Disruption of the MYC-miRNA-EZH2 loop to suppress aggressive B-cell lymphoma survival and clonogenicity.

c-MYC (hereafter MYC) overexpression has been recognized in aggressive B-cell lymphomas and linked to adverse prognosis. MYC activation results in widespread repression of micro-RNA (miRNA) expression and associated with lymphoma aggressive progression. Our recent study identified a MYC-miRNA-EZH2 feed-forward loop linking overexpression of MYC, EZH2 and miRNA repression. Here, using a novel small-molecule BET bromodomain inhibitor, JQ1, and the EZH2 inhibitor, DZNep, we demonstrated that combined treatment of JQ1 and DZNep cooperatively disrupted MYC activation, resulting in a greater restoration of miR-26a expression and synergistically suppressed lymphoma growth and clonogenicity in aggressive lymphoma cells. Furthermore, CHIP assay demonstrated that MYC recruited EZH2 to miR-26a promoter and cooperatively repressed miR-26a expression in aggressive lymphoma cell lines, as well as primary lymphoma cells. Loss- or gain-of-function approaches revealed that miR-26a functioned as a tumor suppressor miRNA and mediated the combinatorial effects of JQ1 and DZNep. These findings represent a novel promising approach for silencing MYC-miRNA-EZH2 amplification loop for combinatorial therapy of aggressive B-cell lymphomas.

Knowledge, attitudes and practice concerning tuberculosis in a growing industrialised area in Myanmar.

SETTING: Factories in industrial zones in Yangon, Myanmar, one of the 22 high tuberculosis (TB) burden countries. OBJECTIVES: To assess workers' knowledge about TB, their health-seeking behaviour, acceptability of TB screening and predictors for approval of the dismissal of TB patients. DESIGN: In a cross-sectional survey, structured interviews with 349 factory workers were followed by 27 in-depth interviews and two focus group discussions with employers. RESULTS: Among 349 workers, 95% perceived TB as being curable, 50% correctly reported air as the main mode of transmission and 68% were aware of free treatment. Although 88% perceived screening before employment as necessary, only 14% underwent screening; 96% were willing to undergo contact screening for TB, but only 55% could afford it; 33% agreed with the dismissal of workers with TB, which was associated with lower education, shorter time in employment, not having a history of TB contact and unwillingness to work with an index TB case due to fear and lack of knowledge. CONCLUSION: More effective communication strategies towards factory workers are needed to increase workers' knowledge about transmission and reduce stigma. Employers should be sensitised to protect employees with TB and invest in preventive activities.

Myc represses miR-15a/miR-16-1 expression through recruitment of HDAC3 in mantle cell and other non-Hodgkin B-cell lymphomas.

Our recent study demonstrated miR-15a/16-1 downregulation in mantle cell lymphoma (MCL). Here, we investigated mechanisms of miR-15a/16-1 transcriptional repression and its epigenetic regulation by c-Myc and histone deacetylase (HDAC) in MCL. c-Myc expression was detected in MCL cell lines and in the primary MCL samples, and pri-miR-15a/16-1 mRNAs were significantly upregulated in Mino and Jeko-1 cells with c-Myc knockdown by small interfering RNAs (siRNAs). Our co-immunoprecipitation analysis showed that c-Myc interacted with HDAC3. Moreover, using chromatin immunoprecipitation, we demonstrated that both c-Myc and HDAC3 co-localized to the two promoters of the miR-15a/16-1 cluster gene, DLEU2, and inhibition of HDAC3 increased histone acetylation of the DLEU2 promoters. Luciferase reporter assay confirmed the dependence of Myc-mediated DLEU2 transcriptional repression on HDAC3. Treatment with the pan-HDAC inhibitor, suberoylanilide hydroxamic acid and HDAC3 siRNA resulted in increased miR-15a/16-1 expression. The regulatory mechanism of miR-15a/16-1 was further demonstrated in Burkitt lymphoma and Myc overexpressing cell lines. These findings highlight the role of HDAC3 in Myc-induced miR-15a/16-1 changes and reveal novel mechanisms for c-Myc-driven microRNA suppression and malignant transformation in aggressive B-cell malignancies.

