In an extensive ethnobotanical survey (130 informants) of the medicinal plants of Israel, 16 species were found to be used for hypoglycaemic treatments. The list includes Achillea fragrantissima (Forssk.) Sch.-Bip, Ammi visnaga (L.) Lam, Atriplex halimus L., Capparis spinosa L., Ceratonia siliqua L., Cleome droserifolia (Forssk.) Del., Eryngium creticum Lam., Inula viscosa (L.) Ait., Matricaria aurea (Loefl.) Sch.-Bip, Origanum syriaca L., Paronychia argentea Lam, Prosopis farcta (Banks et Sol.) Macbride, Salvia fruticosa Mill., Sarcopoterium spinosum (L.) Sp., and Teucrium polium L.; eight of them (marked with an asterisk) are first recorded here as used for this purpose.

Glioblastoma multiform is the most common and aggressive type of brain tumor. The overexpression of ecto-5'-nucleotidase/CD73 (ecto-5'-NT/CD73), an adhesion molecule and the main enzymatic source of extracellular adenosine, has been reported in tumor cells, and it is emerging as a component of glioma progression. Here, we evaluated the involvement of ecto-5'-NT/CD73 in cell adhesion through its interaction with different components of the extracellular matrix in the human U138MG glioma cell line. The results indicated that adenosine induced an increase in glioma cell adhesion. The treatment of glioma cells with adenosine receptor antagonists, APCP (α,β-methylene ADP) and dipyridamole prevented the adenosine effect, indicating the participation of extracellular and intracellular signaling pathways in cell adhesion mediated by adenosine. The ECM protein laminin (lam) and chondroitin sulfate (ChS) modulated the ecto-5'-NT/CD73 activity and glioma adhesion in a parallel manner, suggesting the involvement of purinergic signaling in the effects mediated by the extracellular matrix. Taken together, these results suggest that ecto-5'-NT/CD73, an important producer of extracellular adenosine, may modulate glioma cell adhesion and tumor cell-extracellular matrix interactions.

BACKGROUND

The rapid increase in consumption of herbal remedies worldwide has been stimulated by several factors, including the notion that all herbal products are safe and effective. However, over the past decade, several news-catching episodes in developed communities indicated adverse effects, sometimes life-threatening, allegedly arising as a consequence to taking herbal products or traditional medicines from various ethnic groups. Despite the popular use of Moringa oleifera for treating various disorders, there is limited or no scientific data available regarding safety aspects of this remedy, nor are there any documented toxicological studies that can be used to ascertain the safety index of its herbal preparation. Therefore, this present study aimed to carry out extensive toxicological evaluation of the aqueous leaf extract of Moringa oleifera.

METHODS

In an acute toxicity test, male Wistar albino mice were orally administered an aqueous extract up to 6400 mg/kg and intraperitoneally up to 2000 mg/kg. A sub-chronic toxicity test was performed by daily administration with the extract at 250, 500 and 1500 mg/kg orally for 60 days. Control rats received distilled water. Sperm quality was analyzed, haematological and biochemical (liver enzymes, urea and creatinine) parameters were determined and a histopathological examination was carried out.

RESULTS

The LD(50) was estimated to be 1585 mg/kg. The extract did not elicit any significant difference (P≥0.05) in sperm quality, haematological and biochemical parameters in the treated rats compared to the control. Moreover, there was no significant difference in weight gain of the control and treated animals although there was a dose-dependent reduction in food consumption of the animals treated with 250 to 1500 mg/kg extract.

CONCLUSIONS

Results obtained in this study suggest that the aqueous leaf extract of Moringa oleifera is relatively safe when administered orally.

BACKGROUND

The optimal oral anti-viral agent to use in patients with decompensated HBV cirrhosis remains unclear.

OBJECTIVE

We performed a meta-analysis of the oral nucleos(t)ide analogues in patients with decompensated HBV cirrhosis.

METHODS

One year efficacy and safety outcomes in 22 studies published in English between '95 and 2010 were analysed.

