Tenofovir is a new effective treatment option for patients with chronic hepatitis B, but could be potentially hampered by mutations in the hepatitis B virus (HBV) polymerase conferring drug resistance. Drug resistance may occur preferentially if long-term administration is required, for example, in patients with hepatitis B e antigen (HBeAg)-negative HBV infection bearing precore (PC) and basal core promoter (BCP) mutations. The rtA194T polymerase mutation has been found in HBV/HIV coinfected patients during tenofovir treatment and may be associated with tenofovir resistance. We generated replication-competent HBV constructs harboring rtA194T alone or in addition to lamivudine (LAM) resistance (rt180M + rtM204V), PC mutations, and BCP mutations and assessed their replicative capacity after transient transfection in human hepatoma cells. The rtA194T polymerase mutation alone or in conjunction with LAM resistance reduced the replication efficiency as compared with wild-type (WT) HBV. In contrast, combination of rtA194T (+/- LAM resistance) with HBeAg-negative PC or BCP mutants increased the replication capacity of the drug-resistant polymerase mutants, thereby restoring the viral replication to similar levels as WT clones. Clones harboring rtA194T showed partial resistance to tenofovir in vitro and also to LAM but remained susceptible to telbivudine and entecavir.

CONCLUSIONS

The rtA194T polymerase mutation is associated with partial tenofovir drug resistance and negatively impacts replication competence of HBV constructs. Viral replication, however, can be restored to WT levels, if these polymerase mutations occur together with precore or basic core promoter substitutions as found in HBeAg-negative hepatitis B. Patients with HBeAg-negative chronic HBV infection may therefore be at particular risk when developing drug resistance to tenofovir. Telbivudine or entecavir should be considered as effective alternative treatment options for these patients.

The nucleolus is a plurifunctional, nuclear organelle, which is responsible for ribosome biogenesis and many other functions in eukaryotes, including RNA processing, viral replication and tumour suppression. Our knowledge of the human nucleolar proteome has been expanded dramatically by the two recent MS studies on isolated nucleoli from HeLa cells [Andersen, Lyon, Fox, Leung, Lam, Steen, Mann and Lamond (2002) Curr. Biol. 12, 1-11; Scherl, Coute, Deon, Calle, Kindbeiter, Sanchez, Greco, Hochstrasser and Diaz (2002) Mol. Biol. Cell 13, 4100-4109]. Nearly 400 proteins were identified within the nucleolar proteome so far in humans. Approx. 12% of the identified proteins were previously shown to be nucleolar in human cells and, as expected, nearly all of the known housekeeping proteins required for ribosome biogenesis were identified in these analyses. Surprisingly, approx. 30% represented either novel or uncharacterized proteins. This review focuses on how to apply the derived knowledge of this newly recognized nucleolar proteome, such as their amino acid/peptide composition and their homologies across species, to explore the function and dynamics of the nucleolus, and suggests ways to identify, in silico, possible functions of the novel/uncharacterized proteins and potential interaction networks within the human nucleolus, or between the nucleolus and other nuclear organelles, by drawing resources from the public domain.

We have previously reported that group V secretory phospholipase A2 (sPLA2) amplifies the action of cytosolic phospholipase A2(cPLA2) alpha in regulating eicosanoid biosynthesis by mouse peritoneal macrophages stimulated with zymosan (Satake, Y., Diaz, B. L., Balestrieri, B., Lam, B. K., Kanaoka, Y., Grusby, M. J., and Arm, J. P. (2004) J. Biol. Chem. 279, 16488-16494). To further understand the role of group V sPLA2, we studied its localization in resting mouse peritoneal macrophages before and after stimulation with zymosan and the effect of deletion of the gene encoding group V sPLA2 on phagocytosis of zymosan. We report that group V sPLA2 is present in the Golgi apparatus and recycling endosome in the juxtanuclear region of resting peritoneal macrophages. Upon ingestion of zymosan by mouse peritoneal macrophages, group V sPLA2 is recruited to the phagosome. There it co-localizes with cPLA2alpha, 5-lipoxygenase, 5-lipoxygenase-activating protein, and leukotriene C4 synthase. Using immunostaining for the cysteinyl leukotrienes in carbodiimide-fixed cells, we show, for the first time, that the phagosome is a site of cysteinyl leukotriene formation. Furthermore, peritoneal macrophages from group V sPLA2-null mice demonstrated a >50% attenuation in phagocytosis of zymosan particles, which was restored by adenoviral expression of group V sPLA2 but IIA not group sPLA2. These data demonstrate that group V sPLA2 contributes to the innate immune response both through regulation of eicosanoid generation in response to a phagocytic stimulus and also as a component of the phagocytic machinery.

