the single most frequent isolate (26/62). The oligonucleotide chip was highly specific and sensitive in detecting the causative agents of bacteremia directly from positive blood cultures.
The clinical course of prostate cancer is grouped into two broad phases. The first phase, which is the growth of the androgen-dependent cancer (AD-Ca) responds well to androgen depletion treatment while the second phase, that could be termed as androgen depletion-independent cancer (ADI-Ca) does not. We used two separate prostate tumors, one AD-Ca and one ADI-Ca from the conditional Pten deletion mouse model to generate from each a pair of cell lines. The AD-Ca cell lines (E2 and E4) and the ADI-Ca cell lines (cE1 and cE2) display bi-allelic deletion at the Pten gene locus, an event which is specific for the prostate epithelium for this mouse model, and a fairly similar level of expression of the androgen receptor (AR). The ADI-Ca cell lines (cE series) grow well in the absence of androgen, display increased AR transcription under androgen-deprived environment, and retain the sensitivity to increased proliferation when androgen is supplemented. The AD-Ca cell lines (E series) grow slowly in the absence of androgen, and, unlike cE cells, do not show increased AR expression when maintained in the absence of androgen. The detection of epithelial cell markers, such as CK8, CK14, CK18 and E-cadherin in the cE series is conforming with the polygonal epithelial morphology of these cells in culture. The E cells also present mostly polygonal-shaped morphology with a small percent of cells with fibroblastoid morphology, and produce little or very low levels of cytokeratins, but increased levels of vimentin, Twist and Slug, the markers known to be associated with epithelial-mesenchymal transition. Each of the cell lines, when inoculated subcutaneously into male or female NOD.SCID mice induced tumors within eight weeks with 100% incidence. Histopathological examinations of the tumor sections, however, led to noticeable biological differences. The cE series engenders adenocarcinomas, particularly in male hosts, and the E series induces sarcomatoid carcinomas (positively stained for CK8 and AR as well as vimentin expression) in either male or female hosts. These new cell lines are promising models for the elucidation of the androgen metabolism and their role in prostate cancer.
Conditional Pten-deletion mouse model of prostate adenocarcinoma; mouse prostate cancer cell lines; androgen-dependent and androgen depletion-independent prostate cancer; prostate sarcomatoid carcinoma
The LKB1 – AMPK signaling pathway serves as a critical cellular sensor coupling energy homeostasis to cell growth, proliferation and survival. However, how tumor cells suppress this signaling pathway to gain growth advantage under conditions of energy stress is largely unknown. Here, we show that AMPK activation is suppressed in melanoma cells with the B-RAF V600E mutation and that down-regulation of B-RAF signaling activates AMPK. We find that in these cells LKB1 is phosphorylated by ERK and Rsk, two kinases downstream of B-RAF, and that this phosphorylation compromises the ability of LKB1 to bind and activate AMPK. Furthermore, expression of a phosphorylation-deficient mutant of LKB1 allows activation of AMPK and inhibits melanoma cell proliferation and anchorage-independent cell growth. Our findings provide a molecular linkage between the LKB1-AMPK and the RAF-MEK-ERK pathways and suggest that suppression of LKB1 function by B-RAF V600E plays an important role in B-RAF V600E-driven tumorigenesis.
γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses.
Autophagy (‘self-eating’, lysosome-dependent degradation and recycling of the intracellular components in response to stress) and apoptosis (‘self-killing’, cells commit suicide in response to stress) are important host defense mechanisms against viral infections. γ-herpesvirus 68 (γHV68) encodes a Bcl-2 family protein, vBcl-2, that effectively antagonizes both autophagy and apoptosis and is required for chronic viral infection and pathogenesis. However, the relative contributions of the vBcl-2-mediated evasion of autophagy and apoptosis to γHV68 persistent infection remain largely unknown. Here, we characterized a series of vBcl-2 mutants to genetically and functionally distinguish these closely related activities of vBcl-2 in vitro and in vivo. We have found that the inhibition of autophagy by vBcl-2 is important for maintaining latent infections, while the anti-apoptotic activity of vBcl-2 is largely involved in efficient viral reactivation from latency. Our findings thus reveal a novel paradigm for the vBcl-2-mediated evasion of autophagy and apoptosis during chronic viral infection, identifying a vital role for autophagy in controlling γHV68 latent infection.
Prostate cancer is the second leading cause of cancer-related deaths in men. Activation of MAP kinase signaling pathway has been implicated in advanced and androgen-independent prostate cancers, although formal genetic proof has been lacking. In the course of modeling malignant melanoma in a tyrosinase promoter transgenic system, we developed a genetically-engineered mouse (GEM) model of invasive prostate cancers, whereby an activating mutation of BRAFV600E–a mutation found in ∼10% of human prostate tumors–was targeted to the epithelial compartment of the prostate gland on the background of Ink4a/Arf deficiency. These GEM mice developed prostate gland hyperplasia with progression to rapidly growing invasive adenocarcinoma without evidence of AKT activation, providing genetic proof that activation of MAP kinase signaling is sufficient to drive prostate tumorigenesis. Importantly, genetic extinction of BRAFV600E in established prostate tumors did not lead to tumor regression, indicating that while sufficient to initiate development of invasive prostate adenocarcinoma, BRAFV600E is not required for its maintenance.