Follicular dendritic cell-induced microRNA-mediated upregulation of PRDM1 and downregulation of BCL-6 in non-Hodgkin's B-cell lymphomas.

B-cell lymphoma 6 (BCL6) and PR domain containing 1 (PRDM1) are considered as master regulators for germinal center (GC) formation and terminal B-cell differentiation. Dysregulation of BCL6 and PRDM1 has been associated with lymphomagenesis. Here, we show for the first time that direct cell-cell contact between follicular dendritic cells (FDC) and B-lymphocytes, by influencing the expression of a set of microRNAs (miRNAs), regulates the expression of BCL6 and PRDM1. We identify that, on cell adhesion to FDC, FDC induces upregulation of PRDM1 expression through downregulation of miR-9 and let-7 families and induces downregulation of BCL-6 through upregulation of miR-30 family in B-lymphocytes and lymphoma cells. We further demonstrate that the miR-30 family directly controls BCL-6 expression and miR-9-1 and let-7a directly control PRDM-1 expression through targeting their 3'UTR, mediating the FDC effect. Our studies define a novel regulatory mechanism in which the FDC, through induction of miRNAs in B-lymphocytes, orchestrates the regulation of transcription factors, promotes germinal center B-cell survival and differentiation. Dysregulation of miRNAs may interfere with B-cell survival and maturation, thus representing a novel molecular mechanism, as well as a potential therapeutic target in B-cell lymphomas.

Lymphoma cell adhesion-induced expression of B cell-activating factor of the TNF family in bone marrow stromal cells protects non-Hodgkin's B lymphoma cells from apoptosis.

This study explores whether lymphoma cell adhesion-induced B cell-activating factor (BAFF) expression in bone marrow stromal cells (BMSCs) protects B lymphoma cells from apoptosis. We first showed protection of lymphoma cells from apoptosis by conditioned medium of a stromal cell-lymphoma cell coculture, either spontaneous or induced by mitoxantrone, implying a role for soluble factor(s) in lymphoma cell survival. Addition of BAFF counteracted mitoxantrone-induced apoptosis and elicited a reduction in spontaneous apoptosis in primary lymphomas, suggesting a role of BAFF in sustaining B-cell survival. Abundant BAFF was detected in the BMSC cell line (HS-5) and primary BMSCs by flow cytometry, RT-PCR and immunoblotting. BAFF levels were 20- to 200-fold higher in BMSCs than in lymphoma cells, and lymphoma cell adhesion to BMSCs augmented BAFF secretion twofold through upregulation of BAFF gene expression. Finally, neutralization of BAFF by TACI-Ig or depletion of BAFF by small hairpin RNA (shRNA) in BMSCs significantly enhanced lymphoma cell response to chemotherapy and overcame stroma-mediated drug resistance, suggesting that lymphoma cells use BMSC-derived BAFF as a survival factor. These findings support the hypothesis that lymphoma cells interact with BMSCs, resulting in stromal niches with high BAFF concentration, and identify BMSC-derived BAFF as a functional determinant for B lymphoma cell survival in the bone marrow environment.

Bone marrow stromal cells prevent apoptosis of lymphoma cells by upregulation of anti-apoptotic proteins associated with activation of NF-kappaB (RelB/p52) in non-Hodgkin's lymphoma cells.

Stromal cells are an essential component of the bone marrow microenvironment that regulate or supports tumor survival. In this study we therefore studied the role of stromal cells in lymphoma cell survival. We demonstrated that adhesion of the B-cell lymphoma cell lines SUDH-4 and 10 to bone marrow stroma inhibited mitoxantrone-induced apoptosis. This adhesion-dependent inhibition of mitoxantrone-induced apoptosis correlated with decreased activation of caspases-8 and 9, and cleavage of caspase 3 and PARP. Electrophoretic mobility shift assays (EMSA) analysis demonstrated significantly increased NF-kappaB binding activity in lymphoma cells adhered to stroma cells compared to lymphoma cells in suspension. This DNA binding activity could be attributed to cell adhesion-mediated proteolysis of the NF-kappaB precursor, p100 (NF-kappaB2). This resulted in the generation of active p52, which translocated to the nucleus in complex with p65 and RelB. Coculture with stromal cells also induced expression of the NF-kappaB-regulated anti-apoptotic molecules, XIAP, cIAP(1) and cIAP(2). Inhibition of NF-kappaB significantly suppressed HS-5-induced protection against apoptosis in lymphoma cell lines as well as in primary lymphoma cells. Thus, bone marrow stroma protects B-cell lymphoma cells against apoptosis, at least in part through activation of NF-kappaB dependent mechanism involving up-regulation of NF-kappaB regulated antiapoptotic proteins. Consequently, this study suggests a new approach to decrease the resistance of lymphoma to chemotherapy.