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.

Tuberculosis remains a major global health problem. Laboratory diagnostic methods that allow effective, early detection of cases are central to management of tuberculosis in the individual patient and in the community. Since the 1880s, laboratory diagnosis of tuberculosis has relied primarily on microscopy and culture. However, microscopy fails to provide species- or lineage-level identification and culture-based workflows for diagnosis of tuberculosis remain complex, expensive, slow, technically demanding and poorly able to handle mixed infections. We therefore explored the potential of shotgun metagenomics, sequencing of DNA from samples without culture or target-specific amplification or capture, to detect and characterise strains from the Mycobacterium tuberculosis complex in smear-positive sputum samples obtained from The Gambia in West Africa. Eight smear- and culture-positive sputum samples were investigated using a differential-lysis protocol followed by a kit-based DNA extraction method, with sequencing performed on a benchtop sequencing instrument, the Illumina MiSeq. The number of sequence reads in each sputum-derived metagenome ranged from 989,442 to 2,818,238. The proportion of reads in each metagenome mapping against the human genome ranged from 20% to 99%. We were able to detect sequences from the M. tuberculosis complex in all eight samples, with coverage of the H37Rv reference genome ranging from 0.002X to 0.7X. By analysing the distribution of large sequence polymorphisms (deletions and the locations of the insertion element IS6110) and single nucleotide polymorphisms (SNPs), we were able to assign seven of eight metagenome-derived genomes to a species and lineage within the M. tuberculosis complex. Two metagenome-derived mycobacterial genomes were assigned to M. africanum, a species largely confined to West Africa; the others that could be assigned belonged to lineages T, H or LAM within the clade of "modern" M. tuberculosis strains. We have provided proof of principle that shotgun metagenomics can be used to detect and characterise M. tuberculosis sequences from sputum samples without culture or target-specific amplification or capture, using an accessible benchtop-sequencing platform, the Illumina MiSeq, and relatively simple DNA extraction, sequencing and bioinformatics protocols. In our hands, sputum metagenomics does not yet deliver sufficient depth of coverage to allow sequence-based sensitivity testing; it remains to be determined whether improvements in DNA extraction protocols alone can deliver this or whether culture, capture or amplification steps will be required. Nonetheless, we can foresee a tipping point when a unified automated metagenomics-based workflow might start to compete with the plethora of methods currently in use in the diagnostic microbiology laboratory.

OBJECTIVE

In settings of high HIV prevalence, tuberculosis control and patient management are hindered by lack of accurate, rapid tuberculosis diagnostic tests that can be performed at point-of-care. The Determine TB LAM Ag (TB LAM) test is a lateral flow immunochromatographic test for detection of mycobacterial lipoarabinomannan (LAM) in urine. Our objective was to determine sensitivity and specificity of the TB LAM test for tuberculosis diagnosis.

METHODS

Prospective diagnostic accuracy study.

METHODS

Hospital and outpatient settings in Uganda and South Africa.

METHODS

HIV-infected adults with tuberculosis symptoms and/or signs.

METHODS

Participants provided a fresh urine specimen for TB LAM testing, blood for mycobacterial culture, and 2 respiratory specimens for smear microscopy and mycobacterial culture.

METHODS

For the TB LAM test, sensitivity in participants with culture-positive tuberculosis and specificity in participants without tuberculosis.

