OBJECTIVE

To determine the factors associated with virological response (VR), HBeAg loss or the emergence of adefovir (ADV)-related mutations in ADV-treated chronic hepatitis B (CHB) patients with lamivudine (LAM) resistance.

METHODS

Fifty-four LAM-resistant CHB patients (46% HBeAg-positive) were treated with ADV monotherapy (n=28) or ADV plus LAM (n=26) for a mean of 30.4 months.

RESULTS

Thirty-eight patients (70.4%) achieved VR defined as HBV-DNA levels <10(4)copies/ml within the first 12 months of treatment. Six (24%) of 25 HBeAg-positive patients exhibited HBeAg loss and 20% seroconverted to anti-HBe. Eight patients (14.8%) developed ADV-related mutations. In the multivariate analysis, female gender (HR=0.20, 95% CI: 0.05-0.76, p=0.018), HBeAg-negative (HR=0.37, 95% CI: 0.14-0.96, p=0.040) and low baseline HBV-DNA levels (HR=0.65, 95% CI: 0.45-0.95, p=0.027) were independent predictors of VR, whereas low HBV-DNA levels (HR=0.36, 95% CI: 0.11-1.20, p=0.095) and HBV-genotype D (HR=0.06, 95% CI: 0.004-0.84, p=0.037) independently predicted HBeAg loss.

CONCLUSIONS

ADV therapy suppresses viral replication in more than 70% of LAM-R patients. Factors associated with virologic response are female gender, HBeAg-negative status and low baseline serum HBV-DNA levels. Genotype D HBV infection and low baseline HBV-DNA levels independently predict HBeAg loss.

Cerebral autoregulation adjusts cerebrovascular resistance in the face of changing perfusion pressures to maintain relatively constant flow. Results from several studies suggest that cardiac output may also play a role. We tested the hypothesis that cerebral blood flow would autoregulate independent of changes in cardiac output. Transient systemic hypotension was induced by thigh-cuff deflation in 19 healthy volunteers (7 women) in both supine and seated positions. Mean arterial pressure (Finapres), cerebral blood flow (transcranial Doppler) in the anterior (ACA) and middle cerebral artery (MCA), beat-by-beat cardiac output (echocardiography), and end-tidal Pco(2) were measured. Autoregulation was assessed using the autoregulatory index (ARI) defined by Tiecks et al. (Tiecks FP, Lam AM, Aaslid R, Newell DW. Stroke 26: 1014-1019, 1995). Cerebral autoregulation was better in the supine position in both the ACA [supine ARI: 5.0 ± 0.21 (mean ± SE), seated ARI: 3.9 ± 0.4, P = 0.01] and MCA (supine ARI: 5.0 ± 0.2, seated ARI: 3.8 ± 0.3, P = 0.004). In contrast, cardiac output responses were not different between positions and did not correlate with cerebral blood flow ARIs. In addition, women had better autoregulation in the ACA (P = 0.046), but not the MCA, despite having the same cardiac output response. These data demonstrate cardiac output does not appear to affect the dynamic cerebral autoregulatory response to sudden hypotension in healthy controls, regardless of posture. These results also highlight the importance of considering sex when studying cerebral autoregulation.

Pulmonary lymphangioleiomyomatosis (LAM) is characterized by the proliferation of abnormal smooth muscle cells (LAM cells) and destruction of alveolar structure. Immunohistochemical studies suggest that excess matrix metalloproteinases (MMPs) synthesized by LAM cells function in the proteolytic mechanisms of this disease. We postulated MMP levels in the blood are elevated in LAM patients. Serum samples were collected from 36 LAM patients and 25 controls, and gelatinolytic activities were semi-quantified by gelatin zymography. The reliability of serum data for MMP-9 was confirmed by the measurement of MMP-9 concentration in plasma by enzyme-linked immunosorbent assay as well as by gelatin zymography. Serum levels of MMP-9 (0.7+/-0.1 AU), but not MMP-2, were significantly elevated in LAM patients compared with controls (0.1+/-0 AU). Plasma and serum levels of MMP-9 significantly correlated. These results suggest the involvement of MMP-9 in LAM.

