Tests that detect Mycobacterium tuberculosis antigens in clinical specimens could provide rapid direct evidence of active disease. We performed a systematic review to assess the diagnostic accuracy of antigen detection tests for active tuberculosis (TB) according to standard methods and summarized test performance using bivariate random effects meta-analysis. Overall, study quality was a concern. For pulmonary TB (47 studies, 5,036 participants), sensitivity estimates ranged from 2% to 100% and specificity from 33% to 100%. Lipoarabinomannan (LAM) was the antigen most frequently targeted (23 studies, 49%). The pooled sensitivity of urine LAM was higher in HIV-infected than HIV-uninfected individuals (47%; 95% confidence interval [CI], 26 to 68% versus 14%; 95% CI, 4 to 38%); pooled specificity estimates were similar: 96%; 95% CI, 81 to 100% and 97%; 95% CI, 86 to 100%, respectively. For extrapulmonary TB (21 studies, 1,616 participants), sensitivity estimates ranged from 0% to 100% and specificity estimates from 62% to 100%. Five studies targeting LAM, ESAT-6, Ag85 complex, and the 65-kDa antigen in cerebrospinal fluid, when pooled, yielded the highest sensitivity (87%; 95% CI, 61 to 98%), but low specificity (84%; 95% CI, 60 to 95%). Because of the limited number of studies targeting any specific antigen other than LAM, we could not draw firm conclusions about the overall clinical usefulness of these tests. Further studies are warranted to determine the value of LAM detection for TB meningitis in high-HIV-prevalence settings. Considering that antigen detection tests could be translated into rapid point-of-care tests, research to improve their performance is urgently needed.

The human pulmonary surfactant protein A (hSP-A) has been implicated in the early capture and phagocytosis of the pathogenic Mycobacterium tuberculosis by alveolar macrophages. In this report, we examined the interaction of alveolar proteinosis patient hSP-A with Mycobacterium bovis BCG, the vaccinating strain, as a model of pathogenic mycobacteria, and Mycobacterium smegmatis, a nonpathogenic strain. We found that hSP-A binds to the surface of M. bovis BCG, but also to a slightly lesser extent, to M. smegmatis, indicating that hSP-A does not discriminate between virulent and nonpathogenic strains. Among the various glycoconjugates isolated from the mycobacterial envelope, we found that the best ligands are the two major lipoglycans: the mannosylated lipoarabinomannan (ManLAM) and the lipomannan. In contrast, the mannose-capped arabinomannan, structurally close to the ManLAM, as well as the LAMs from the non pathogenic M. smegmatis are poorly recognized by hSP-A. These results clearly show that the presence of both the terminal mannose residues and the phophatidyl-myo-inositol anchor are necessary to achieve the highest binding affinity. Selective removal of either the terminal mannose or the acyl residues esterifying the glycerol moiety of the ManLAM abrogates the interaction with hSP-A, further supporting the notion that the hSP-A recognition of the carbohydrate epitopes of the lipoglycans is dependent of the presence of the fatty acids.

Despite the complexity of tuberculosis (TB) serology, antibodies (Abs) remain attractive biomarkers for TB. Recent evidence of a mycobacterial capsule that consists mainly of the polysaccharides arabinomannan (AM) and glucan provides new options for serologic targets. For this study, Ab responses to AM and glucan for 47 U.S. TB patients (33 HIV negative [HIV(-)], 14 HIV positive [HIV(+)]), 42 healthy controls, and 38 asymptomatic HIV(+) controls were evaluated by enzyme-linked immunosorbent assays (ELISAs). The results were compared with Ab responses to the mycobacterial glycolipid cell wall antigen lipoarabinomannan (LAM) and to the proteins malate synthase (MS) and MPT51. We found that the main immunoglobulin (Ig) isotype response to polysaccharides was IgG, predominantly of subclass IgG2. IgG responses to AM were significantly higher for HIV(-) and HIV(+) TB cases than for controls (P, <0.0001 and <0.01, respectively); significantly higher for HIV(-) than for HIV(+) TB cases (P, <0.01); and significantly higher in sputum smear-positive than smear-negative patients in both HIV(-) and HIV(+) cases (P, 0.01 and 0.02, respectively). In both TB groups, titers of Ab to glucan were significantly lower than titers of Ab to AM (P, <0.0001). IgG responses to AM and MS or to AM and MPT51 did not correlate with each other in HIV(-) TB patients, while they correlated significantly in HIV(+) TB patients (P, 0.01 and 0.05, respectively). We conclude that Ab responses to AM could contribute to the serodiagnosis of TB, especially for HIV(-) TB patients. This study also provides new and important insights into the differences in the profiles of Abs to mycobacterial antigens between HIV(-) and HIV(+) TB patients.