RESULTS

A total of 1013 participants were enrolled. Among culture-positive tuberculosis patients, the TB LAM test identified 136/367 (37.1%) overall and 116/196 (59.2%) in the group with CD4 ≤100 cells per cubic millimeter. The test was specific in 559/573 (97.6%) patients without tuberculosis. Sensitivity of the urine TB LAM test plus sputum smear microscopy was 197/367 (53.7%) overall and 133/196 (67.9%) among those with CD4 ≤100. CD4 ≤50 [adjusted odds ratio (AOR), 6.2; P < 0.001] or 51-100 (AOR, 7.1; P < 0.001), mycobacteremia (AOR, 6.1; P < 0.01) and hospitalization (AOR, 2.6; P = 0.03) were independently associated with a positive TB LAM test.

CONCLUSIONS

In HIV-positive adults with CD4 ≤100, the TB LAM urine test detected over half of culture-positive tuberculosis patients, in <30 minutes and without the need for equipment or reagents.

Serum immunoglobulin (Ig) G responses to a variety of mycobacterial antigens were measured in children from the UK, in children with tuberculosis from Hyderabad, India and Dhaka, Bangladesh, classified according to whether the disease was disseminated or localized, and in non-tuberculous controls. Anti-lipoarabinomannan (LAM) IgG responses in UK children showed a marked trough between 6 months and 3 years coincident with the reported peak incidence of disseminated tuberculosis. Geometric mean IgG responses to sonicates of slow-growing mycobacteria (rich in LAM) in 36 children with disseminated tuberculosis were markedly lower than in 99 children with localized tuberculous lesions (for Mycobacterium scrofulaceum P < 0.01, for M. tuberculosis P < 0.01, and for M. vaccae P < 0.01). Responses to purified LAM were also lower in the disseminated tuberculosis group (P < 0.05) but there was no difference between the groups in their response to mycobacterial 65 kDa protein. Multiple regression analysis showed that the reduced response to sonicated mycobacterial antigens and to LAM in children with disseminated disease was independent of age, nutritional status, skin test reactivity, duration of previous symptoms, and city of origin. There was no evidence for sequestration of antibody to immune complexes. These findings are compatible with the hypothesis that children with low levels of antibody to sonicated mycobacterial antigen and to LAM, or those who cannot mount an antibody response, are predisposed to dissemination. A role for antibody in preventing disseminated forms of tuberculosis in childhood has implications for the development of improved vaccines and for the optimum timing of vaccination with bacille Calmette-Guérin.

OBJECTIVE

A low-cost point-of-care urine assay for lipoarabinomannan (LAM) used for screening patients prior to antiretroviral therapy (ART) rapidly diagnoses a proportion of tuberculosis (TB) cases. We determined the characteristics and outcomes of such patients.

METHODS

Adults enrolling in a South African township ART clinic were systematically screened for pulmonary TB by testing paired sputum samples using microscopy, liquid culture and Xpert MTB/RIF in a centralized laboratory. Stored urine samples were retrospectively tested for LAM using the Determine TB-LAM assay, but results did not inform treatment. Patients were followed up in the routine ART service and early (90-day) programmatic outcomes were determined. Analysis was restricted to those with CD4 cell counts below 200 cells/μl.

RESULTS

Of patients with CD4 cell counts below 200 cells/μl and complete results (n=325), 59 (18.2%) had culture-positive TB. Of these, 23 (39%) patients tested urine LAM-positive and 36 (61%) urine LAM-negative. Patients with LAM-positive TB had much lower CD4 cell counts, higher plasma viral loads, lower haemoglobin concentrations and lower BMIs compared to those with LAM-negative TB. They also had evidence of higher mycobacterial load, more frequently testing sputum smear-positive, Xpert-positive (sputum and urine) and having a shorter time to sputum culture positivity. Of five (8.5%) patients who died, four did so before TB treatment was started. All five retrospectively tested LAM-positive.

CONCLUSIONS

A low-cost point-of-care urine test for LAM rapidly diagnoses a sub-group of cases with advanced HIV-associated TB and poor prognosis. If used in combination with laboratory-based diagnostics, treatment delays would decrease and survival might be improved.