The genes erm(B), mef(A), and both erm(B) and mef(A) were identified in 42.6, 10.1, and 47.3%, respectively, of the erythromycin-resistant Streptococcus pneumoniae isolates. Of the strains, 3.8% were nonsusceptible to levofloxacin and had 1 to 6 amino acid changes in the quinolone resistance-determining region, including a new mutation, Asn94Ser, in the product of parC. Levofloxacin with reserpine was highly specific for efflux screening.
As the incidence of nontuberculous mycobacterial infection has been increasing recently in Korea, the importance of drug susceptibility test for clinical isolates of mycobacteria has become larger. In this study we determined the antimicrobial susceptibility patterns of clinical isolates of M. fortuitum and M. abscessus in Korea, and evaluated the efficacy of a modified broth microdilution method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC), in terms of its ability to provide accurate and easy-to-read minimal inhibitory concentration (MIC) endpoints for the susceptibility testing of rapidly growing mycobacteria. Most isolates of M. fortuitum and M. abscessus in Korea are susceptible or intermediately susceptible to amikacin, cefoxitin, ciprofloxacin, and clarithromycin. Many isolates of M. fortuitum are susceptible to doxycycline, sulfamethoxazole, and imipenem, while many M. abscessus isolates are resistant to these drugs. In the present study, the modified broth microdilution method using STC was found to be reliable, easy to read, and inexpensive for M. fortuitum and M. abscessus susceptibility testing. The modified colorimetric MIC testing method using STC was proven to be a useful surrogate for RGM antibiotic susceptibility testing.
Mycobacteria, Atypical; Mycobacterium Fortuitum; Mycobacterium Abscessus; Microbial Sensitivity Tests; Colorimetry
An oligonucleotide chip (Combichip Mycobacteria chip) detecting specific mutations in the rpoB, katG, and inhA genes of Mycobacterium tuberculosis was compared with conventional antimicrobial susceptibility results. The probes detecting drug resistance were as follows: 7 wild-type and 13 mutant probes for rifampin and 2 wild-type and 3 mutant probes for isoniazid. Target DNA of M. tuberculosis was amplified by PCR, followed by hybridization and scanning. Direct sequencing was performed to verify the results of the oligonucleotide chip. One-hundred seven of 115 rifampin-resistant strains (93%) had mutations in the rpoB gene. Eighty-five of 119 isoniazid-resistant strains (71%) had mutations in the katG gene or inhA gene. The diagnostic oligonucleotide chip with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of resistance against rifampin and isoniazid in M. tuberculosis isolates.
Kaposi sarcoma–associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA’s contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis.
The viral immediate-early transactivator Rta/Orf50 is necessary and sufficient to initiate Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) reactivation from latently infected cells. Since Rta/Orf50 is conserved among all known gamma-2-herpesviruses, we investigated whether the murine gamma-68-herpesvirus (MHV-68) and rhesus monkey rhadinovirus (RRV) homologs can functionally substitute for KSHV Rta/Orf50. (i) Our comparison of 12 KSHV promoters showed that most responded to all three Rta/Orf50proteins, but three promoters (vGPCR, K8, and gB) responded only to the KSHV Rta/Orf50 transactivator. Overall, the activation of KSHV promoters was higher with KSHV Rta than with the RRV and MHV-68 Rta. (ii) Only the primate Rta/Orf50 homologs were able to interfere with human p53-depedent transcriptional activation. (iii) Transcriptional profiling showed that the KSHV Rta/Orf50 was more efficient than it's homologs in inducing KSHV lytic transcription from the latent state. These results suggest that the core functionality of Rta/Orf50 is conserved and independent of its host, but the human protein has evolved additional, human-specific capabilities.
Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a human-oncogenic herpesvirus. Cells from KSHV-associated tumors, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), are of endothelial and B-cell origin, respectively. KSHV persists indefinitely in these cell lineages during latent infection. Indeed, cellular latency is a hallmark of all herpesviruses that is intimately linked to their pathogenesis. We previously characterized the promoter for the KSHV latency-associated nuclear antigen LANA/orf73. LANA is required for latent episome maintenance and has also been implicated in oncogenesis. Hence, regulation of LANA expression is critical to KSHV persistence. We find that a region extending to bp −1299 upstream of the LANA transcription start site is able to drive lacZ-reporter gene expression in several lines of transgenic mice. In agreement with KSHV's natural tropism, we detected reporter gene expression in CD19-positive B cells but not in CD3-positive T cells. We also detected expression in the kidney and, at a lower level, in the liver. In contrast to KS tumors, transgene expression was localized to kidney tubular epithelium rather than vascular endothelial cells. This suggests that our promoter fragment contains all cis-regulatory elements sufficient for B-cell specificity but not those required for endothelial specificity. Alternatively, while the trans-acting factors required for LANA expression in B cells are evolutionarily conserved, those that regulate endothelial cell-specific expression are unique to humans. Our in vivo studies address a conundrum in KSHV biology: in culture, KSHV is able to infect a variety of cell types indiscriminately, while in healthy latent carriers KSHV is found in B lymphocytes. The transgenic-mouse experiments reported here suggest that tissue-restricted LANA gene expression could explain B-cell-specific viral persistence.