BACKGROUND

Nucleotide analogues have recently been approved for the treatment of patients with hepatitis B virus (HBV) infection. However, it is still controversial whether the decrease of HBV-DNA amount induced by treatment with nucleotide analogues can reduce the risk of hepatocellular carcinoma (HCC) development in HBV patients.

METHODS

A total of 293 HBV patients without HCC who were treated with lamivudine (LAM) were enrolled in a multicenter trial. The incidence of HCC was examined after the start of LAM therapy, and the risk factors for liver carcinogenesis were analyzed. The mean follow-up period was 67.6 ± 27.4 months.

RESULTS

On multivariate analysis for HCC development in all patients, age ≥50 years, platelet count <14.0 × 10(4)/mm(3), cirrhosis, and median HBV-DNA levels of ≥4.0 log copies/ml during LAM treatment were significant risk factors. The cumulative carcinogenesis rate at 5 years was 3% in patients with chronic hepatitis and 30% in those with cirrhosis. For the chronic hepatitis patients, the log-rank test showed the significant risk factors related to HCC development to be age ≥50 years, platelet count <14.0 × 10(4)/mm(3), and hepatitis B e antigen negativity, but median HBV-DNA levels of <4.0 log copies/ml (maintained viral response, MVR) did not significantly suppress the development of HCC. In cirrhosis patients, however, the attainment of MVR during LAM treatment was revealed to reduce the risk of HCC development.

CONCLUSIONS

These results suggest that the incidence of HCC in HBV patients with cirrhosis can be reduced in those with an MVR induced by consecutive LAM treatment.

We estimate the correlation coefficient between two variables with repeated observations on each variable, using linear mixed effects (LME) model. The solution to this problem has been studied by many authors. Bland and Altman (1995) considered the problem in many ad hoc methods. Lam, Webb and O'Donnell (1999) solved the problem by considering different correlation structures on the repeated measures. They assumed that the repeated measures are linked over time but their method needs specialized software. However, they never addressed the question of how to choose the correlation structure on the repeated measures for a particular data set. Hamlett et al. (2003) generalized this model and used Proc Mixed of SAS to solve the problem. Unfortunately, their method also cannot implement the correlation structure on the repeated measures that is present in the data. We also assume that the repeated measures are linked over time and generalize all the previous models, and can account for the correlation structure on the repeated measures that is present in the data. We study how the correlation coefficient between the variables gets affected by incorrect assumption of the correlation structure on the repeated measures itself by using Proc Mixed of SAS, and describe how to select the correlation structure on the repeated measures. We also extend the model by including random intercept and random slope over time for each subject. Our model will also be useful when some of the repeated measures are missing at random.

Lymphangioleiomyomatosis (LAM), a rare multisystem disease found primarily in women of childbearing age, is characterized by the proliferation of abnormal smooth muscle-like cells, LAM cells, that form nodules in the pulmonary interstitium. Proliferation of LAM cells results, in part, from dysfunction in tuberous sclerosis complex (TSC) genes TSC1 (hamartin) and/or TSC2 (tuberin). Identification of LAM cells in donor lungs, their isolation from blood, and their presence in urine, chylous ascites, and pleural effusions are consistent with their ability to metastasize. Here, we investigated the presence on LAM cells of the hyaluronic acid receptor CD44 and its splice variants associated with metastasis. The heterogeneous populations of cells grown from lungs of 12 LAM patients contain cells expressing mRNA for the variant CD44v6. Histologically, CD44v6 was present in LAM lung nodules, but not in normal vascular smooth muscle cells. CD44v6-positive sorted cells showed loss of heterozygosity at the TSC2 locus; binding of CD44v6 antibody resulted in loss of cell viability. Levels of CD44 were higher in cultured Eker rat (Tsc2-/-) cells than in Tsc2+/+ cells, but unlike human LAM cells, the Tsc2-/- Eker rat cells did not contain CD44v6 splice variant mRNA. CD44 splicing and signaling is regulated by osteopontin. Plasma from LAM patients contained higher concentrations of osteopontin than plasma of healthy, age-, and sex-matched volunteers (P = 0.00003) and may be a biomarker for LAM. The cell surface receptor CD44 and its splice variant CD44v6 may contribute to the metastatic potential of LAM cells.