L-selectin (also termed LAM-1, Leu-8,TQ-1, gp90MEL-14, peripheral lymph node homing receptor and LECAM-1) is an adhesion protein thought to be important in leukocyte entry into lymphoid tissues and sites of inflammation. We, as well as others, have shown that leukocyte activation by chemotactic factors results in rapid shedding (release) of L-selectin from the cell surface. Here we have used flow cytometry, enzyme-linked immunosorbent assay, and SDS-PAGE/Western blot analysis to determine whether cross-linking of L-selectin in the absence of activation causes shedding. We found that rapid loss of leukocyte L-selectin expression (down-regulation) could be induced by treating cells with a chemical cross-linker [bis (sulfosuccinimidyl) suberate]. L-selectin down-regulation via cross-linking could occur at 4 degrees C and in the absence of detectable cell activation (increased expression of CD11b/18). The loss of L-selectin expression was due to shedding of the molecule from the leukocyte cell surface. Cross-linking of L-selectin with specific monoclonal antibodies also caused loss of surface expression of L-selectin at 37 degrees C. Finally, shed L-selectin was detected in the plasma of healthy adults whose peripheral blood leukocytes demonstrated no obvious signs of activation. Our results suggest that activation-independent shedding of leukocyte L-selectin may occur in vivo and a possible mechanism could involve cross-linking of leukocyte L-selectin. This provides a novel mechanism for rapid regulation of expression of a leukocyte-endothelial cell adhesion receptor.

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.

Lipoarabinomannan (LAM) is a major structural surface component of mycobacteria. Arabinomannan (AM) oligosaccharides derived from LAM of Mycobacterium tuberculosis H37Rv were isolated and covalently conjugated to tetanus toxoid (TT) or to short-term culture filtrate proteins (antigen 85B (Ag85B) or a 75kDa protein) from M. tuberculosis strain Harlingen. The different AM oligosaccharide (AMOs)-protein conjugate vaccine candidates proved to be highly immunogenic, inducing boosterable IgG responses against the AMOs portion of the conjugates in rabbits and guinea-pigs. Proliferation of T-cells from C57BL/6 mice immunized with the conjugates was seen upon in vitro stimulation with PPD. In C57BL/6 mice subcutaneous immunization with the AMOs-antigen 85B conjugate in alum provided significant protection compared to sham (alum only) immunized mice (P < 0.021) as estimated by long term survival against intravenous challenge with 10(5) M. tuberculosis H37Rv. Subcutaneous immunization followed by nasal boost with an AMOs-TT conjugate in Eurocine L3 adjuvant provided high (P < 0.025) protection as determined by long term survival after intranasal challenge with 10(5) virulent M. tuberculosis strain Harlingen. This level of protection was comparable to that obtained with the conventional live attenuated BCG vaccine. In guinea-pigs, immunization with AMOs-Ag85B in Eurocine L3 adjuvant followed by aerogenic challenge with M. tuberculosis H37Rv resulted in increased survival and reduced pathology in lungs and spleens relative to non-immunized animals.

High rates and transmission of multidrug-resistant (MDR) tuberculosis (TB) have been associated with the Mycobacterium tuberculosis complex (MTBC) Beijing lineage, pointing to the importance of pathogen genetic factors for the modulation of infection outcome and epidemiology. We present here an in-depth analysis of the population structure of MTBC strains from the Republic of Georgia, a high-incidence setting at the Black Sea Coast. Phylogenetic lineages were identified based on 24-locus MIRU-VNTR (for mycobacterial interspersed repetitive unit-variable number tandem repeat) and spoligotyping analysis. Clusters of strains with identical genotyping profiles were determined as an indicator for the rate of recent transmission. Among the 183 M. tuberculosis isolates investigated, the most prominent lineage found was Beijing (26%), followed by the LAM (18%), Ural (12%), and Haarlem (5%) strains. A closely related previously undefined phylogenetic group (62 strains) showed a genotyping pattern similar to laboratory strain H37RV and was denominated as "Georgia-H37RV-like." Although isoniazid resistance was found among strains of different lineages, MDR TB was nearly completely restricted to Beijing strains (P < 0.0001). Approximately 50% of the isolates were grouped in clusters, indicating a high rate of recent transmission. Our data indicate that, in addition to the confirmation of the importance of Beijing genotype strains for the TB epidemiology in former Soviet Union countries, a high-population diversity with strains of the LAM, Ural, Haarlem, and a previously undefined lineage represents nearly two-thirds of the strains found in Georgia. Higher rates among previously treated and MDR TB patients point to a higher potential of lineage Beijing to escape therapy and develop MDR TB.