BACKGROUND

Imidacloprid and Thiacloprid are two neonicotinoid insecticides whose use have been raising exponentially. Both act selectively as agonist of the insect nicotinic-Acetylcholine Receptor (nAChR) and therefore, by definition, they hold the same mode of action. Notwithstanding the growing attention to the ecotoxicological effects of neonicotinoids, there is a lack of information on their toxicodynamics and their mixture effects, in particular, in aquatic organisms.

OBJECTIVE

The main objectives of this work were: (i) assess sublethal effects of two neonicotinoids-Imidacloprid and Thiacloprid-in the tissues of the marine mussel (Mytilus galloprovincialis Lam.); (ii) identify the molecular dynamics elicited by the two chemicals through gene/protein expression profiling and a functional genomics approach; (iii) assess the effects of a neonicotinoid binary mixture.

METHODS

Sublethal effects were measured by means of digestive gland lysosomal membrane stability (LMS) and gill acetylcholinesterase (AChE) activity. Gene expression profiles were evaluated in the digestive gland using a 1.7K cDNA microarray and quantitative-PCR (Q-PCR). Proteome profiling was performed by means of two-dimensional electrophoresis of digestive gland cytosolic proteins. Functional genomics was based on the over-representation of Gene Ontology (GO) terms.

CONCLUSIONS

Our results showed that (i) biomarkers responded in the micromolar range; (ii) Imidacloprid and Thiacloprid elicited distinct toxicodynamics as depicted by the different transcriptomic and proteomic profiles and the opposite trend of AChE activity; (iii) at biomarkers level, the joint effects of the two chemicals appeared to fulfill the principle of independence, but this was less evident at molecular level where a novel specific molecular signature took place.

CONCLUSIONS

These findings imply that different toxicodynamics may occur also as a response of chemicals with the same mode of action. Our results unveil also the incongruousness of the actual concept of pesticide mode of action in the context of ecological risk assessment analysis of chemical mixtures.

Neural substrates for processing constant speed visual motion have been extensively studied. Less is known about the brain activity patterns when the target speed changes continuously, for instance under the influence of gravity. Using functional MRI (fMRI), here we compared brain responses to accelerating/decelerating targets with the responses to constant speed targets. The target could move along the vertical under gravity (1g), under reversed gravity (-1g), or at constant speed (0g). In the first experiment, subjects observed targets moving in smooth motion and responded to a GO signal delivered at a random time after target arrival. As expected, we found that the timing of the motor responses did not depend significantly on the specific motion law. Therefore brain activity in the contrast between different motion laws was not related to motor timing responses. Average BOLD signals were significantly greater for 1g targets than either 0g or -1g targets in a distributed network including bilateral insulae, left lingual gyrus, and brain stem. Moreover, in these regions, the mean activity decreased monotonically from 1g to 0g and to -1g. In the second experiment, subjects intercepted 1g, 0g, and -1g targets either in smooth motion (RM) or in long-range apparent motion (LAM). We found that the sites in the right insula and left lingual gyrus, which were selectively engaged by 1g targets in the first experiment, were also significantly more active during 1g trials than during -1g trials both in RM and LAM. The activity in 0g trials was again intermediate between that in 1g trials and that in -1g trials. Therefore in these regions the global activity modulation with the law of vertical motion appears to hold for both RM and LAM. Instead, a region in the inferior parietal lobule showed a preference for visual gravitational motion only in LAM but not RM.