RESULTS

Substantial heterogeneity was noted in the inclusion/exclusion criteria, controls, and sensitivity of the HBV DNA assay used. Pooled 1-year data showed benefit favouring lamivudine (LAM) vs. untreated controls for Child-Turcotte-Pugh (CTP) score improvement by ≥2 (OR: 117 (15 921), P ≤ 0.0001) and transplant-free survival (OR: 3.2 (1.2, 9), P = 0.022). Adefovir (ADV) led to undetectable HBV DNA at 1-year in 41% compared to 83% with LAM and 80% with entecavir (ETV). Overall, 1-year transplant-free survival rates varied from 78% with LAM to 95% and 94% with Tenofovir (TDF) and Telbivudine (TBV), respectively. The 1-year incidence of drug resistant HBV was 0% with ADV, ETV and TDF and 11% with LAM although TBV was associated with a 29% incidence at 2 years. Drug-related adverse events were infrequently reported.

CONCLUSIONS

All the oral anti-viral agents were associated with improved virological, biochemical and clinical parameters at 1-year. However, the efficacy of lamivudine and telbivudine is limited by drug resistance, and adefovir is limited by its potency and slower onset of action. Additional studies of tenofovir and entecavir are needed to determine the optimal agent(s) for treatment naïve patients and in those with drug-resistant decompensated HBV cirrhosis.

BACKGROUND

After orthotopic liver transplantation (OLT) in chronic hepatitis B (HBV), adequate prophylaxis for recurrence of HBV in the graft is mandatory.

OBJECTIVE

Evaluate safety of HBV prophylaxis with tenofovir and emtricitabine (TDF/FTC) after cessation of hepatitis B immunoglobulin (HBIG) after OLT in chronic HBV.

METHODS

In 17 consecutive patients after OLT in chronic HBV we started TDF/FTC after cessation of HBIG. All had received HBIG >6 months. 15/17 were HBsAg negative and 16/17 had undetectable HBV-DNA.

RESULTS

After mean follow-up of 2 years 16/17 patients were alive, one died due to urosepsis. All 16 with undetectable HBV-DNA remained HBV-DNA negative. From 15 HBsAg negative patients at start, in one seroconversion to positive HBsAg occurred, without detectable HBV-DNA. Liver biochemistry remained within the normal ranges. There were no cases of drug discontinuation. No major side effects were reported. TDF/FTC use saves €16,262/year over standard-of-care (HBIG+LAM). This prospective follow-up study shows that in liver transplantation for chronic hepatitis B, after initial treatment including HBIG for at least 6 months combined with or followed by (dual) nucleos(t)ide analog therapy, TDF/FTC provides adequate prophylaxis against recurrent HBV infection without major side effects and leads to substantial cost savings over a regimen with HBIG.

CONCLUSIONS

Combined prophylaxis with TDF/ETV nucleoside plus nucleotide analogs and cessation of immunoglobulin after liver transplantation in chronic hepatitis B is safe and effective.

Advances in fluorescence-activated cell sorter technology have brought about multicolor analysis of cell phenotypes. To clarify the phenotypes of human hematopoietic stem cells (HSCs), we initially prepared novel antibodies against CD34 and labeled one of them (4A1) with allophycocyanin (APC). With this, we analyzed the phenotypes of CD34+ HSCs and showed that primitive HSCs or CD34+CD33- cells expressed adhesion molecules such as CD43, CD44, CD11a, CD11c, CD18, and leukocyte adhesion molecule (LAM-1). The more primitive hematopoietic cells or CD34+CD38- cells also expressed CD11a and CD18 with an incidence of 20% to 30%. To clarify the role of adhesion molecules in HSCs, we examined the colony forming capacity after long-term culture with allogeneic irradiated stromal layers. Among CD34+CD33- cells, CD18+ cells gave rise to colony-forming cells (CFCs) on stromal layers, but reached a maximum at week 2, after which the number of generated CFCs decreased. On the other hand, CD18- cells generated less CFCs than CD18+ cells at 2 to 3 weeks, but increased after 4 weeks of culture. When CD18 or CD11a antibody was added to a coculture system of CD34+CD33- cells with stromal layers, the number of generated CFCs decreased significantly compared with the no antibody control. Leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) was expressed on some populations of hematopoietic cells and contributed to the proliferation by interacting with stromal cells. However, more primitive cells capable of reconstituting hematopoiesis did not express LFA-1. These data provide a rationale for the administration of anti-LFA-1 antibody after bone marrow transplantation for reducing the graft failure.