Efforts in prevention and control of tuberculosis suffer from the lack of detailed knowledge of the mechanisms used by pathogenic mycobacteria for survival within host cell macrophages. The exploitation of host cell signaling pathways to the benefit of the pathogen is a phenomenon that deserves to be looked into in detail. We have tested the hypothesis that lipoarabinomannan (LAM) from the virulent species of Mycobacterium tuberculosis possesses the ability to modulate signaling pathways linked to cell survival. The Bcl-2 family member Bad is a proapoptotic protein. Phosphorylation of Bad promotes cell survival in many cell types. We demonstrate that man-LAM stimulates Bad phosphorylation in a phosphatidylinositol 3-kinase (PI-3K)-dependent pathway in THP-1 cells. Man-LAM activated PI-3K. LAM-stimulated phosphorylation of Bad was abrogated in cells transfected with a dominant-negative mutant of PI-3K (Delta p85), indicating that activation of PI-3K is sufficient to trigger phosphorylation of Bad by LAM. Since phosphorylation of Bad occurred at serine 136, the target of the serine/threonine kinase Akt, the effect of LAM on Akt kinase activity was tested. Man-LAM could activate Akt as evidenced from phosphorylation of Akt at Thr(308) and by the phosphorylation of the exogenous substrate histone 2B. Akt activation was abrogated in cells transfected with Deltap85. The phosphorylation of Bad by man-LAM was abrogated in cells transfected with a kinase-dead mutant of Akt. These results establish that LAM-mediated Bad phosphorylation occurs in a PI-3K/Akt-dependent manner. It is therefore the first demonstration of the ability of a mycobacterial virulence factor to up-regulate a signaling pathway involved in cell survival. This is likely to be one of a number of virulence-associated mechanisms by which bacilli control host cell apoptosis.

In previous work we found loss of heterozygosity (LOH) of the wild-type TSC2 allele in the abnormal pulmonary smooth muscle cells and renal angiomyolipoma cells from patients with sporadic pulmonary lymphangiomyomatosis (LAM). Here we report TSC2 LOH in microdissected pulmonary LAM cells from a patient with tuberous sclerosis complex (TSC), demonstrating for the first time that the two-hit tumor suppressor gene model applies to the TSC-associated, as well as sporadic LAM. We also compared the chromosome 16 LOH region between angiomyolipoma and pulmonary LAM from two patients with sporadic LAM. Previously we found that these patients had TSC2 mutations and TSC2 LOH in their angiomyolipomas and pulmonary LAM cells but not in normal lung or kidney cells. This suggests that pulmonary LAM may result from the migration of smooth muscle cells from renal angiomyolipomas to the lung. In this case, one would predict that the angiomyolipoma and LAM cells would have identical LOH patterns. We found that at each chromosome 16 marker, the results were concordant between angiomyolipoma and LAM. This is consistent with a model in which pulmonary LAM cells and angiomyolipoma cells have a common genetic origin.