UDP-N-acetylglucosamine-3-O-acyltransferase (UDP-GlcNAc acyltransferase) catalyzes the first step of lipid A biosynthesis (M. S. Anderson and C. R. H. Raetz, J. Biol. Chem. 262:5159-5169, 1987). We here report the isolation of the lpxA gene of Pseudomonas aeruginosa from a library of Pseudomonas strain PAO1 expressed in Escherichia coli LE392 (J. Lightfoot and J. S. Lam, J. Bacteriol. 173:5624-5630, 1991). Pseudomonas lpxA encodes a 10-carbon-specific UDP-GlcNAc acyltransferase, whereas the E. coli transferase is selective for a 14-carbon acyl chain. Recombinant cosmid 1137 enabled production of a 3-hydroxydecanoyl-specific UDP-GlcNAc acyltransferase in E. coli. It was identified by assaying lysozyme-EDTA lysates of individual members of the library with 3-hydroxydecanoyl-acyl carrier protein (ACP) as the substrate. Cosmid 1137 contained a 20-kb insert of P. aeruginosa DNA. The lpxA gene region was localized to a 1.3-kb SalI-PstI fragment. Sequencing revealed that it contains one complete open reading frame (777 bp) encoding a new lpxA homolog. The predicted Pseudomonas LpxA is 258 amino acids long and contains 21 complete hexapeptide repeating units, spaced in approximately the same manner as the 24 repeats of E. coli LpxA. The P. aeruginosa UDP-GlcNAc acyltransferase is 54% identical and 67% similar to the E. coli enzyme. A plasmid (pGD3) containing the 1.3-kb SalI-PstI fragment complemented E. coli RO138, a temperature-sensitive mutant harboring lpxA2. LpxA assays of extracts of this construct indicated that it is>> 1,000-fold more selective for 3-hydroxydecanoyl-ACP than for 3-hydroxymyristoyl-ACP. Mass spectrometry of lipid A isolated from this strain by hydrolysis at pH 4.5 revealed [M-H]- 1,684.5 (versus 1,796.5 for wild-type lipid A), consistent with 3-hydroxydecanoate rather than 3-hydroxymyristate at positions 3 and 3'.

Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of beta-lactamase (beta laM) to the C-terminus. Neither of these changes affected multimerization. PulG precursor could be post-translationally processed by prepilin peptidase (PulO), indicating that the N-terminus of prePulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopulation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure.

The ability of 17 recombinant mycobacterial proteins, native antigen 85 complex, lipoarabinomannan (LAM), and Mycobacterium tuberculosis lysate to detect antibody responses induced by bacille Calmette-Guérin (BCG) vaccination and active tuberculosis infection were studied in enzyme-linked immunosorbent assays. Only LAM-reactive serum immunoglobulin G responses were significantly increased in both BCG-vaccinated patients and patients with active tuberculosis (P<.05), and oral BCG vaccination also induced significant increases in LAM-reactive secretory immunoglobulin A (P<.05). LAM-reactive antibody assays can serve as markers of humoral and mucosal immunity in future trials of BCG and newer attenuated mycobacterial vaccines.

Lymphangioleiomyomatosis (LAM) is a rare disease which afflicts young women of childbearing age. Recently, it has been listed as an indication for lung transplantation. We describe a case of recurrent LAM in a 31-year-old woman occurring in the allograft of a male donor after single lung transplantation. Nonisotopic in situ hybridization shows that the smooth muscle cell proliferation is of donor origin.

BACKGROUND

X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-).

METHODS

A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone.

RESULTS

Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation.

CONCLUSIONS

This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.