Lymphangioleiomyomatosis (LAM) is a multisystem disease of women, characterized by proliferation of abnormal smooth muscle-like LAM cells, leading to the formation of lung cysts, fluid-filled cystic structures in the axial lymphatics (eg, lymphangioleiomyomas), and renal angiomyolipomas. LAM is caused by mutations of the TSC1 or TSC2 genes, which encode, respectively, hamartin and tuberin, two proteins with a major role in control of the mammalian target of rapamycin (mTOR) signaling pathway. LAM occurs sporadically or in association with tuberous sclerosis complex, an autosomal-dominant syndrome characterized by widespread hamartomatous lesions. LAM may present with progressive dyspnea, recurrent pneumothorax, or chylothorax. Pulmonary function tests show reduced flow rates (forced expiratory volume in the first second) and diffusion capacity. Exercise testing may reveal gas exchange abnormalities, ventilatory limitation, and hypoxemia. The severity and progression of disease may be assessed by lung histology scores, quantification of computed tomography, pulmonary function testing, 6-minute walk tests, cardiopulmonary exercise testing, and measurement of serum vascular endothelial growth factor D levels. Sirolimus and everolimus, two mTOR inhibitors, are effective in stabilizing lung function and reducing the size of chylous effusions, lymphangioleiomyo-mas, and angiomyolipomas. However, inhibition of mTOR complex 1 increases autophagy, possibly enhancing LAM cell survival. Inhibition of autophagy with hydroxychloroquine, in combination with sirolimus, has been proposed as a possible treatment for LAM. Deficiency of tuberin results in increased RhoA GTPase activity and cell survival, an effect that is mediated through mTOR complex 2 signaling. Because sirolimus and everolimus only affect the activity of mTOR complex 1, therapies targeting RhoA GTPases with simvastatin, which inhibits Rho GTPases and promotes apoptosis, are being investigated. As in the case of cancer, LAM may be best treated with multiple drugs targeting signaling pathways considered important in the pathogenesis of disease.

Acinetobacter spp. are responsible for an increasing number of opportunistic, nosocomial infections. They have been isolated from diverse inanimate objects in the hospital environment and are resistant to most of the commonly used antibiotics. Existing media for the isolation of Acinetobacter spp. are either nonselective, allowing the growth of unwanted bacteria, or too inhibitory, inhibiting the growth of many Acinetobacter strains. For the rapid isolation and effective control of Acinetobacter infection, a new selective and differential medium, Leeds Acinetobacter Medium (LAM), has been developed to isolate Acinetobacter spp. from clinical and environmental sources. The concentration of antibiotics and other ingredients in this medium have been determined according to the results of MIC and viable counts performed for these ingredients. LAM was compared with other selective and differential media for the isolation of Acinetobacter spp. from a local hospital environment and proved to be better in terms of recovery and selectivity.

Protein phosphorylation is an important posttranslational modification process that plays a crucial role in signal transduction. There are many protein kinases involved in cell signaling. However, substrate motifs of many protein kinases in signal transduction are not well-known. Traditional methodologies for identifying these motifs are often difficult and inefficient. In the present study, we developed a novel approach for discovering linear substrate motifs of protein kinases. This method is based on the screening of random synthetic combinatorial peptide libraries on beads where each bead expresses only one peptide entity [Lam et al. (1991) Nature 354, 82-84]. We chose cyclic AMP-dependent protein kinase (cAPK) as a model system in the present study since it is a well-studied enzyme. Random pentapeptide and heptapeptide libraries were screened with the addition of [gamma-32P]ATP and cAPK. 32P-Labeled peptide-beads were then isolated for microsequencing. The identified peptide motif for cAPK was RRXS and is identical to that reported in the literature. Kinetic studies of the best three peptides indicate that they are efficient substrates for cAPK discovered from random synthetic combinatorial peptide libraries. Our results also suggest that this method is potentially useful for identifying substrate motifs of various protein kinases with high sensitivity and specificity. In addition, this method can also be used as a general method for identifying linear substrate motifs for various posttranslational modifications.