X-ray phase imaging of biological soft tissue using a direct-sensing x-ray HARP tube camera.

A HDTV camera having a direct-sensing x-ray high-gain avalanche rushing amorphous photoconductor (HARP) tube was used, for the first time, to acquire x-ray phase maps. The tube can achieve a high sensitivity as a result of the avalanche multiplication process in the HARP target. A beryllium plate, rather than a glass plate, was used as the face plate of the tube to minimize the loss of x-rays due to absorption, and a 15 microm thick HARP target was directly formed on it. In the experiment, the x-ray phase shifts produced by a rat liver were measured using synchrotron x-rays (lambda = 0.0766 nm) and a triple Laue-case (LLL) x-ray interferometer. Interference patterns produced by the sample were observed with the direct-sensing x-ray HARP tube camera. A voltage of 1300 V was applied to the HARP target to give an output signal gain of two. The camera was operated in 1125 scanning-line mode, and real-time images were stored on a workstation at a rate of 30 images/s with an image format of 960 (H) x 1100 (V) pixels. A phase-map image of the sample was successfully obtained using the fringe scanning method and phase unwrapping. The observed phase shifts ranged from 50 degrees to 200 degrees . Trees of blood vessels in the rat liver were clearly depicted without using a contrast agent. The spatial resolution of the x-ray camera was estimated to be better than 35 microm in the vertical direction and 100 microm in the horizontal direction.

National anti-tuberculosis drug resistance survey, 2002, in Myanmar.

SETTING: Thirty townships of Myanmar. OBJECTIVES: To determine the proportions of drug-resistant tuberculosis (TB) in new and previously treated pulmonary tuberculosis (PTB) cases in Myanmar. DESIGN: A cross-sectional study. Drug susceptibility was tested by the proportion method at the National Tuberculosis Reference Laboratory, Yangon. RESULTS: Of 874 TB patients included from 30 sites, 849 isolates obtained from individual patients (733 from new cases and 116 from previously treated cases) were tested for susceptibility to four primary anti-tuberculosis drugs. Of 733 isolates tested from new TB patients, 10% were resistant to any one of the anti-tuberculosis drugs, 6.5% to isoniazid (INH), 4.6% to rifampicin (RMP) and 4.0% were multidrug-resistant (MDR). Of the 116 previously treated patients, 30.2% were resistant to any one of the drugs, 26.7% to INH, 15.5% to RMP and 15.5% were MDR. Previous anti-tuberculosis treatment of more than 1 month was strongly associated with the development of MDR-TB (adjusted OR 4.8, 95% CI 2.5-9.1). CONCLUSION: The first national drug resistance survey in Myanmar revealed 4% and 15.5% MDR-TB among new and retreatment cases, respectively. Previous antituberculosis treatment was an important risk factor for MDR-TB. Continuous monitoring of drug resistance trends is needed

Determination of aliphatic amines in mineral flotation liquors and reagents by high-performance liquid chromatography after derivatization with 4-chloro-7-nitrobenzofurazan.

