Although the combination of lamivudine (LAM) and high-dose intravenous (IV) hepatitis B immunoglobulin (HBIG) is very effective in preventing hepatitis B virus (HBV) recurrence after liver transplantation (LT), the major limitation of this regimen is its high cost. A more cost-effective, convenient and widely accepted regimen is urgently needed. We evaluated the safety and efficacy of another strategy using LAM and individualized low-dose intramuscular (IM) HBIG. Between May 2002 and December 2009, a total of 254 adult patients undergoing LT for HBV-related benign end-stage liver diseases received this regimen in our center. The mean follow-up of these patients was 41.2 +/- 22.7 months. Their 1-, 3- and 5-year survival rates were 85.3%, 77.4% and 76.4%, respectively, and 1-, 3- and 5-year HBV recurrence rates were 2.3%, 6.2% and 8.2%. Fourteen patients experienced posttransplant HBV recurrence. Pretransplant high viral load and posttransplant prednisone withdrawal time were observed to be associated with recurrence. In conclusion, combination therapy with LAM and individualized low-dose IM HBIG provides a safe and effective prophylaxis against HBV recurrence after LT at about 5% of the cost of conventional high-dose IV HBIG regimens.

The mechanisms by which T lymphocytes escape apoptosis during their activation are still poorly defined. In this study, we elucidated the intracellular signaling pathways through which beta1 integrins modulate Fas-mediated apoptosis in T lymphocytes. In experiments done in Jurkat T cells and activated peripheral blood T lymphocytes, engagement of alpha2beta1 integrin with collagen type I (Coll I) was found to significantly reduce Fas-induced apoptosis and caspase-8 activation; Annexin V binding and DNA fragmentation were reduced by approximately 42 and 38%, respectively. We demonstrated that the protective action of Coll I does not require new protein synthesis but was dependent on the activation of the MAPK/Erk pathway. Furthermore, we found that activation of protein phosphatase 2A (PP2A) by Coll I was required for both Coll I-mediated activation of Erk, and inhibition of Fas-induced caspase-8 activation and apoptosis. Other ligands of beta1 integrins, fibronectin (Fbn), and laminin (Lam), did not sustain significant Erk activation and had no effect on Fas-induced apoptosis. Taken together, these results provide the first evidence of a PP2A-dependent activation of the MAPK/Erk pathway downstream of alpha2beta1 integrin, which has a functional role in regulating Fas-mediated apoptosis in T lymphocytes. As such, this study emphasizes the potential importance that Coll I interactions may have on the control of T lymphocyte homeostasis and their persistence in chronic inflammatory diseases.

OBJECTIVE

More than 100 cases of nephropathy over the last 10 years caused by the systemic and longer term application of Chinese snakeroot (Aristolochia fangchi) highlighted the risk of using preparations which contain aristolochic acids. On the other hand anecdotal evidence highlights the widespread use of Aristolochia species (Aristolochiaceae) in many regions of the world. Therefore, it was our objective to systematically assess the scientific literature available on the local and traditional use of Aristolochia spp. on a worldwide scale. Our review identifies core species which need to be investigated and which may need monitoring (esp. in national and international trade).

METHODS

An extensive review of the literature available in libraries in London on the uses of species of Aristolochia was undertaken. Relevant information was extracted and entered into a database for analysis.

RESULTS

Based on the assessment of 566 reference sources 685 individual sets of data were recorded. Seven species--Aristolochia indica L. (Asia), Aristolochia serpentaria L. (North America), Aristolochia debilis Sieb & Zucch. (China), Aristolochia acuminata Lam (India), Aristolochia trilobata L. (Central/South America, Caribbean), Aristolochia clematitis L. (Europe) and Aristolochia bracteolata Lam. (Africa)--are reported widely as being used medicinally. The medical uses vary, but of particular interest are uses in case of gastrointestinal problems, which is likely to result in repeated exposure to the botanical drugs by an individual. About half of all records relate to uses of Aristolochia species in Asia, one-third to the Americas, a continent which has so far received practically no attention in terms of assessing the risk of using species of Aristolochia. Of the 99 species (plus several identified at genus level only) for which we were able to summarise ethnobotanical information, preliminary phytochemical information is only available for 24 species and some of the most common ones including Aristolochia acuminata have so far not been studied phytochemically.