A flocculating protein from the seeds of Moringa oleifera Lam. was isolated by extraction with phosphate buffer followed by cation exchange chromatography. The molecular mass of the protein determined by SDS-PAGE was about 6.5 kDa, the isoelectric point was above pH 10. Amino acid analysis and sequencing showed high contents of glutamine, arginine and proline, and a total of 60 residues. The amino terminus is blocked by pyroglutamate. The flocculant capacity, determined in glass powder suspension, is comparable to that of a cationic polymer on polyacrylamide basis. Flocculation activity may be explained by the patch charge mechanism due to low molecular weight and high charge density.

Nystatin isolated from Streptomyces is a polyene antibiotic that is frequently used in the treatment and prophylaxis of fungal infections. Here, the fractional sterol concentration dependencies of the partition coefficient for partitioning of nystatin into ergosterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), cholesterol/DMPC, ergosterol/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylcholine (POPC), and ergosterol/POPC/1-palmitoyl-2-oleoyl-L-alpha-phosphatidylethano lam ine (POPE) multilamellar vesicles have been determined fluorometrically at 37 degrees C using approximately 0.3-1.0 mol % sterol concentration increments over a wide concentration range (e.g., 18-54 mol % sterol). This unconventional approach of varying membrane sterol content, in contrast to previous studies using large sterol concentration increments (e.g., 10 mol %), leads to a striking observation. The partition coefficient of nystatin changes dramatically with membrane sterol content in a well-defined alternating manner, displaying a local minimum at or very close to the critical sterol mole fractions (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % sterol) predicted for sterols regularly distributed in either hexagonal or centered rectangular superlattices. In ergosterol/DMPC bilayers, for example, there is a >3-fold increase in nystatin partitioning with a minute change (approximately 1 mol %) in sterol content on either side of the critical sterol mole fraction, 25.0 mol %. These results provide semifunctional evidence supporting the sterol regular distribution model [Chong, P. L.-G. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10069-10073]. More importantly, these results reveal a new membrane phenomenon, that is, that nystatin partitioning is affected by the extent of sterol regular distribution in the plane of the membrane. This phenomenon occurs not only in saturated (e.g., DMPC) but also in unsaturated (e.g., POPC) lipid membranes, and persists in the presence of polar headgroup heterogeneity (e.g., POPC/POPE). This membrane property points to a new method for studying the interactions of polyene antibiotics with sterol-containing membranes, and the need to consider the membrane sterol content of the target cells when administering nystatin or other polyene antibiotics.

OBJECTIVE

Lymphangioleiomyomatosis (LAM) is characterized by the abnormal growth of smooth muscle-like cells (LAM cells) and cystic destruction of the lung parenchyma. LAM cell-derived matrix metalloproteinases (MMPs) are thought to play a prominent role in the tissue destruction. The aim of this study was to determine whether doxycycline, a known MMP inhibitor, can inhibit LAM cell proliferation or mitochondrial function and/or modulate MMPs and their tissue inhibitors (TIMPs).

METHODS

Wild-type and tuberous sclerosis complex-2 (TSC2)-null mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% fetal bovine serum (FBS). Human LAM cells were derived from the lungs of LAM patients and airway smooth muscle cells from control subjects. Cells were stimulated with FBS with or without doxycycline for up to 9 days. Proliferation was assessed by manual cell counts and MTT assay, MMP production by zymography and ELISA, and TIMP production using elisa.

RESULTS

Doxycycline did not change FBS-induced proliferation in MEFs or human cells. However, doxycycline did reduce metabolic activity of both wild-type and TSC2-null MEFs and LAM cells, but had no effect on control cells. Furthermore, doxycycline reduced MMP-2 from MEFs and decreased active-MMP-2 from LAM cells but had no effect on TIMP-1 and TIMP-2 from human LAM cells.

CONCLUSIONS

Doxycycline decreased MMP levels and cell metabolic activity, which raises the possibility of therapeutic efficacy in LAM.

Lymphangioleiomyomatosis is a rare disease which afflicts young women of childbearing age. It is sufficiently uncommon that randomization or any other systematic evaluation of regimens of treatment has been difficult. Review of scattered case reports implies that a number of hormonal manipulations may be equally effective. A comprehensive review of the literature revealed 30 cases of LAM treated with eight regimens of treatment. Evaluation with predetermined criteria (meta-analysis) shows that administration of progesterone or oophorectomy or both are the most effective treatments, resulting in improvement or stabilization of the disease in the majority of cases.