The method described here fulfils the need for a suitable analytical method to determine the concentrations of single and mixed aliphatic amines in the range from hexylamine (C6) to octadecylamine (C18) in flotation test solutions and in commercial flotation collectors. Amines do not have a UV-vis spectrum in aqueous solution but by reacting an amine-containing solution with 4-chloro-7-nitrobenzofurazan solution (chloro-NBD), derivatized products (amino-NBDs) are formed which have absorbance maxima at 470nm. Excess chloro-NBD and the amino-NBDs can be separated from each other by high-performance liquid chromatography (HPLC) and their concentrations measured with a UV-vis detector. Important variables in the derivatization stage are pH, temperature, chloro-NBD concentration, and reaction time, all of which interact with each other. A three-stage statistical procedure was used to determine the optimum conditions. In each stage, an 8-test design was used in which a high and low limit was set for each variable, and the chromatogram peak area of the derived amino-NBD was measured. The optimum derivatization conditions established were pH 8.9, chloro-NBD concentration 0.20% (w/v), temperature 70 degrees C, and reaction time 60 min. Optimum elution conditions for chromatography were an eluent containing 80% (v/v) acetonitrile in aqueous solution containing 40mM acetic acid at pH 4.5. With a flow rate of 2.0 ml/min, dodecylamine had a retention time of about 3 min, whereas octadecylamine had a retention time of 44 min. Straight-line calibration curves were obtained up to at least 200 ppm of amine in solution. The lower limit of detection was estimated to be 0.05 microM (10ppb) with a signal to noise ratio of 3. No interfering substances were found. The method was successfully applied to the analysis of solutions from an actual flotation test and to a solid commercial amine.

A lower jaw of Pondaungia cotteri from the Late Middle Eocene Pondaung Formation (Myanmar) confirms its anthropoid status.

Pondaungia cotteri is the largest primate known from the Late Middle Eocene Pondaung Formation, Myanmar. Its taxonomic status has been the subject of much debate because of the fragmentary nature of its remains. Initially described as an anthropoid, some authors recently have associated it with adapid primates. These debates have been fueled not only by the incompleteness of the fossils attributed to Pondaungia but also by the reticence of many authors to regard Asia as an important evolutionary theater for Eocene anthropoids. During the November 1998 Myanmar-French Pondaung Expedition, a right lower jaw was discovered that yields the most nearly complete dentition of Pondaungia cotteri ever found: it shows the complete horizontal ramus, alveoli for the second incisor and canine, three premolars, and three molars. The symphysis showed all characteristics of anthropoids but was unfused. The canine root is large, the first premolar is absent, and the second premolar is single-rooted, reduced, and oblique in the tooth row, as in anthropoids. The premolars show a reduced mesio-distal length compared with the tooth row, and their morphology is very similar to that of Amphipithecus mogaungensis. Therefore, the two Pondaung taxa appear to be closely related to each other, with Siamopithecus as their sister taxon.

A new primate from the Middle Eocene of Myanmar and the Asian early origin of anthropoids.

A new genus and species of anthropoid primate, Bahinia pondaungensis gen. et sp. nov., is described from the Yashe Kyitchaung locality in the Late Middle Eocene Pondaung Formation (Myanmar). It is related to Eosimias, but it is represented by more complete remains, including upper dentition with associated lower jaw fragment. It is interpreted as a new representative of the family Eosimiidae, which corresponds to the sister group of the Amphipithecidae and of all other anthropoids. Eosimiidae are now recorded from three distinct Middle Eocene localities in Asia, giving support to the hypothesis of an Asian origin of anthropoids.

HIV transmission in sexual partners of persons with HIV/AIDS attending the Infectious Diseases Hospital, Yangon.

A study was conducted in the Infectious Diseases Hospital, Yangon, for one year from August 1996 to 1997, to assess the extent and the factors related to HIV transmission among sexual partners of HIV/AIDS cases. It was a cross-sectional comparative study on 67 (61 males, 6 females) HIV positive individuals with or without AIDS, and their sexual partners. Separate interviews of index cases and partners were done, and clinical examination and laboratory tests for HIV and sexually transmitted diseases (STDs) were performed. HIV transmission was found in 41.8% of the partners. Male-to-female transmission was 39.3% (n=61) and female to male transmission was 66.7% (n=6), Seven and one half percent of the partners were suffering from AIDS. There were 4 (6.6%) pregnant mothers and 1 (25%) was HIV positive. Though 75% of the partners did not use condoms during their married life, HIV transmission was significantly reduced in the condom users (odds ratio (OR) = 0.18, 95% confidence interval (CI) 0.02-0.98 p = < 0.05). The seropositive men who were less than 30 years of age had greater HIV transmission (OR = 5.67, 95% CI 1.13-36.46). However, socio-demographic factors, number of marital partners and age of first sex partners, duration of marriage, number of sexual relationships between these couples, duration of HIV positivity and AIDS infection, immunological status of the index group and STD positivity among partners had no significant association with the transmission of HIV. This study demonstrated that the transmission of HIV was high among the sexual partners of persons with HIV infection. It also highlighted the requirement of effective counseling and preventive measures against HIV infection among the couples.