CONCLUSIONS

Species of Aristolochia are used medicinally in many regions of the world and both from an ethnopharmacological and a public health perspective this poses a risk. A systematic assessment of the content of aristolochic acids in the most widely used species is needed to evaluate whether their uses pose a potential health risk. In China and Europe species of Aristolochia have been associated with nephropathy and it is important to evaluate whether nephropathy occurs in other parts of the world, especially India and Central America where the use of species of Aristolochia are reported to be commonly used in traditional medicine.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Without effective prophylaxis, liver transplantation for hepatitis B virus (HBV)-related liver disease is frequently complicated by severe and rapidly progressive HBV recurrence. Combination prophylaxis with hepatitis B immune globulin (HBIG) and lamivudine (LAM) reduces long-term recurrence rates below 10%; however, HBIG is costly and inconvenient to administer. We, therefore, conducted a multicenter, prospective study of outcomes with an HBIG-sparing regimen of LAM plus adefovir dipivoxil (ADV) initiated at the time of listing for liver transplantation and continued after transplantation. Twenty-six patients were recruited into this study at the time of listing for transplantation, and 20 subsequently underwent transplantation. Twelve of the 26 patients had LAM exposure before the study baseline, but none had LAM resistance. The median HBV viral load before the institution of antiviral therapy was approximately 4.0 log(10) IU/mL (range=2.3-7.5 log(10) IU/mL). To the 20 patients who underwent transplantation, 800 IU of intramuscular HBIG was given immediately after transplantation and daily for 7 days only (total HBIG dose=6400 IU). All transplant patients remained alive without HBV recurrence (they were negative for hepatitis B surface antigen, and HBV DNA was undetectable) after a median follow-up of 57 months after transplantation (range=27-83 months). The median serum creatinine level in these patients rose from 81 to 119 μmol/L over the course of the study. No patient required dose reduction or cessation. After the completion of this prospective study, the regimen was modified so that no perioperative HBIG was administered if the pretransplant serum HBV DNA level was suppressed below 3 log(10) IU/mL. Another 28 patients with HBV-related liver disease underwent transplantation (18 without HBIG). All remained alive and well without HBV recurrence after a median follow-up of 22 months after transplantation (range=10-58 months). In conclusion, a combination of LAM and ADV initiated at the time of wait listing provides safe and effective protection against recurrent HBV infection without the high costs and inconvenience associated with long-term HBIG therapy.

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either 'core candidates' (common to the two lines) or 'line specific'. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log2 ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.

Lymphangioleiomyomatosis (LAM) is a rare cystic lung disease that primarily affects women. The purpose of these guidelines is to provide recommendations for the diagnosis and treatment of LAM.

Systematic reviews were performed to summarize evidence pertinent to our questions. The evidence was summarized and discussed by a multidisciplinary panel. Evidence-based recommendations were then formulated, written, and graded using the Grading of Recommendations, Assessment, Development, and Evaluation approach.

After considering the panel's confidence in the estimated effects, the balance of desirable (i.e., benefits) and undesirable (i.e., harms and burdens) consequences of treatment, patient values and preferences, cost, and feasibility, recommendations were formulated for or against specific interventions. These included recommendations for sirolimus treatment and vascular endothelial growth factor D testing and recommendations against doxycycline and hormonal therapy.

Evidence-based recommendations for the diagnosis and treatment of patients with LAM are provided. Frequent reassessment and updating will be needed.