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR). A presumed critical step in the biosynthesis of ManLAM, and other mannose-containing glycoconjugates, is the conversion of mannose-6-phosphate to mannose-1-phosphate, by a phosphomannomutase (PMM), to produce GDP-mannose, the primary mannose-donor in mycobacteria. We have identified four M. tb H37Rv genes with similarity to known PMMs. Using in vivo complementation of PMM and phosphoglucomutase (PGM) deficient strains of Pseudomonas aeruginosa, and an in vitro enzyme assay, we have identified both PMM and PGM activity from one of these genes, Rv3257c (MtmanB). MtmanB overexpression in M. smegmatis produced increased levels of LAM, lipomannan, and phosphatidylinositol mannosides (PIMs) compared with control strains and led to a 13.3 +/- 3.9-fold greater association of mycobacteria with human macrophages, in a mannan-inhibitable fashion. This increased association was mediated by the overproduction of higher order PIMs that possess mannose cap structures. We conclude that MtmanB encodes a functional PMM involved in the biosynthesis of mannosylated lipoglycans that participate in the association of mycobacteria with macrophage phagocytic receptors.

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.

OBJECTIVE

We retrospectively compared the antiviral effect of tenofovir disoproxil fumarate (TDF) with that of adefovir dipivoxil (ADV) for patients with chronic hepatitis B (CHB) who developed resistance to lamivudine (LAM).

METHODS

One hundred nine patients (86 males), all Asian-American except 1 Caucasian male, with LAM resistance received TDF or ADV. HBV DNA levels were measured every 3 months. The HBeAg loss and ALT normalization were assessed at 12 months on therapy.

RESULTS

Forty-four patients (37 males) received TDF (12 with LAM) and 65 (49 males) received ADV (18 with LAM). Median ages (years) for TDF and ADV were 49 (32-68) and 45 (22-68), respectively. Median duration of therapy was 13 months (6-38) and 17 months (6-34) for the TDF and ADV groups. Baseline HBV DNA levels (log(10) copies/ml) were 6.2 +/- 1.7 for the TDF and 6.5 +/- 1.6 for ADV groups. Baseline ALT (IU/l) levels were 77.0 +/- 86.0 and 100 +/- 195 for the TDF and ADV (P = 0.46) groups, respectively. At 12 months, mean levels of log(10) HBV DNA were 1.5 +/- 1.0 and 4.3 +/- 2.2 for TDF and ADV (P = 0.01). HBeAg loss and ALT normalization at 12 months showed no differences. Using a single factor, ANOVA (2-tailed P value), 4 groups, TDF (n = 32), TDF + LAM (12), ADV (47), and ADV + LAM (18), were compared. HBV DNA reduction at 12 months was the greatest for TDF + LAM (P < 0.001).

CONCLUSIONS

Our results suggest that for LAM-resistant HBV, TDF, alone or combined with LAM exerts greater viral reduction than ADV. However, no difference in HBeAg loss was observed. It appears that stronger HBV DNA reduction may not necessarily accelerate HBeAg loss.

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

Particulate enzymes from suspension-cultured ryegrass (Lolium multiflorum Lam.) endosperm cells incorporated glucosyl residues from UDP-glucose and GDP-glucose into beta-glucans. Three types of beta-glucans were produced from UDP-glucose: 1,3-beta-glucan; 1,4-beta-glucan; and mixed-linkage 1,3;1,4-beta-glucan. As in other systems, relatively more 1,4-beta-glucan was produced from a low (10 micromolar) UDP-glucose concentration, and relatively more 1,3-beta-glucan was produced from a high (1 millimolar) UDP-glucose concentration. However, in ryegrass, 1,3;1,4-beta-glucan represented a major proportion of the products at both low and high UDP-glucose concentrations. The arrangement of linkages in the 1,3;1,4-beta-glucan was different at the two concentrations; at the low UDP-glucose concentration, more sequences of three consecutive 1,4-linkages were produced.The effects of pH, temperature, and metal ion concentrations on incorporation were dependent on the UDP-glucose concentration. At the low UDP-glucose concentration, incorporation into all three types of beta-glucan increased with increasing pH. At the high UDP-glucose concentration, 1,3-beta-glucan was the major product at pH 7 and below; 1,4-beta-glucan synthesis was optimal at pH 8; and synthesis of 1,3;1,4-beta-glucan was greatest above pH 8.With 10 micromolar GDP-glucose as substrate, 1,4-beta-glucan, but no 1,3;1,4-beta-glucan, was produced. Incorporation from either UDP-glucose or GDP-glucose was not influenced by the presence of the other.