In the dentate gyrus of adult female meadow voles, a high dose of estradiol benzoate (EB) increases (within 4 h) then decreases (within 48) the number of dividing progenitor cells (Ormerod BK, Galea LAM. 2001. Reproductive status regulates cell proliferation within the dentate gyrus of the adult female meadow vole: A possible regulatory role for estradiol. Neurosci 2:169-179). We investigated whether time-dependent EB exposure differentially influences the number of new granule cells produced in the adult female rat dentate gyrus and whether EB-stimulated adrenal activity mediates the decrease in cell proliferation. Ovariectomized rats received either an EB (10 microg in 0.1 mL) or vehicle (0.1 mL) injection either 4 or 48 h (Experiment 1) before a BrdU injection (200 mg/kg) and were perfused 24 h later to assess the number of new cells. Relative to vehicle, the number of new cells increased following a 4 h exposure (p < or = 0.04) but decreased following a 48 h exposure (p < or = 0.006) to EB. In Experiment 2, the number of new cells within the dentate gyrus of ovariectomized and adrenalectomized females did not significantly differ between groups exposed to EB versus vehicle for 48 h prior to BrdU administration, suggesting the decreased number of new cells observed within the dentate gyrus of adrenal-intact adult female rats is mediated by EB-stimulated adrenal activity. We conclude that estradiol dynamically regulates cell proliferation within the dentate gyrus of adult female rats in the time-dependent manner observed previously in voles and suppresses cell proliferation by influencing adrenal steroids. Investigating how estradiol dynamically regulates neurogenesis could provide insight into the mechanisms by which the proliferation of progenitor cells is controlled within the adult rodent hippocampus.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.

The combination of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogues [NA(s)] is considered as the standard of care for prophylaxis against HBV recurrence after liver transplantation (LT), but the optimal protocol is controversial. We evaluated the efficacy of the newer NAs with high genetic barrier (hgbNA) [i.e. entecavir (ETV) or tenofovir (TDF)] with or without HBIG as prophylaxis against HBV recurrence after LT. In total, 519 HBV liver transplant recipients from 17 studies met the inclusion criteria and they were compared to those under lamivudine (LAM) and HBIG who had been selected in our previous review. Patients under HBIG and LAM developed HBV recurrence (115/1889 or 6.1%): (a) significantly more frequently compared to patients under HBIG and a hgbNA [1.0% (3/303), p < 0.001], and (b) numerically but not significantly more frequently compared to the patients who received a newer NA after discontinuation of HBIG [3.9% (4/102), p = 0.52]. The use of a hgbNA without any HBIG offered similar antiviral prophylaxis compared to HBIG and LAM combination, if the definition of HBV recurrence was based on HBV DNA detectability [0.9% vs. 3.8%, p = 0.11]. Our findings favor the use of HBIG and a hgbNA instead of HBIG and LAM combined prophylaxis against HBV recurrence after LT.

Natural killer (NK) cells keep viral infections under control at the early phase by directly killing infected cells. Influenza is an acute contagious respiratory viral disease transmitted from host-to-host in the first few days of infection. The evasion of host innate immune defenses including NK cells is important for its success as a viral pathogen of humans and animals. NK cells encounter influenza virus within the microenvironment of infected cells. It therefore is important to investigate the direct effects of influenza virus on NK cell activity. Recently we demonstrated that influenza virus directly infects human NK cells and induces cell apoptosis to counter their function (H. Mao, W. Tu, G. Qin, H. K. W. Law, S. F. Sia, P.-L. Chan, Y. Liu, K.-T. Lam, J. Zheng, M. Peiris, and Y.-L. Lau, J. Virol. 83:9215-9222, 2009). Here, we further demonstrated that both the intact influenza virion and free hemagglutinin protein inhibited the cytotoxicity of fresh and interleukin-2 (IL-2)-activated primary human NK cells. Hemagglutinin bound and internalized into NK cells via the sialic acids. This interaction did not decrease NKp46 expression but caused the downregulation of the zeta chain through the lysosomal pathway, which caused the decrease of NK cell cytotoxicity mediated by NKp46 and NKp30. The underlying dysregulation of the signaling pathway involved zeta chain downregulation, leading to decreased Syk and ERK activation and granule exocytosis upon target cell stimulation, finally causing reduced cytotoxicity. These findings suggest that influenza virus developed a novel strategy to evade NK cell innate immune defense that is likely to facilitate viral transmission and also contribute to virus pathogenesis.