This study compared virologic response to entecavir monotherapy and de novo lamivudine plus adefovir (LAM + ADV) combination therapy in patients with chronic hepatitis B (CHB) with high viral load (HVL). Hepatitis B e antigen (HBeAg)-positive patients [hepatitis B virus (HBV) DNA levels >1 × 10(7) copies/ml] were assigned to LAM + ADV or entecavir treatment. The primary efficacy endpoint measure of the multicenter prospective cohort study was proportion of patients with CHB with virologic response, defined as HBV DNA <300 copies/ml at week 48. During treatment, 39.1 % (18/46) of patients in the LAM + ADV group and 48.1 % (25/52) of those in the entecavir group achieved virologic response in week 48 (P = 0.37). A baseline alanine aminotransferase (ALT) level ≥5 × ULN (upper limit of normal) or baseline serum HBV DNA level <8 log10 IU/ml could predict virologic response at week 48 (P = 0.025). The mean reduction in HBV DNA was comparable (P = 0.45); no significant difference was found in the proportion of ALT normalization (P = 0.46) or HBeAg seroconversion (P = 0.88). Two cases of genotypic resistance were found (rtM204 V + rtL180 M and rtA181T/V) in the LAM + ADV group, with a resistance rate of 4.3 %; there was no genotypic resistance in the entecavir group (P = 0.13). De novo LAM + ADV combination therapy is as effective as entecavir monotherapy in HBeAg-positive patients with CHB with HVL. Moreover, genotypic resistance was only found in the LAM + ADV group at week 48. Baseline ALT levels ≥5 ULN or baseline serum HBV DNA levels <8 log10 IU/ml were favorable predictors of virologic response in CHB with HVL.

The participation of attentional and associative mechanisms in extinction, spontaneous recovery, external disinhibition, renewal, reinstatement, and reacquisition was evaluated through computer simulations with an extant computational model of classical conditioning (N. A. Schmajuk, Y. Lam, & J. A. Gray, 1996; N. A. Schmajuk & J. A. Larrauri, 2006). The model assumes that attention to stimuli (controlled by environmental novelty) and associations between stimuli interact during memory storage (learning) and retrieval (performance). Computer simulations indicated that a combination of attentional and associative mechanisms might be sufficient to describe most of the properties of extinction. However, configural mechanisms seem necessary to describe the properties of cues that precede the target stimulus during extinction (extinction cues) and might improve the description of some experimental results regarding the associative properties of the extinction context. These configural mechanisms can be easily integrated into the present version of the model.

OBJECTIVE

HBV recurrence increases morbidity and mortality in HBsAg+ patients undergoing liver transplantation. We aimed to estimate the relative efficacy of combined therapy with hepatitis B immunoglobulin (HBIG) and lamivudine (LAM) versus HBIG monotherapy for preventing HBV-related morbidity and mortality in this setting.

METHODS

We performed a meta-analysis of clinical trials that met the prespecified criteria and provided data for risk estimation of HBV recurrence in HBsAg+ liver transplant patients receiving HBIG and LAM versus HBIG alone. Databases searched until May 2007 included MEDLINE (Ovid), PubMed, Embase, Toxnet, Scopus, and Web of Science. Literature search and data extraction were conducted independently by 2 study investigators; then 2 other investigators reviewed and screened eligible studies. Odds ratios (ORs) for the risk reduction with HBIG and LAM versus HBIG alone were calculated by using a random-effects model.

RESULTS

Two prospective and 4 retrospective studies were included in the meta-analysis. The OR showing risk reduction in HBV recurrence with HBIG and LAM (n = 193) versus HBIG alone (n = 124) was 0.08 (95% confidence interval [CI], 0.03-0.21). HBV-related death and all-cause mortality could only be assessed in 3 studies each. The ORs showing HBV-related death and all-cause mortality reduction with HBIG and LAM versus HBIG alone were 0.08 (95% CI, 0.02-0.33) and 0.02 (95% CI, 0.06-0.82), respectively.

CONCLUSIONS

Although this meta-analysis was limited by small studies and varying levels of immunosuppression, it is apparent that adding LAM to HBIG improved HBV-related morbidity and mortality in HBsAg+ recipients of liver transplants.