Lipoarabinomannan (LAM) and lipid-free arabinomannan (AM) were prepared from Mycobacterium paratuberculosis. Purification of LAM was done by ultracentrifugation of the phenol-water-extracted crude polysaccharide, followed by affinity and anion exchange chromatography. AM was purified from the supernatant of the ultracentrifuged polysaccharide or from alkaline-extracted material by gel filtration and anion exchange chromatography. Chemical analysis revealed arabinose and mannose in LAM (1.4:1) and AM (3.5:1) and the presence of palmitic, stearic, and tuberculostearic acids for a total of 7.8% lipid in LAM. Traces of phosphorus were found in the AMs, particularly LAM (0.05%). Nuclear magnetic resonance confirmed the presence of alpha-arabinosyl residues and the acylated nature of LAM. LAM exhibited lipid-dependent aggregation, as indicated by a Triton-induced decrease in molecular weight. By using bovine sera, LAM was found to be active in the complement fixation test, whereas AM was inactive and inhibited this activity. Thus, the presence of AM in crude polysaccharide could explain the variable complement fixation results. Triton-dissociated LAM exhibited a precipitin (Cl) in common with that of AM, confirming shared determinants. LAM in its lipid-dependent aggregated form, however, exhibited a second precipitin (C2), which may be due to the disparity in antigen size or a novel epitope. The lipid content of LAM rendered it 100 times more effective for coating plates in the enzyme immunoassay than lipid-free AM.

CD19 expressed on the surface of B lymphocytes is a key member of a cell surface signal transduction complex that includes TAPA-1, Leu 13 and CD21. The human CD19 protein is composed of 540 amino acids with a cytoplasmic domain of 242 amino acids. Although the cytoplasmic domain of CD19 has no sequence homology with other proteins, the cytoplasmic domain of human, mouse, and guinea pig CD19 is highly conserved suggesting that this region of CD19 is at least partially responsible for signaling activity. In this study, the regions of CD19 required for intermolecular associations and signal transduction were determined by comparing a series of carboxyl-terminal cytoplasmic tail deletion mutants and a CD19/L-selectin chimera with native CD19. CD19 expressed in the human Rex T cell line and the K562 erythroleukemia cell line generated transmembrane signals and also associated with endogenous TAPA-1. Deletion of 95% of the CD19 cytoplasmic domain did not affect the ability of CD19 to be expressed or to associate with TAPA-1. However, replacement of the CD19 transmembrane and cytoplasmic domains with those of L-selectin (CD19-LAM) resulted in the loss of CD19 complex formation, suggesting that the membrane spanning domain is critical for this association. Similarly, the induction of homotypic adhesion through CD19 or truncated CD19 was equivalent, whereas homotypic adhesion was not induced via the CD19-LAM chimera. In addition, the cytoplasmic domain was not necessary for CD19 mAb-mediated growth inhibition or internalization. In contrast, the CD19 cytoplasmic domain was required for optimal mAb-induced increases in [Ca2+]i in CD19 cDNA-transfected Rex cells. Thus, the CD19 cytoplasmic domain is responsible for the induction of increased [Ca2+]i, and the transmembrane region is required for cell surface associations with the other members of the CD19 complex and most signaling events. Therefore, mAb binding to CD19 is likely to initiate multiple intracellular signal transduction cascades either through CD19 directly, or through other members of the CD19 complex.