Variability in the classification of radiographs using the 1980 International Labor Organization Classification for Pneumoconioses.

This study describes the extent of agreement in classification of chest radiographs using the International Labor Organization (ILO) classification among six readers from the United States and Canada. A set of 119 radiographs was created and read by three Canadian and three US readers. The two ratings of interest were profusion (scored from 0/- to 3/+) and pleural abnormalities consistent with pneumoconiosis (scored with the ILO system, then collapsed into a yes/no). We used a number of approaches to evaluate interreader agreement on profusion and pleural changes, determining concordance, observed agreement, kappa statistic, and a new measure to approximate sensitivity and specificity. This study found that five of six readers had good fair to good agreement for pleural findings and for profusion as a dichotomous variable (> or = 1/0 vs < or = 0/1) using the kappa statistic, while a sixth reader had poor agreement. We found that concordance, expressed as percent agreement, was higher for normal radiographs than for ones that showed disease, and describe the use of the kappa statistic to control for this finding. This analysis adds to the existing literature with the use of the kappa statistic, and by presenting a new measure for "underreading" and "overreading" tendencies.

Augmentation of lipopolysaccharide-induced thymocyte apoptosis by interferon-gamma.

The role of interferon (IFN)-gamma on thymocyte apoptosis in response to lipopolysaccharide (LPS) was investigated. The administration of LPS into mice induced marked apoptosis of thymocytes in vivo, but the simultaneous injection of anti-IFN-gamma antibody with LPS completely prevented thymocyte apoptosis. Pretreatment of mice with IFN-gamma markedly enhanced LPS-induced thymocyte apoptosis. Thymocyte apoptosis augmented by IFN-gamma occurred in the thymic cortex, and target cells undergoing apoptosis were CD4+8+ immature thymocytes. IFN-gamma itself did not induce thymocyte apoptosis in vivo and in vitro. IFN-gamma exhibited no synergistic action with effector molecules, such as tumor necrosis factor (TNF)-alpha and glucocorticoids. Further, it was shown that IFN-gamma did not enhance the susceptibility of thymocytes to apoptosis. Pretreatment of mice with IFN-gamma significantly augmented the serum TNF-alpha level and the serum cortisol level in response to LPS. Therefore, we suggest that IFN-gamma might augment LPS-induced thymocyte apoptosis through elevating serum TNF-alpha and cortisol levels.

Marked reduction of mouse peritoneal CD5+ B cells by intraperitoneal administration of lipopolysaccharide.

Intraperitoneal administration of lipopolysaccharide to mice induced a marked reduction of CD5+ B cells in the peritoneal cavity. The reduction was not induced by intravenous, subcutaneous, or oral administration of lipopolysaccharide. The reduction continued for about 10 days after the injection, and the CD5+ B-cell count recovered to the normal state about 14 days after the injection. The reduction of peritoneal CD5+ B cells might be caused by apoptotic cell death. Injection of lipopolysaccharide did not result in production of antibody to lipopolysaccharide. On the other hand, intraperitoneal injection of heat-killed bacteria did not induce a reduction of peritoneal CD5+ B cells and elicited the definite production of antibody to lipopolysaccharide.

Effects of cleaning, chemical disinfection, and sterilization procedures on the mechanical properties of endodontic instruments.

The aim of this study was to test and compare the values of torsional moment, torsional angular deflection, bending moment, and permanent angular deflection of three designs of root canal files (Unifile, Flexofile, and H-File) before and after cross-infection treatment procedures, according to ANSI/ADA specification no. 28. An increase in value for all mechanical properties tested was observed after the treatment procedures, with the exception of Flexofile wherein a decrease in permanent angular deflection was evident. Unifile showed a decrease in torsional moment and bending moment. The changes in mechanical properties after treatment procedures ranged from 0.1 to 63% from the control groups. Generally, the changes in values observed were insignificant and still well within ANSI/ADA specification no. 28. Thus, they do not have any clinical significance.