OBJECTIVE

The purpose of this study was to determine the levels of isoniazid and ethionamide resistance and to identify associated mutations in endemic multidrug-resistant (MDR) strains of Mycobacterium tuberculosis from the Lisbon metropolitan area, Portugal.

METHODS

Seventeen clinical MDR tuberculosis (TB) strains were characterized by standard and semi-quantitative drug susceptibility testing to assess the level of isoniazid and ethionamide resistance. The genes katG, inhA, ethA and ndh were screened for mutations. All strains were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) analysis.

RESULTS

All strains showed high-level resistance to both isoniazid (>1 mg/L) and ethionamide (>25 mg/L). MIRU-VNTR typing revealed the presence of two main clusters, Lisboa3 and Q1, in 16/17 strains, all of which showed the C-15T mutation in the promoter region of the inhA gene. The 16 strains belong to the Latino-American-Mediterranean (LAM) genotype and the other strain belongs to the Beijing genotype. Sequencing of the inhA open reading frame revealed that the 16 strains also had mutations in the structural region of the gene, leading to the S94A substitution in 9 strains and the I194T substitution in 7 strains.

CONCLUSIONS

The results reveal that the presence of a mutation in the inhA regulatory region together with a mutation in the inhA coding region can lead to the development of high-level isoniazid resistance and cross-resistance to ethionamide among the MDR-TB strains circulating in Lisbon. This mutational pattern also hints to a possible involvement of strain-specific factors that could be a feature of the Portuguese MDR-TB strains where the LAM family is the major circulating genotype.

Hepatitis B virus (HBV) recurrence following liver transplantation (LTx) has been controllable primarily with the use of hepatitis B immune globulin (HBIg) and lamivudine (LAM). However, HBV resistance to LAM and/or HBIg has become an increasing problem prompting the use of newer antiviral agents. The purpose of our study was to investigate the association between therapy, HBV breakthrough, and allograft / patient survival in HBV-positive liver transplant recipients. We performed a retrospective review of the medical records of patients that were transplanted for HBV from June 1994 to May 2003. A total of 92 patients, positive for either hepatitis B surface antigen (HBsAg) or HBV deoxyribonucleic acid (DNA) pretransplant, received LAM monotherapy or HBIg (6 months) plus LAM therapy post-liver transplant. HBV breakthrough post-LTx was noted in 14 patients. All patients had detectable HBV DNA prior to liver transplantation; none of the patients that were HBV DNA negative prior to transplant had detectable HBV DNA posttransplant. Of these 14, 9 patients (64%) were switched from LAM to adefovir dipivoxil (ADF) and 5 patients (36%) to tenofovir disoproxil fumarate (TNV). In conclusion, pre-LTx HBV viremia should be considered in planning post-LTx prophylaxis. Trials to evaluate oral antiviral agents in combination with or without HBIg therapy are needed.

Heteropolymeric B-band lipopolysaccharide in Pseudomonas aeruginosa PAO1 is synthesized via the so-called Wzy-dependent pathway, requiring a functional Wzy for polymerization of O-antigen repeat units in the periplasm. Wzy is an integral inner membrane protein for which the detailed topology has been mapped in a recent investigation (Islam, S. T., Taylor, V. L., Qi, M., and Lam, J. S. (2010) mBio 1, e00189-10), revealing two principal periplasmic loops (PL), PL3 and PL5, each containing an RX(10)G motif. Despite considerable sequence conservation between the two loops, the isoelectric point for each peptide displayed marked differences, with PL3 exhibiting a net-positive charge and PL5 showing a net-negative charge. Data from site-directed mutagenesis of amino acids in each PL have led to the identification of several key Arg residues within the two RX(10)G motifs that are important for Wzy function, of which Arg(176), Arg(290), and Arg(291) could not be functionally substituted with Lys. These observations support the proposed role of each PL in a catch-and-release mechanism for Wzy-mediated O-antigen polymerization.