Performance for a variety of visual tasks improves with practice. The purpose of this study was to determine the nature of the processes underlying perceptual learning of identifying letters in peripheral vision. To do so, we tracked changes in contrast thresholds for identifying single letters presented at 10 degrees in the inferior visual field, over a period of six consecutive days. The letters (26 lowercase Times-Roman letters, subtending 1.7 degrees) were embedded within static two-dimensional Gaussian luminance noise, with rms contrast ranging from 0% (no noise) to 20%. We also measured the observers' response consistency using a double-pass method on days 1, 3 and 6, by testing two additional blocks on each of these days at luminance noise of 3% and 20%. These additional blocks were the exact replicates of the corresponding block at the same noise contrast that was tested on the same day. We analyzed our results using both the linear amplifier model (LAM) and the perceptual template model (PTM). Our results showed that following six days of training, the overall reduction (improvement across all noise levels) in contrast threshold for our seven observers averaged 21.6% (range: 17.2-31%). Despite fundamental differences between LAM and PTM, both models show that learning leads to an improvement of the perceptual template (filter) such that the template is more capable of extracting the crucial information from the signal. Results from both the PTM analysis and the double-pass experiment imply that the stimulus-dependent component of the internal noise does not change with learning.

Pulmonary lymphangioleiomyomatosis (LAM) is a rare disease that affects women in the reproductive years. It is occasionally associated with tuberous sclerosis, especially in the incomplete form. As it is likely that oestrogen plays a central role in disease progression, exogenous oestrogen will cause a deterioration in LAM. However, the early stage of this disease is easy to miss unless the physician is a specialist. Although there have been some reports in menopausal women given exogenous oestrogen for osteoporosis, this is the first report of pulmonary LAM caused by exogenous oestrogen used for the treatment of infertility.

Lipoarabinomannan (LAM) is the major arabinose- and mannose-containing phosphorylated lipopolysaccharide (LPS) in mycobacterial cell walls. LAM preparations from a virulent strain (Erdman) (LAM(Erdman)) and an attenuated strain (H37Ra) (LAMH37Ra) of Mycobacterium tuberculosis, as well as from M. leprae (a virulent mycobacterium), were analyzed for their effects on various macrophage (M phi) effector functions. LAMH37Ra, like gram-negative LPS, exhibited a dose-dependent ability to induce tumor necrosis factor alpha (TNF-alpha) production in normal M phi, and gamma interferon (IFN-gamma) priming of the M phi greatly augmented the levels of TNF-alpha. However, the effects of LAMH37Ra were unaffected by polymyxin B, which totally abrogated the effects of LPS. LAM(Erdman) and LAM from M. leprae, on the other hand, induced virtually no TNF-alpha production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of production. Analysis of M phi mRNA by reverse transcription-polymerase chain reaction revealed that the levels of TNF-alpha mRNA induced by the various preparations correlated with the levels of TNF-alpha protein detected. Interestingly, both LAMH37Ra and LAM(Erdman) could block subsequent IFN-gamma- and LPS-induced M phi activation, a previously reported measure of the potent ability of LAM to down-regulate M phi effector functions. Two lines of evidence suggested, however, that M phi cyclooxygenase products did not play a role in this down-regulation. LAMH37Ra and LPS could induce the production of NO2- in both normal and IFN-gamma-primed M phi, whereas LAM(Erdman) could stimulate NO2- production only in primed M phi. Both LAMH37Ra and LAM(Erdman) could substitute for LPS as a triggering signal for IFN-gamma-primed M phi in a toxoplasma killing assay. The triggering ability of LAM(Erdman), however, was abrogated by an anti-TNF-alpha antibody, suggesting that sufficient TNF-alpha production was stimulated by LAM(Erdman) to drive a M phi function relevant in host resistance. Thus, mycobacterial LAM is a potent regulator of M phi functions, a fact that may have important consequences in mycobacterial disease.