Entecavir (ETV) is currently recommended as a rescue therapy purely for adefovir (ADV)-resistant chronic hepatitis B virus (HBV) infections. We evaluated the efficacy of ETV in patients who were resistant to lamivudine (LAM)/ADV sequential therapy and in those resistant to LAM monotherapy. Fifty LAM/ADV-resistant and 38 LAM-resistant patients who received ETV 1 mg/day for at least 48 weeks were enrolled. Mean baseline serum HBV DNA and alanine aminotransferase (ALT) levels were significantly lower in the LAM/ADV-resistant group, compared with the LAM-resistant group (6.90 versus 7.62 log(10) copies/mL and 102.6 versus 160.2 IU/L; both P < 0.05); hepatitis B e antigen (HBeAg) status and LAM-resistant mutation patterns were similar in the two groups. At week 48, mean reductions in HBV DNA and ALT levels were significantly less in the LAM/ADV-resistant group (-2.96 versus -4.86 log(10) copies/mL and -68.3 versus -128.9 IU/L; both P < 0.05). Achievement of undetectable HBV DNA was also less common in the LAM/ADV-resistant group (10.0% versus 34.2%; P = 0.006), although the rates of HBeAg loss and ALT normalization did not differ between the two groups. Resistance to both LAM and ADV was an independent risk factor for failure of HBV DNA negativity at week 48 (odds ratio, 0.138; P = 0.019). In both LAM/ADV-resistant and LAM-resistant groups, primary responders >> or =1 log decline in HBV DNA at week 12) achieved a significantly greater decrease in HBV DNA levels over the 48-week period, compared with primary nonresponders (-4.18 versus -0.97 and -5.37 versus -2.15 log(10) copies/mL, respectively; both P < 0.05).

CONCLUSIONS

The 48-week ETV treatment was less effective in LAM/ADV-resistant than in LAM-resistant patients. Continuing ETV monotherapy could be determined based on the virological response at 12 weeks in LAM/ADV-resistant patients.

We examined the mechanisms involved in neutrophil adherence to cultured human umbilical vein endothelial cells (HEC) induced by direct stimulation of the neutrophils by phorbol myristate acetate (PMA), formylmethionyl-leucyl-phenylalanine (FMLP), or the calcium ionophore A23187 (neutrophil-dependent adherence), or by pretreatment of HEC with interleukin-1 (IL-1), tumour necrosis factor (TNF) or lipopolysaccharide (LPS) (endothelial-dependent adherence). Two distinct mechanisms for neutrophil adherence to HEC were demonstrated by performing adherence assays: (i) at 37 degrees versus 4 degrees; (ii) in the presence of Ca2+ only versus Mg2+ only; and (iii) in the presence or absence of monoclonal antibodies (mAb) to the CD11/CD18 adhesion complex of neutrophils. A CD11/CD18-dependent mechanism (i.e. inhibited by anti-CD18 mAb) was identified that was active in the presence of Mg2+ only but not of Ca2+ only, and at 37 degrees but not at 4 degrees. A CD11/CD18-independent mechanism (i.e. not inhibited by anti-CD18 mAb) was active at 4 degrees and at 37 degrees, and in the presence of Ca2+ only and of Mg2+ only. Neutrophil-dependent adherence induced by FMLP or PMA occurred solely via the CD11/CD18-dependent mechanism, whereas endothelial-dependent adherence induced by a 4-hr pretreatment with IL-1, TNF, or LPS involved both CD11/CD18-dependent and/independent mechanisms. CD11/CD18-deficient neutrophils isolated from a patient with leucocyte adherence deficiency (LAD) maintained the ability to adhere to LPS-pretreated HEC in the presence of Ca2+ only, indicating that this mechanism of adherence involves a receptor on the neutrophil distinct from CD11/CD18. Furthermore, the disappearance of the CD11/CD18-independent, but not of the CD11/CD18-dependent mechanism of adherence, in HEC treated with TNF for 24 hr suggests that the two mechanisms of neutrophil adherence also involve distinct inducible endothelial-leucocyte adhesion molecules (E-LAM).