Selectins (E- and P-selectin) and other endothelial expressed leukocyte adhesion molecules (ELAMs) are potential targets for site-specific delivery of therapeutics to the vascular endothelium due to their specific and highly regulated expression in vascular disease. It was recently shown that degradable microspheres coated with antibodies against E-selectin or other ELAMs can target inflammation in vivo. However, targeting ELAMs alone cannot differentiate between normal and diseased state, as a basal level of these LAMs are expressed on endothelium in healthy tissues. Furthermore, leukocytes usually employ two separate adhesion molecules in parallel to home to diseased tissues, and we recently quantified the advantages of a two-receptor display for the targeting of leukocyte mimetics (Eniola AO, Willcox PJ, Hammer DA. Interplay between rolling and firm adhesion elucidated with a cell-free system engineered with two distinct receptor-ligand pairs. Biophys J 2003;85:2720-31). Here, we describe a leukocyte mimetic for targeting therapeutics to the vasculature in inflammatory diseases via two receptors, selectin and intercellular cell adhesion molecule-1 (ICAM-1), where biodegradable, polymer microspheres were co-functionalized with the selectin ligand, sialyl Lewis(X) (sLe(X)), and an antibody against ICAM-1, anti-ICAM-1 (aICAM-1). These two-receptor targeted particles, at given ratios of sLe(X)/aICAM-1, firmly adhere to substrate surface in flow only when both targeting ligands can interact with their respective receptors, mimicking the multi-step in vivo leukocyte adhesion in inflammation. Thus, we have faithfully recreated the specificity and extent of leukocyte adhesion in a platform that can allow for local delivery of therapeutics.

Highly-regulated signals surrounding stem cells, such as growth factors at specific concentrations and matrix mechanical stiffness, have been implicated in modulating stem cell proliferation and maturation. However, tight control of proliferation and lineage commitment signals is rarely achieved during growth outside the body, since the spectrum of biochemical and mechanical signals that govern stem cell renewal and maturation are not fully understood. Therefore, stem cell control can potentially be enhanced through the development of material platforms that more precisely orchestrate signal presentation to stem cells. Using a biomimetic interfacial interpenetrating polymer network (IPN), we define a robust synthetic and highly-defined platform for the culture of adult neural stem cells. IPNs modified with two cell-binding ligands, CGGNGEPRGDTYRAY from bone sialoprotein [bsp-RGD(15)] and CSRARKQAASIKVAVSADR from laminin [lam-IKVAV(19)], were assayed for their ability to regulate self-renewal and differentiation in a dose-dependent manner. IPNs with >5.3 pmol/cm(2) bsp-RGD(15) supported both self-renewal and differentiation, whereas IPNs with lam-IKVAV(19) failed to support stem cell adhesion and did not influence differentiation. The IPN platform is highly tunable to probe stem cell signal transduction mechanisms and to control stem cell behavior in vitro.

The TSC1 and TSC2 proteins, which function as a TSC1/TSC2 tumor suppressor complex, are associated with lymphangioleiomyomatosis (LAM), a genetic disorder characterized by the abnormal growth of smooth muscle-like cells in the lungs. The precise molecular mechanisms that modulate LAM cell growth remain unknown. We demonstrate that TSC2 regulates LAM cell growth. Cells dissociated from LAM nodules from the lungs of five different patients with LAM have constitutively activated S6K1, hyperphosphorylated ribosomal protein S6, activated Erk, and increased DNA synthesis compared with normal cells from the same patients. These effects were augmented by PDGF stimulation. Akt activity was unchanged in LAM cells. Rapamycin, a specific S6K1 inhibitor, abolished increased LAM cell growth. The full-length TSC2 was necessary for inhibition of S6 hyperphosphorylation and DNA synthesis in LAM cells, as demonstrated by co-microinjection of the C-terminus, which contains the GTPase activating protein homology domain, and the N-terminus, which binds TSC1. Our data demonstrate that increased LAM cell growth is associated with constitutive S6K1 activation, which is extinguishable by TSC2 expression. Loss of TSC2 GAP activity or disruption of the TSC1/TSC2 complex dysregulates S6K1 activation, which leads to abnormal cell proliferation associated with LAM disease.

Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, characterized by proliferation of abnormal smooth muscle-like cells (LAM cells) that can metastasize, leading to the formation of lung cysts, fluid-filled cystic structures in the axial lymphatics (e.g., lymphangioleiomyomas), and angiomyolipomas, benign tumors usually involving the kidneys, comprising LAM cells and adipocytes, intermixed with incompletely developed vascular structures. LAM occurs sporadically or in association with tuberous sclerosis complex, an autosomal dominant syndrome characterized by hamartoma-like tumor growths. LAM may present with progressive dyspnea, recurrent pneumothorax, chylothorax, or abdominal hemorrhage. Computed tomography scans show thin-walled cysts scattered throughout the lungs, abdominal angiomyolipomas, and lymphangioleiomyomas. Pulmonary function tests show reduced flow rates (FEV(1)) and diffusion capacity (DL(CO)). Exercise testing may reveal gas exchange abnormalities, ventilatory limitation, and hypoxemia, which can occur with near-normal lung function. Methods used to grade the severity of disease are the LAM histology score, semiquantitative and quantitative computer tomography, pulmonary function testing, and cardiopulmonary exercise testing. Currently, progression of disease is best assessed by serial measurements of FEV(1), DL(CO), and exercise performance. New quantitative radiographic techniques that may offer advantages over physiologic testing are now available. Several potential biomarkers, such as LAM cells in peripheral blood, urine, and chyle and chemokines, vascular endothelial growth factors, and matrix metalloproteinases, may be useful as diagnostic tools or markers of organ involvement, disease severity, and progression.

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M(r) of its subunit was 77,000. The cells converted [(14)C]-l-phenylalanine into [(14)C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M(r) 77, 137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology.

Pseudomonas aeruginosa coexpresses two distinct lipopolysaccharide (LPS) molecules known as A band and B band. B band is the serospecific LPS, while A band is the common LPS antigen composed of a D-rhamnose O-polysaccharide region. An operon containing eight genes responsible for A-band polysaccharide biosynthesis and export has recently been identified and characterized (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, unpublished data; H. L. Rocchetta, J. C. Pacan, and J. S. Lam, unpublished data). In this study, we report the characterization of two genes within the cluster, designated wzm and wzt. The Wzm and Wzt proteins have predicted sizes of 29.5 and 47.2 kDa, respectively, and are homologous to a number of proteins that comprise ABC (ATP-binding cassette) transport systems. Wzm is an integral membrane protein with six potential membrane-spanning domains, while Wzt is an ATP-binding protein containing a highly conserved ATP-binding motif. Chromosomal wzm and wzt mutants were generated by using a gene replacement strategy in P. aeruginosa PAO1 (serotype 05). Western blot analysis and immunoelectron microscopy using A-band- and B-band-specific monoclonal antibodies demonstrated that the wzm and wzt mutants were able to synthesize A-band polysaccharide, although transport of the polymer to the cell surface was inhibited. The inability of the polymer to cross the inner membrane resulted in the accumulation of cytoplasmic A-band polysaccharide. This A-band polysaccharide is likely linked to a carrier lipid molecule with a phenol-labile linkage. Chromosomal mutations in wzm and wzt were found to have no effect on B-band LPS synthesis. Rather, immunoelectron microscopy revealed that the presence of A-band LPS may influence the arrangement of B-band LPS on the cell surface. These results demonstrate that A-band and B-band O-antigen assembly processes follow two distinct pathways, with the former requiring an ABC transport system for cell surface expression.

A quick, simple and reliable method of extracting DNA from sweetpotato (Ipomoea batatas (L.) Lam.) has been developed. The method was applied successfully for extraction of total DNA from leaves and total RNA from leaves and various tissues. The yield of DNA extracted by this procedure was high (about 1 mg/g leaf tissue). The extracted DNA was completely digested by restriction endonucleases indicating the absence of common contaminating compounds. The absorbancy ratios of A260/A230 and A260/A280 of isolated RNA were approx. 2 and the yield was about 0.2 mg/g fresh wt. CIPK and tublin genes were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total DNA and RNA isolated by this method was of sufficient quality for subsequent molecular analysis.

BACKGROUND

Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages.

RESULTS

We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAMLAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission.

CONCLUSIONS

Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.