No study has reported on the comparative effect of adefovir (ADV) add-on lamivudine (LAM) versus switching to entecavir (ETV) in LAM-resistant patients with chronic hepatitis B. From October 2007 to September 2008, 92 consecutive LAM-resistant patients were enrolled (47 LAM+ADV and 45 ETV 1 mg). All patients were followed for at least 12 months. The parameters assessed included normalization of ALT, HBeAg seroconversion, undetectable HBV DNA, reduction of HBV DNA, and predictors of virologic response. In the LAM+ADV and ETV groups, the baseline DNA levels were 7.61 (5.19-9.49) and 7.10 (5.43-9.74)log(10)copies/ml, respectively. At month 12, a virologic response occurred in 18/47 (38.3%) and 11/45 (24.4%; P=0.182) patients; ALT normalization, in 39/41 (95.1%) and 36/40 (90.0%; P=0.432); HBeAg seroconversion, in 5.1% and 2.4% (P=0.606); and virologic breakthrough, in 2.1% and 11.1% (P=0.107), respectively. The mean reduction from the baseline HBV DNA level was greater in the LAM+ADV group at month 12 (3.80 ± 1.12 vs. 2.7 ± 1.32 log(10)copies/ml; P<0.001). In the multivariate analysis, the independent parameters related to a virologic response at month 12 were baseline ALT (OR=1.003, 95% CI=1.000-1.006, P=0.026) and baseline HBV DNA (OR=0.495, 95% CI=0.298-0.823, P=0.007). Compared with switching to ETV monotherapy, ADV add-on LAM therapy was more effective at reducing the viral load in patients with LAM resistance, and the baseline HBV DNA and ALT levels were independent predictors of the virologic response. However, ADV add-on therapy had limitations in patients with a higher baseline HBV DNA in LAM rescue therapy.

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAMLAMLAMLAM-1. Blocking of PPME or fucoidin binding by LAMLAMLAMLAMLAMLAMLAMLAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.

OBJECTIVE

To develop and retrospectively analyze an instrument that rapidly characterizes pretreatment stroke severity for use in prehospital acute stroke clinical trials.

METHODS

The Los Angeles Motor Scale (LAMS) was constructed by assigning point values to Los Angeles Prehospital Stroke Screen (LAPSS) items of facial weakness, arm strength, and grip to yield a total 0-5 scale. The concurrent, discriminant, and predictive validities of the LAMS were probed using data from 90 patients enrolled in acute stroke clinical trials. Predictive performance of the LAMS was compared with that of the initial full National Institutes of Health Stroke Scale (NIHSS) and the five-item shortened NIHSS (sNIHSS) in projecting long-term outcomes on standard functional end points.

RESULTS

<em>LAMS</em> score at entry averaged mean 2.6, median 2. Entry <em>LAMS</em> scores correlated closely with entry NIHSS scores (r=0.75). <em>LAMS</em> score correlations with three-month functional outcome measures were robust. Receiver operator curve analyses (c statistic) for performance in predicting three-month outcomes were: three-month modified Rankin-<em>LAMS</em> 0.75, sNIHSS 0.69, NIHSS 0.74; three-month Barthel Index-<em>LAMS</em> 0.77, sNIHSS 0.76, NIHSS 0.82; three-month NIHSS-<em>LAMS</em> 0.76, sNIHSS 0.62, NIHSS 0.70; and three-month GOS-<em>LAMS</em> 0.55, sNIHSS 0.67, NIHSS 0.76. Considering dichotomized three-month measures, entry <em>LAMS</em> scores were markedly lower in patients destined for excellent outcome, e.g., three-month modified Rankin score<or=1, mean entry <em>LAMS</em> score 2.2 versus 3.7, p=.0002.

CONCLUSIONS

A motor score derived from the LAPSS rapidly quantifies stroke severity in the field and predicts functional outcomes with accuracy comparable to that of the full NIHSS and the sNIHSS.

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).

BACKGROUND

Among tuberculosis (TB) high incidence regions, Sub-Saharan Africa is particularly affected with approx. 1.6 million new cases every year. Besides this dramatic situation, data on the diversity of Mycobacterium tuberculosis complex (MTBC) strains causing this epidemic in this area are only sparsely available. Here we analyzed the population structure of strains from Sierra Leone with a special focus on the prevalence of M. africanum.

RESULTS

A total of 97 strains isolated from smear positive cases registered for re-treatment in the Western Area and Kenema districts in years 2003/2004 were investigated by susceptibility testing (first line drugs) and molecular typing (IS6110 fingerprinting, spoligotyping, and MIRU-VNTR typing). Among the strains analyzed, 32 were resistant to isoniazid, and 11 were multidrug resistant (at least resistant to isoniazid and rifampin). The population diversity was high with two previously described M. africanum lineages (West African-1, n = 6; West African-2, n = 17) and seven M. tuberculosis lineages (Haarlem, n = 14; LAM, n = 15; EAI, n = 4; Beijing, n = 4; S-type, n = 4, X-type, n = 1; Cameroon, n = 4). Furthermore, two new M. tuberculosis genotypes Sierra Leone-1 (n = 7) and -2 (n = 10) were found. Strain classification according to a 7 bp deletion in pks1/15 revealed that the majority of M. tuberculosis strains belonged to the Euro American lineage (66 out of 74).