The outcome of patients with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) referred for liver transplantation (LT) is unknown. A high frequency of lamivudine-resistant (LAM-R) HBV infection may increase the risk of liver-related death pre-transplantation and prophylaxis failure post-transplantation. We evaluated the association of LAM-R HBV on pre-transplant survival and post-transplant outcomes in 35 consecutive HIV-HBV coinfected patients referred for LT between July 2000 and September 2002. At the time of referral, the median CD4 count was 273/mm, MELD was 14, and LAM-R HBV infection was present in 67%. Among these referred patients, 26% were listed, 29% not listed due to relative/absolute contraindications; 26% not listed as too early for LT; 9% not listed as too sick for LT; and 11% died during transplant evaluation. Of the 9 listed patients, 4 remained listed, 1 died 18 months post-referral, and 4 were transplanted (11% of total) 3 to 40 months after listing. Of 17 evaluated but not listed patients, 5 died (p=0.38 compared to listed group) and all deaths were liver-related. All the HBV-HIV coinfected patients, who were transplanted, are HBsAg negative and have undetectable HBV DNA levels on prophylactic therapy using hepatitis B immune globulin (HBIG) plus lamivudine, with and without tenofovir or adefovir, with median 33.1 months follow-up. Late referral and the presence of LAM-R HBV pre-transplantation are common in referred HIV-HBV patients. In HIV-HBV coinfected patients undergoing LT, HBV recurrence is successfully prevented with combination prophylaxis using HBIG and antivirals.

Lymphangioleiomyomatosis (LAM), a multisystem disease found in middle-aged women, is characterized by cystic lung destruction and abdominal tumors (e.g., angiomyolipomas, lymphangioleimyomas), resulting from proliferation of abnormal-appearing, smooth muscle-like cells (LAM cells). The LAM cells, in combination with other cells, form nodular structures within the lung interstitium and in the walls of the cysts. LAM cells contain mutations in the tuberous sclerosis complex TSC1 and/or TSC2 genes, which lead to dysregulation of the mammalian target of rapamycin, affecting cell growth and proliferation. Proliferation and migration of vascular smooth muscle cells and production of angiogenic factors are regulated, in part, by angiotensin II. To determine whether a LAM-specific renin-angiotensin system might play a role in the pathogenesis of LAM, we investigated the expression of genes and gene products of this system in LAM nodules. mRNA for angiotensinogen was present in RNA isolated by laser-captured microdissection from LAM nodules. Angiotensin I-converting enzyme and chymase-producing mast cells were present within the LAM nodules. We detected renin in LAM cells, as determined by the presence of mRNA and immunohistochemistry. Angiotensin II type 1 and type II receptors were identified in LAM cells by immunohistochemistry and immunoblotting of microdissected LAM nodules. Angiotensin II is localized in cells containing alpha-smooth muscle actin (LAM cells). A LAM-specific renin-angiotensin system appears to function within the LAM nodule as an autocrine system that could promote LAM cell proliferation and migration, and could represent a pharmacologic target.

Rhodococcus equi is a major cause of foal morbidity and mortality. We have investigated the presence of lipoglycan in this organism as closely related bacteria, notably Mycobacterium tuberculosis, produce lipoarabinomannans (LAM) that may play multiple roles as virulence determinants. The lipoglycan was structurally characterized by gas chromatography-mass spectrometry following permethylation, capillary electrophoresis after chemical degradation, and (1)H and (31)P and two-dimensional heteronuclear nuclear magnetic resonance studies. Key structural features of the lipoglycan are a linear alpha-1,6-mannan with side chains containing one 2-linked alpha-d-Manp residue. This polysaccharidic backbone is linked to a phosphatidylinositol mannosyl anchor. In contrast to mycobacterial LAM, there are no extensive arabinan domains but single terminal alpha-d-Araf residue capping the 2-linked alpha-d-Manp. The lipoglycan binds concanavalin A and mannose-binding protein consistent with the presence of t-alpha-d-Manp residues. We studied the ability of the lipoglycans to induce cytokines from equine macrophages, in comparison to whole cells of R. equi. These data revealed patterns of cytokine mRNA induction that suggest that the lipoglycan is involved in much of the early macrophage cytokine response to R. equi infection. These studies identify a novel LAM variant that may contribute to the pathogenesis of disease caused by R. equi.

BACKGROUND

Accurate, simple and cost-effective diagnostic tests are needed for diagnosis of active tuberculosis (TB). Serodiagnosis is attractive as it can be harnessed for point-of-care tests.