Retroviral vectors are an efficient and widely employed means of introducing an exogenous expression cassette into target cells. These vectors have been shown to integrate semi-randomly into the cellular genome, and can be associated with genotoxicity due to impact on expression of proximate genes. Therefore, efficient and accurate integration site analysis, while quantifying contributions of individual vector-containing clones, is desirable. Linear amplification-mediated polymerase chain reaction (LAM-PCR) is a widely used technique for identifying integrated proviral and host genomic DNA junctions. However, LAM-PCR is subject to selection bias inherent in the reliance of the assay on the presence of a restriction enzyme-cutting site adjacent to a retrievable integration site, and it is further limited by an inability to discriminate prior to sequencing between the flanking genomic DNA of interest and uninformative internal vector DNA. We report a modified restriction enzyme-free LAM-PCR (Re-free LAM-PCR) approach that is less time and labor intensive compared to conventional LAM-PCR, but in contrast to some other nonrestrictive methods, compares in efficiency and sensitivity, excludes retrieval of uninformative internal vector sequences, and allows retrieval of integration sites unbiased by the presence of nearby restriction sites. However, we report that Re-free LAM-PCR remains inaccurate for quantitation of the relative contributions of individual integration site-containing clones in a polyclonal setting, suggesting that bias in LAM-PCR retrieval of integration sites is not wholly explained by restriction enzyme-related factors.

Famciclovir (FCV) and lamivudine (LAM) reduce viral replication in patients with recurrent hepatitis B virus (HBV) infection after orthotopic liver transplantation (OLT). Eighteen of 20 patients with insufficient response to FCV were treated with 100 mg LAM daily after OLT. These patients had shown nonresponse (n = 5), partial response (n = 7), or breakthrough (n = 6) during FCV therapy. Despite passive immunoprophylaxis with hepatitis B immunoglobulin after liver transplantation, HBV reinfection had occurred in 14 of 15 transplanted patients. HBV-DNA levels and the regions A to E of the HBV-DNA polymerase gene were analyzed before and after treatment failure to either therapy. Within 4 weeks on LAM, all but 1 patient showed a 95% average reduction of the HBV-DNA level. As with FCV, we did not observe any severe side-effects attributable to LAM. However, 7 patients developed a breakthrough within 12, 29 (n = 2), 32, 37, 54, and 145 weeks under treatment with LAM associated with the methionine-to-valine signature mutation (M552V) in the YMDD motif in all. With FCV, no unique, but a dominant, resistance pattern with the L528M mutation was identified for patients with breakthrough under FCV. In contrast, nonresponders or patients with partial response to FCV did not exhibit such mutations. Our results indicate that the L528M mutation is a risk factor for LAM breakthrough, because breakthrough during LAM occurred earlier in patients with this mutation (50 +/- 10 weeks vs. 120 +/- 21 weeks). Because breakthrough on either treatment is frequent for this specific group of patients, the use of combination therapy should be explored.

Human alveolar macrophages from bronchoalveolar lavage are usually poor accessory cells for antigen-induced T-lymphocyte proliferation and poor stimulators of allogeneic mixed leukocyte reactions (MLR) when compared to peripheral blood monocytes. In contrast, cells harvested from minced lungs are good stimulators of a MLR. We have characterized the accessory cells obtained after enzymatic digestion of human lung tissue. Pulmonary mononuclear cells were separated from the dissociated lung cell mixture on Ficoll-Hypaque. Loosely adherent cells (LAM) were obtained after an overnight incubation on plastic culture dishes of initially adherent mononuclear cells. LAM were significantly more effective than were pulmonary mononuclear cells (p less than 0.05), firmly adherent cells (p less than 0.05), alveolar macrophages obtained by bronchoalveolar lavage (p less than 0.05), or monocytes (p less than 0.05) in stimulating allogeneic resting T-cells. Addition of indomethacin and catalase markedly improved T-cell proliferation induced by LAM. Enrichment for Fc receptor negative or for nonphagocytic cells further enhanced the MLR-stimulating capacity of LAM. Phase-contrast studies demonstrated an enrichment in cells compatible with dendritic cells in LAM as compared to firmly adherent cells. We conclude that there are potent accessory cells in human lung that are loosely adherent, Fc receptor negative, and poorly phagocytic, and thus are dissimilar from classic macrophages. We hypothesize that cells similar to dendritic cells might play a role in the initiation of immune responses in lung parenchyma.