CONCLUSIONS

Resistance rates in Sierra Leone have reached an alarming level. The population structure of MTBC strains shows an intriguing diversity raising the question of possible consequences for TB epidemic and for the introduction of new diagnostic tests or treatment strategies in West Africa.

BACKGROUND

In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

RESULTS

During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

CONCLUSIONS

The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination.

Lipoarabinomannans (LAMs) are major Ags of the mycobacterial cell envelope where they apparently insert through a glycosylphosphatidylinositol (GPI) anchoring structure. LAMs induce host macrophages to secrete TNF-alpha, IL-1, and IL-6 and inhibit T cell proliferative responses. The mechanisms by which LAMs mediate these effects remain poorly understood. We show that LAMs were efficiently inserted into the plasma membranes of human and murine lymphomonocytic cells through their GPI anchor. Prior deacylation of LAMs abrogated this event. Phosphatidylinositol hexamannoside (PIM6), the GPI anchor of all LAMs, competitively inhibited LAM insertion. Deacylated PIM6 was not inhibitory. The hexamannoside glycan of PIM6 appears to be important for LAM insertion, since phosphatidylinositol from soybean, lacking the glycan core, was not as efficient an inhibitor. Interaction of LAM with target cells was influenced by the gel/fluid phase distribution of membrane lipids, suggesting a direct interaction of the LAM-GPI anchor with the membrane bilayer. The inserted LAMs were mobile in the plane of the membrane and interfered with Ab-mediated mobilization of the GPI-anchored Thy-1 molecules. Further, LAMs were preferentially incorporated into isolated plasma membrane vesicles enriched in Thy-1. Our results strongly suggest that 1) interaction of LAMs with host lymphomonocytic cells is mediated through a preferential integration of LAM-GPI anchor into specialized plasma membrane domains enriched in endogenous GPI-anchored molecules, and 2) both the acyl chains and the mannoside core glycan of the LAM-GPI anchor contribute to the specificity of integration.

Little observational data exist describing telbivudine (LdT) or lamivudine (LAM) use in late pregnancy for preventing hepatitis B mother-to-child transmission (MTCT) in real-world settings. During the period of January 2009 to March 2011, we enrolled hepatitis B e antigen-positive mothers with HBV DNA >6 log10 copies/mL in China. At gestation week 28, the mothers received LdT or LAM until postpartum week 4 or no treatment (NTx). The study endpoints were the safety of LdT/LAM use and MTCT rates. Of the 700 mothers enrolled, 648 (LdT/LAM/NTx=252/51/345) completed the 52-week study with 661 infants (LdT/LAM/NTx=257/52/352). On treatment, viral rebound occurred in 1.6% of mothers, all resulting from medication noncompliance. There was no genotypic mutation detected. At delivery, significantly lower HBV DNA levels were noted in mothers who received LdT or LAM versus NTx. Alanine aminotransferase flares were observed in 17.1% of treated mothers versus 6.3% of untreated mothers (P < 0.001). At birth, hepatitis B surface antigen (HBsAg) was detected in 20% and 24% of newborns in the treated and NTx groups, respectively. At week 52, an intention-to-treat analysis indicated 2.2% (95% confidence [CI]: 0.6-3.8) of HBsAg+ infants from the treated group versus 7.6% (95% CI: 4.9-10.3) in the NTx group (P50.001) and no difference of HBsAg+ rate between infants in the LdT and LAM groups(1.9% vs. 3.7%; P=0.758). On-treatment analysis indicated 0% of HBsAg+ infants in the treated group versus 2.84% in the NTx group (P=0.002). There were no differences for gestational age or infants' height, weight, Apgar scores, or birth defect rates between infants from the treated and untreated groups.

CONCLUSIONS

LdT and LAM use in late pregnancy for highly viremic mothers was equally effective in reducing MTCT. The treatment was well tolerated with no safety concerns identified.