The studies discussed have established that inflammatory or immune cytokines, such as IL-1, TNF, and LT, as well as bacterial endotoxin, can act directly on vascular endothelial cells to modulate two important functional properties. The first of these, the inducible expression of E-LAMs, provides a mechanism for the local regulation of leukocyte-vessel wall interactions. This endothelial-dependent mechanism may be relevant to a broad spectrum of pathologic processes, including inflammation, delayed hypersensitivity reactions, and atherogenesis. The second, modulation of endothelial tissue factor PCA and fibrinolytic components, has important implications for the local balance of prothrombotic and antithrombotic influences at the blood-vessel wall interface. Thus, under the influence of inflammatory stimuli, vascular endothelial cells may actively contribute to the development and maintenance of intravascular or perivascular fibrin. Although the endothelial effector mechanisms of these functional alterations are distinct, their induction by similar stimuli points to important interrelationships of leukocyte-vessel wall adhesion and thrombosis. Further understanding of the regulation of endothelial expression of E-LAMs and coagulant properties should contribute to our understanding of vascular disease.

A combination of hepatitis B immunoglobulin (HBIG) and nucleos(t)ide analogues (NUCs) is currently recommended as prophylaxis against the recurrence of hepatitis B virus (HBV) after liver transplantation (LT), but the optimal protocol is a matter of controversy. The aim of this study was the identification of factors associated with post-LT HBV recurrence in patients receiving HBIG and NUCs. We searched MEDLINE and PubMed for studies in English about the effectiveness of HBIG and NUCs [lamivudine (LAM) and/or adefovir dipivoxil (ADV)] against post-LT HBV recurrence (January 1998 to June 2010). Forty-six studies, which included 2162 HBV LT recipients, met the selection criteria. Patients receiving HBIG and LAM experienced HBV recurrence more frequently than patients receiving HBIG and ADV with or without LAM [6.1% (115/1889) versus 2.0% (3/152), P = 0.024], although they also were more frequently treated with indefinite HBIG prophylaxis (90% versus 57%, P < 0.001). For patients receiving HBIG and LAM, a lower frequency of HBV recurrence was associated with a high HBIG dosage (≥10,000 IU/day) versus a low HBIG dosage (<10,000 IU/day) during the first week after LT [3.2% (14/440) versus 6.5% (80/1233), P = 0.016], but the HBIG protocol had no impact on HBV recurrence in patients receiving HBIG and ADV. In conclusion, in comparison with the combination of HBIG and LAM, the combination of HBIG and ADV is associated with a lower rate of HBV recurrence after LT. Patients receiving HBIG and LAM should be given a high dosage of HBIG during the first week after LT, but a lower dosage can be used safely in patients receiving HBIG and ADV. Further studies with newer and more potent anti-HBV agents are definitely required.

We have investigated the expression pattern conferred by a cis-regulatory element (-212 to -154) from the upstream region of the octopine synthase (ocs) gene in transgenic tobacco plants. Analysis of beta-glucuronidase expression driven by the ocs regulatory element revealed a pattern that is tissue-specific and developmentally regulated. In young seedlings, expression is confined primarily to root tips. In older seedlings, expression is stronger and becomes apparent also in the shoot apex. Insertion of a 16-base pair palindromic sequence (-193 to -178), which is included in the regulatory element, into an rbcS promoter results in the expression of rbcS in roots. The 16-base pair palindrome binds activation sequence factor (ASF)-1, a factor from tobacco nuclear extracts that interacts with the sequence between -83 to -63, designated as activation sequence (as)-1, of the cauliflower mosaic virus 35S promoter [Lam et al. (1989). Proc. Natl. Acad. Sci. USA 86, in press]. The in vivo expression patterns and in vitro binding properties of the ocs palindromic sequence are remarkably similar to those of the as-1 element of the cauliflower mosaic virus 35S promoter. These results suggest the involvement of ASF-1 in the transcriptional regulation of the ocs promoter and the 35S promoter.

OBJECTIVE

To describe the abdominal computed tomographic (CT) and ultrasonographic (US) findings in patients with thoracic lymphangioleiomyomatosis (LAM) and to relate the prevalence of the findings to the severity of pulmonary disease.