Drug resistance among children with culture-confirmed tuberculosis (TB) provides an accurate measure of transmitted drug resistance within the community. We describe the genotype diversity in children with culture-confirmed TB and investigate the relationship between genotype and drug resistance. A prospective study was conducted from March 2003 through August 2005 at Tygerberg Children's Hospital, in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed TB were included. Genotype analysis and phenotypic drug susceptibility testing were performed on the first culture-positive isolate from each patient. Mutation analysis was performed on all drug-resistant isolates. Spoligotyping was successfully performed on isolates from 391/399 (98%) children diagnosed with culture-confirmed TB. Drug susceptibility testing was also performed on 391 isolates; 49 (12.5%) were resistant to isoniazid, and 20 (5.1%) of these were resistant to both isoniazid and rifampin. Beijing was the most common genotype family, identified in 130/391 (33.2%) cases, followed by LAM in 114/391 (29.2%) cases. The presence of both Beijing and Haarlem genotype families was significantly associated with drug resistance (26/49 [53.1%] versus 113/342 [33.0%]; odds ratio, 1.7; 95% confidence interval, 1.0 to 2.9). The high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrates their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.

BACKGROUND

The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood.

OBJECTIVE

The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM.

METHODS

In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness.

METHODS

The study was conducted at an academic medical center.

METHODS

Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007).

METHODS

LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline.

METHODS

Activity of MMP-2 and invasiveness of LAMD-SM cells were measured.

RESULTS

LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs.

CONCLUSIONS

The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with alpha1-->2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc(2)155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell.

BACKGROUND

Acokanthera oppositifolia Lam (family: Apocynaceae) is a shrub or small tree with white latex, and the leaves of this plant are used in the form of a snuff to treat headaches and in infusions for abdominal pains and convulsions and septicaemia. Adenia gummifera Harv of the family Passifloraceae is a distinctive woody climber whose infusions are used as emetics and are said to help with some forms of depression. Lipid peroxidation has gained more importance today because of its involvement in pathogenesis of many diseases. Free radicals are the main agents in lipid peroxidation. Antioxidants thus play an important role of protecting the human body against damage by the free radicals. Plants containing phenolic compounds have been reported to possess strong antioxidant properties.

METHODS

The antioxidant activities and phenolic contents of the methanol extracts of the stems of Acokanthera oppositifolia and Adenia gummifera were evaluated using in vitro standard procedures. Spectrophotometry was the basis for the determinations of total phenol, total flavonoids, flavonols, and proanthocyanidins. Tannins, quercetin and catechin equivalents were used for these parameters. The antioxidant activities of the stem extract of Acokanthera oppositifolia were determined by the 2,2'-azinobis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and ferrous reducing antioxidant property (FRAP) methods.

RESULTS

The results from this study showed that the antioxidant activities of the stem extract of Acokanthera oppositifolia as determined by the 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and ferrous reducing antioxidant property (FRAP) methods, were higher than that of Adenia gummifera. The levels of total phenols and flavonols for A. oppositifolia were also higher. On the other hand, the stem extract of Adenia gummifera had higher level of total flavonoids and proanthocyanidins than that of Acokanthera oppositifolia. The 2, 2'-azinobis-3- ethylbenzothiazoline-6-sulfonic acid (ABTS) activities of the 2 plant extracts were similar and comparable to that of BHT.

CONCLUSIONS

Thus, the present results indicate clearly that the extracts of Acokanthera oppositifolia and Adenia gummifera possess antioxidant properties and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants. This study has to some extent validated the medicinal potential of the stems of Acokanthera oppositifolia and Adenia gummifera.

The domestication history of eggplant (Solanum melongena L.) has long been debated, with studies unable to narrow down where domestication occurred within a broad region of tropical Asia. The most commonly hypothesized region is India, however China has an equally old written record of eggplant use dating ca. 2000 years before present. Both regions have a high diversity of landraces and populations of putatively wild eggplant: Solanum incanum L. in India and Solanum undatum Lam. in SE Asia. An additional complication is that there is taxonomic confusion regarding the two candidate progenitors. Here, we synthesize historic, morphologic, and molecular data (nrITS sequence and AFLP) to interpret the phylogeographic relationships among candidate progenitors and Asian eggplant landraces in order to test theories of domestication. A minimum of two domestication events is supported: one in India and one in southern China/SE Asia. Results also support separate domestication of S. melongena subsp. ovigerum, a group of morphologically distinct eggplants found in SE Asia, and suggest Asian S. incanum and S. undatum may not be genetically distinct. Routes of the spread of eggplant cultivation throughout Asia are proposed, and evolutionary relationships among allied species are discussed.