Medicinal plants have been shown to have both chemopreventive and/or therapeutic effects on cancer and other diseases related to oxidative damage. Moringa oleifera Lam., known in the Hausa and Igala languages of Nigeria as "Zogale" and "Gergedi," respectively, and drumstick in English, is a plant that is used both as food and in folkloric medicine in Nigeria and elsewhere. Different parts of the plant were analyzed for polyphenol content as well as in vitro antioxidant potential. The methanol extract of the leaves of M. oleifera contained chlorogenic acid, rutin, quercetin glucoside, and kaempferol rhamnoglucoside, whereas in the root and stem barks, several procyanidin peaks were detected. With the xanthine oxidase model system, all the extracts exhibited strong in vitro antioxidant activity, with 50% inhibitory concentration (IC(50)) values of 16, 30, and 38 microL for the roots, leaves, and stem bark, respectively. Similarly, potent radical scavenging capacity was observed when extracts were evaluated with the 2-deoxyguanosine assay model system, with IC(50) values of 40, 58, and 72 microL for methanol extracts of the leaves, stem, and root barks, respectively. The high antioxidant/radical scavenging effects observed for different parts of M. oleifera appear to provide justification for their widespread therapeutic use in traditional medicine in different continents. The possibility that this high antioxidant/radical scavenging capacity may impact on the cancer chemopreventive potential of the plant must be considered.

D-Arabinofurans, attached to either a galactofuran or a lipomannan, are the primary constituents of mycobacterial cell wall, forming the unique arabinogalactan (AG) and lipoarabinomannan (LAM), respectively. Emerging data indicate that the arabinans of AG and LAM are distinguished by virtue of the additional presence of linear termini in LAM, which entails some unknown feature of the EmbC protein for proper synthesis. In common with the two paralogous EmbA and EmbB proteins functionally implicated for the arabinosylation of AG, EmbC is predicted to carry 13 transmembrane spanning helices in an integral N-terminal domain followed by a hydrophilic extracytoplasmic C-terminal domain. To delineate the function of this C-terminal domain, the embC knock-out mutant of Mycobacterium smegmatis was complemented with plasmids expressing truncated embC genes. The expression level of serially truncated EmbC protein thus induced was examined by EmbC-specific peptide antibody, and their functional implications were inferred from ensuing detailed structural analysis of the truncated LAM variants synthesized. Apart from critically showing that the smaller arabinans are mostly devoid of the linear terminal motif, beta-D-Araf(1-->2)-alpha-D-Araf(1-->5)-alpha-D-Araf(1-->5)-alpha-D-Araf, our studies clearly implicate the C-terminal domain of EmbC in the chain extension of LAM. For the first time a full range of arabinan chains as large as 18-22 Araf residues and beyond could be released intact by the use of an endogenous endo-D-arabinanase from M. smegmatis, profiled, and sequenced directly by tandem mass spectrometry. In conjunction with NMR studies, our results unequivocally show that the LAM-specific linear termini are an extension on a well defined inner branched Ara-(18-22) core. This hitherto unrecognized feature not only allows a significant revision of the structural model of LAM-arabinan since its first description a decade ago but also furnishes a probable molecular basis of selectivity in biosynthesis, as conferred by the EmbC protein.

Bartell, Pasquale F. (University of Pennsylvania, Philadelphia), Thomas E. Orr, and Grace K. H. Lam. Polysaccharide depolymerase associated with bacteriophage infection. J. Bacteriol. 92:56-62. 1966.-A recently isolated bacteriophage of Pseudomonas aeruginosa was observed, in association with bacteria, to produce a polysaccharide depolymerase. Exposure of slime polysaccharide to the enzyme at the pH optimum of 7.5 for 30 to 60 min resulted in a decreased viscosity of 20 to 25%, and a measurable increase in the levels of hexosamines, hexoses, and reducing substances, distinguishing it from other phage-associated depolymerases. Like egg-white lysozyme, the depolymerase produced a clearing of mature bacterial lawns, but was shown to be devoid of muralytic activity by turbidimetric and paper chromatographic analysis. The depolymerase reacted with polysaccharides of only certain strains of P. aeruginosa, and there appeared to be no correlation with phage susceptibility. The enzyme was not detectable in uninfected cultures, nor was it synthesized when infection was initiated by phages other than phage 2. The available data suggest that the genetic information required for biosynthesis of this enzyme is furnished by the phage 2 genome.