Measurement of the cutting efficiency of endodontic instruments: a new concept.

The cutting efficiency of endodontic instruments was measured using an original experimental technique that incorporates new concepts to simulate clinical conditions. Five designs of #ISO 030 endodontic instruments, K-reamer (Maillefer), Flexofile (Maillefer), Helifile (Micro-mega), K-flex (Kerr), and Unifile (De Trey), were chosen and their cutting efficiency assessed at their full working length of 16 mm on two Plexiglas parallelepipeds tilted to follow the 2% conicity of the instruments. For each instrument, four series of 25 cuts were carried out and each cut made on a new flat, smooth Plexiglas surface with an even hardness of 33 VHN. Instruments were tested under a simulated clinical condition of a quarter clockwise turn ROTARY MOTION followed by a PULL ACTION at 16 mm/s rate, with a fixed load on the instrument of 325 g. Water irrigation at a rate of 85 ml/s was supplied before each procedure. Cutting efficiency was evaluated in terms of mass of Plexiglas cut (using a Mettler analytic balance with accuracy of 3 x 10(-5) g) per unit of energy used by the instrument, i.e. mg/J. Unifile was found to have the best cutting efficiency of 0.80 +/- 0.01 (Mean +/- SD) and lowest cutting efficiency loss followed by Flexofile 0.70 +/- 0.03 then Helifile 0.36 +/- 0.01 then K-flex 0.51 +/- 0.07. K-reamer was found to have the lowest cutting efficiency of 0.16 +/- 0.05.

Effects of cleaning, disinfection, and sterilization procedures on the cutting efficiency of endodontic files.

The effects of various cleaning, chemical disinfection, and sterilization procedures on the cutting efficiency of endodontic instruments Unifile (De Trey, Bois Colombes, France), (Flexofile Maillefer, Ballaigues, Switzerland), and H-File (Maillefer)) were investigated. The cross-infection control treatment procedures investigated were as follows: chemical disinfection--NaOCl (2.5%) for 12 and 48 h, and NH4 (5%) for 1 and 4 h; ultrasonic cleaning for 4 and 16 cycles of 15 min; and sterilization methods with chemiclave for 5 and 10 cycles of 20 min, Poupinel for 5 and 10 cycles of 120 min at 180 degrees C and glass beads for 10 and 40 cycles of 40 at 250 degrees C. Cutting efficiency was evaluated as the mass of Plexiglas cut per unit of energy expended by the instrument in microgram/Joule. The cutting efficiency decreased from 1 to 77%, depending on the file design and type of treatment procedures. Heat sterilization (Poupinel) did not modify the cutting efficiency of Unifile and Flexofile. The decrease in cutting efficiency was independent of frequency and duration of treatment procedures.

Cytokine-independent proliferation of IL-2-nonproducing CTL clones in association with high TCR-signal transduction responses.

Alloreactive CTL clone D2-23 proliferated in response to antigenic cells without IL-2 production. Among subclones of D2-23, the F1 but not F2 clone proliferated in response to soluble aCD3 or PMA, although both clones proliferated in response to immobilized aCD3, antigenic cells or soluble aCD3 plus costimulatory cells. The difference in responsiveness between F1 and F2 was not caused by distinct expression of CD3 or Fe receptors. Cyclosporin A, which totally blocks IL-2 production of Th1 cells, barely or only partially inhibited PMA- or aCD3-induced proliferation of F1. F1 did not produce cytokines for proliferation of F2 in response to soluble aCD3. Tyrosine phosphorylation developed for various proteins of F1 and F2 at the levels apparently correlated to the extent of cell proliferation when the cells were stimulated with soluble aCD3 or PMA. The proliferative responsiveness of F1 and F2 to the described stimulators was maintained by stimulation with IL-2 plus antigenic cells, or even IL-2 alone, but was decreased during resting culture or by stimulation with immobilized aCD3. These results show evidence of a new TCR-linked mechanism for CTL proliferation that is independent of costimulatory cell- or cytokine-mediated signaling, but is originally prepared by prior stimulation with IL-2.