METHODS

We evaluated, in a blinded fashion, the sensitivity and specificity of serologic immunoglobulin (Ig)G, IgA and/or IgM responses to Apa, heat shock protein (HSP) 16.3, HSP20, PE35, probable thiol peroxidase Tpx and lipoarabinomannan (LAM) in 42 HIV-negative South African pulmonary TB patients and 67 control individuals. The status of latent Mycobacterium tuberculosis infection (LTBI) among controls was defined through the TST and IFN-γ release assays (IGRAs). We evaluated 47 definite LTBI (IGRA(+)/LTBI), 8 putative LTBI (IGRA(-)/TST(+)) and 12 TB-uninfected (non-LTBI) subjects.

RESULTS

In contrast to anti-PE35 IgA, anti-PE35 IgG and particularly anti-Apa IgA, performances of anti-LAM IgG and selected anti-protein antibodies were less affected by inclusion of LTBI participants into the analysis. Anti-LAM IgG showed with a sensitivity/specificity of 71.4%/86.6% (p < 0.001) the best discrimination between TB and non-TB subjects. Selected five-antibody-combinations (including anti-LAM IgG, anti-LAM IgA and anti-Tpx IgG) further improved this performance to an accuracy exceeding 86%.

CONCLUSIONS

Antibody responses to some Mycobacterium tuberculosis antigens often also reflect latent infection explaining the poor performance of antibody-based tests for active TB in TB-endemic settings. Our results suggest that rather a combination of serological responses against selected protein and non-protein antigens and different Ig classes should be investigated for TB serodiagnostics.

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5-3-μm-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Vietnam, a developing country, has had comparatively good health and human survival at low cost. The economic reform changed the health care system, and private pharmacies during the last 5 years have taken over a majority of the drug distribution. Problems include weaknesses in drug regulation and reported increases in antibiotic resistance. This case study, a purposive sample of two private pharmacies in Hanoi, explored management, including dispensing, inventory and financing, using the concept of triangulation. Observations and interviews of customers were complemented by stock inventory and interviews of the pharmacy staff. Drugs were classified according to the ATC code and the essential drug list of Vietnam. Pretested protocols were used. In all 1833 encounters were studied during the 2 weeks, out of which 286 were children. Less than 1% of customers came with prescriptions and 94.9% decided by themselves which drugs to buy. Antibiotics represented 17%, of which 90% were broad spectrum. Ampicillin dominated, both in children and adults. Some 50% of the antibiotics were given for 2.5 days or less. Less than 50% of the drugs were essential drugs (ED) on dispensing and even less on inventory. Antibiotics and vitamins were the most commonly sold drugs and, overall, brand names dominated. Little if any drug information was observed. Antibiotics were said to represent the most profitable drugs, according to the pharmacy staff. More than 20% of all products were combination drugs, including irrational and popular products with antibiotics and corticosteroids and combinations of aspirin, phenacetin and caffeine. This study shows an unexpectedly high proportion of customers, being "Tu Lam Bac Sy" (their own doctors), deciding themselves which drugs to buy. Although the "Doi moi" renovation has led to much improved drug availability, at least in urban setting, our case study highlights major problems in need of urgent actions. In particular the prevailing practices regarding antibiotics and combination drugs need to be seriously scrutinized and drug regulatory mechanisms should be enforced.

Pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML) are proliferative lesions that occur in sporadic patients, and at much higher frequency in patients with tuberous sclerosis (TSC). The TSC1 and TSC2 genes play a critical role in their pathogenesis. Here we report a marked decrease in interferon (IFN)-gamma expression in both sporadic and TSC-associated AML and LAM. A marked increase in Stat1 expression and phosphorylation at Ser 727, and in phospho-Tyr705-Stat3 levels, was also seen in both AML and LAM tissues. Our results demonstrate that the IFN-gamma-Jak-Stat pathway is perturbed in TSC-related and sporadic LAM and AML, and suggest that IFN-gamma has potential therapeutic benefit for treatment of those lesions.