METHODS

Eighty patients with LAM underwent chest and abdominopelvic CT and abdominopelvic US. The images were reviewed prospectively by one radiologist, and the abdominal findings were recorded and correlated with the severity of pulmonary disease at thin-section CT.

RESULTS

Sixty-one (76%) of 80 patients had positive abdominal findings. The most common abdominal findings included renal angiomyolipoma (AML) in 43 patients (54%), enlarged abdominal lymph nodes in 31 (39%), and lymphangiomyoma in 13 (16%). Less common findings included ascites in eight (10%), dilatation of the thoracic duct in seven (9%), and hepatic AML in three (4%). A significant correlation (P =.02) was observed between enlarged abdominal lymph nodes and increased severity of lung disease.

CONCLUSIONS

There are characteristic abdominal findings in patients with LAM that, in conjunction with the classic thin-section CT finding of pulmonary cysts, are useful in establishing this diagnosis.

Moringa oleifera is used in Thai traditional medicine as cardiotonic. Recent studies demonstrated its hypocholesterolaemic effect. However, to be clinically useful, more scientific data are needed.

OBJECTIVE

We investigated the antioxidant, hypolipidaemic and antiatherosclerotic activities of Moringa oleifera leaf extract.

METHODS

Scavenging activity of the extract on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), and the inhibitory effect on Cu(2+)-induced low-density lipoprotein (LDL) oxidation were determined in in vitro experiment. The effects of the extract on cholesterol levels, conjugated diene (CD) and thiobarbituric acid reactive substances (TBARS) and plaque formations in cholesterol-fed rabbits were investigated.

RESULTS

We found that in scavenging DPPH radicals the extract and Trolox had IC(50) of 78.15+/-0.92 and 2.14+/-0.12microg/ml, respectively. The extract significantly (P<0.05) prolonged the lag-time of CD formation and inhibited TBARS formation in both in vitro and ex vivo experiments in a dose-dependent manner. In hypercholesterol-fed rabbits, at 12 weeks of treatment, it significantly (P<0.05) lowered the cholesterol levels and reduced the atherosclerotic plaque formation to about 50 and 86%, respectively. These effects were at degrees comparable to those of simvastatin.

CONCLUSIONS

The results indicate that this plant possesses antioxidant, hypolipidaemic and antiatherosclerotic activities and has therapeutic potential for the prevention of cardiovascular diseases.

Basic fibroblast growth factor (bFGF) is a potent mitogenic factor for smooth muscle cells, myofibroblasts, and fibroblasts, proliferation of which is a hallmark of idiopathic pulmonary fibrosis (IPF) and lymphangioleiomyomatosis (LAM). Mast cells produce bFGF and have been associated with pulmonary fibrosis. We hypothesize that smooth muscle cell/myofibroblast-like cells will be spatially associated with bFGF-containing mast cells and that bFGF receptors will be expressed on the effector cells in IPF and LAM. We performed quantitative immunohistochemistry for bFGF, mast cell tryptase, smooth muscle actin for smooth muscle cell/myofibroblast-like cells, and fibroblast growth factor receptors (Flg, Bek) and measured collagen and elastic fiber in lung sections from IPF (n = 14), LAM (n = 9), and control lung (n = 10). IPF and LAM lung contained more smooth muscle cell/myofibroblast-like cells than did control lung. bFGF-containing mast cells were abundant both in IPF and LAM and were associated with collagen, elastic fibers, and smooth muscle cell/myofibroblast-like cells in IPF. Flg was expressed on epithelial cells, endothelial cells, smooth muscle cell/myofibroblast-like cells, and macrophages in IPF. In LAM, Flg was expressed on epithelial cells adjacent to smooth muscle cell/myofibroblast-like cell aggregates. Bek was expressed dominantly on smooth muscle cell/myofibroblast-like cells in LAM and on smooth muscle cell/myofibroblast-like cells as well as neutrophils in IPF. These data suggest that mast cell-derived bFGF might exert fibrogenic, proliferative effects on smooth muscle cell/myofibroblast-like cells through its receptors.