The product of yjeK in Escherichia coli is a homologue of lysine 2,3-aminomutase (LAM) from Clostridium subterminale SB4, and both enzymes catalyze the isomerization of (S)- but not (R)-alpha-lysine by radical mechanisms. The turnover number for LAM from E. coli is 5.0 min(-1), 0.1% of the value for clostridial LAM. The reaction of E. coli LAM with (S)-alpha-[3,3,4,4,5,5,6,6-(2)H8]lysine proceeds with a kinetic isotope effect (kH/kD) of 1.4, suggesting that hydrogen transfer is not rate-limiting. The product of the E. coli enzyme is (R)-beta-lysine, the enantiomer of the clostridial product. Beta-lysine-related radicals are observed in the reactions of both enzymes by electron paramagnetic resonance (EPR). The radical in the reaction of clostridial LAM has the (S)-configuration, whereas that in the reaction of E. coli LAM has the (R)-configuration. Moreover, the conformations of the beta-lysine-related radicals at the active sites of E. coli and clostridial LAM are different. The nuclear hyperfine splitting between the C3 hydrogen and the unpaired electron at C2 shows the dihedral angle to be 6 degrees, unlike the value of 77 degrees reported for the analogous radical bound to the clostridial enzyme. Reaction of (S)-4-thialysine produces a substrate-related radical in the steady state of E. coli LAM, as in the action of the clostridial enzyme. While (S)-beta-lysine is not a substrate for E. coli LAM, it undergoes hydrogen abstraction to form an (S)-beta-lysine-related radical with the same stereochemistry of hydrogen transfer from C2 of (S)-beta-lysine to the 5'-deoxyadenosyl radical as in the action of the clostridial enzyme. The resulting beta-lysyl radical has a conformation different from that at the active site of clostridial LAM. All evidence indicates that the opposite stereochemistry displayed by E. coli LAM is determined by the conformation of the lysine side chain in the active site. Stereochemical models for the actions of LAM from C. subterminale and E. coli are presented.

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (+/-)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardia grandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.

An understanding of the number and contribution of individual pluripotent hematopoietic stem cells (HSCs) to the formation of blood lineages has important clinical implications for gene therapy and stem cell transplantation. We have been able to efficiently mark rhesus macaque long-term repopulating stem and progenitor cells with retroviral vectors, and track their in vivo contributions to hematopoiesis using the linear amplification mediated-polymerase chain reaction (LAM-PCR) technique of insertion site analysis. We assessed the impact of busulfan on contributions of individual retrovirally marked clones to hematopoiesis. There were 2 macaques that received transplants of retrovirally transduced CD34(+) cells 2 years previously that were then treated with 4 mg/kg busulfan. Despite only transient and mild suppression of peripheral blood counts, the numbers of individual stem/progenitor clones contributing to granulocyte production decreased dramatically, by 80% in the first monkey and by 60% in the second monkey. A similar impact was seen on clones contributing to T cells. The clone numbers recovered gradually back toward baseline by 5 months following busulfan in the first monkey and by 3 months in the second monkey, and have remained stable for more than one year in both animals. Tracking of individual clones with insertion-site-specific primers suggested that clones contributing to hematopoiesis prior to busulfan accounted for the majority of this recovery, but that some previously undetected clones began to contribute during this recovery phase. These results indicate that even low-dose busulfan significantly affects stem and progenitor cell dynamics. The clonal diversity of hematopoiesis was significantly decreased after even a single, clinically well-tolerated dose of busulfan, with slow but almost complete recovery over the next several months, suggesting that true long-term repopulating stem cells were not permanently deleted. However, the prolonged period of suppression of many clones suggests that transplanted HSCs may have a marked competitive advantage if they can engraft and proliferate during this time period, and supports the use of this agent in nonmyeloablative regimens