bluuebunny/crossref_metadata_2025 huggingface dataset:
‘The Bad People Go and Speak to Her’: Young People’s Choice and Agency when Accessing Mental Health Support in School
Authors: Spratt Jennifer , Shucksmith Janet , Philip Kate , Watson Cate
Published: June 2009

Concerns about the mental health and well‐being of children and young people have been articulated in health and education policy fields as a call for closer working between schools and providers of mental health support services. Drawing from a Scottish study, this article explores issues of access, when mental health initiatives are sited in formal educational settings. In particular, it focuses on the implications for the agency of children and young people seeking support from those services when and how they choose. The study argues that over‐reliance on teachers as the main referral route to service influences what is deemed to be a problem, who is thought to need support and how the interventions are viewed by the children and young people. Alternative approaches are discussed, which offer opportunities for children and young people to explore the available services and make their own choices about their level of engagement.
A novel DNA vaccine containing multiple TB‐specific epitopes cast in a natural structure elicits enhanced Th1 immunity compared with BCG
Authors: Gao Haifeng , Li Kang , Yu Shanshan , Xiong Sidong
Published: June 2009

Vaccination is expected to make a major contribution to the goal of eliminating tuberculosis worldwide by 2050. Because the protection afforded by the currently available tuberculosis vaccine, BCG, is insufficient, new vaccine strategies are urgently needed. Protective immunity against MTB depends on generation of a Th1‐type cellular immune response characterized by secretion of IFN‐γ from antigen‐specific T cells. Epitope‐driven vaccines are created from sub‐sequences of proteins (epitopes) derived by scanning the protein sequences of pathogens and selecting epitopes with patterns of amino acids which permit binding to human MHC molecules. Guided by the crystal structure of HSP65 and its characteristics, four functional T cell epitopes elaborately elicited from ESAT‐6, Ag85A, CFP‐10 and Ag85B were cast into the intermediate domain of HSP65. A panel of a novel chimeric vaccine, ECANS, expressing HSP65 and combined T cell epitopes was created. Gene cloning and sequencing, DNA vaccination and humoral and cellular responses were studied. After being immunized with DNA vaccine three times, all mice injected with ECANS had specific cellular immune responses. In addition, lymphocytes obtained from the spleen of ECANS immunized mice at week eight exhibited significantly greater specific lymphocyte proliferation, IFN‐γ secretion and CTL activity than those of mice that had been immunized with BCG. DNA vaccine with ECANS can successfully induce enhanced specific cellular immune response to PPD, and further study of its protective effects against Mycobacterium tuberculosis in vivo is needed.
A host‐vector system for molecular study of the intracellular growth ofMycobacterium tuberculosisin phagocytic cells
Authors: Nomoto Mari , Ogawa Midori , Fukuda Kazumasa , Miyamoto Hiroshi , Taniguchi Hatsumi
Published: June 2009

The mechanisms by whichMycobacterium tuberculosissurvives and persists in phagocytic cells remain poorly understood. To study the question, a convenient and safe host‐vector system is indispensable. In this study it has been shown that, in contrast withM.smegmatisstrain mc2155 which has been widely used for molecular analysis,M. smegmatisstrain J15cs is able to survive even at day 6 post‐infection in a murine macrophage cell line, J774. The survivability of J15cs was found to depend on the culture medium used for the bacteria prior to infection. Bacteria precultured on nutrient agar medium showed a high survivability and a characteristic cell wall ultrastructure. A plasmid vector, pYT923hyg, was developed from anEscherichia coli‐ mycobacterium shuttle vector pYT923 (previously constructed in our laboratory) to obtain three drug resistant genes (amp‐, hyg‐ and km‐resistant gene) and cloning sites in the km resistant gene. The vector pYT923hyg exerted no influence onin vitrogrowth of J15cs and intracellular survival in J774 cells, and was stably retained in J15cs after serial subculturing (three subcultures) in Luria‐Bertani broth and at day 5 post‐infection into J774 cells. Furthermore, using this system, the possibility of a relationship between some seemingly essential genes ofM. tuberculosisand intracellular growth was demonstrated.

In this study,M. smegmatisstrain J15cs and pYT923hyg were found to be capable of serving as an appropriate host‐vector system for molecular study of the intracellular growth ofM.tuberculosisin phagocytic cells; this system may be useful as a screening tool forM.tuberculosisgenes.
Effect of vascular endothelial growth factor polymorphisms on survival in advanced‐stage non‐small‐cell lung cancer
Authors: Masago Katsuhiro , Fujita Shiro , Kim Yung Haku , Hatachi Yukimasa , Fukuhara Akiko , Nagai Hiroki , Irisa Kaoru , Ichikawa Masataka , Mio Tadashi , Mishima Michiaki
Published: June 2009

Polymorphisms have been identified in the vascular endothelial growth factor (VEGF) gene that may affect VEGF production. We hypothesized that such polymorphisms may correlate with survival outcomes among advanced‐stage non‐small‐cell lung cancer (NSCLC) patients. We evaluated the association between VEGF polymorphisms and overall survival among patients with advanced NSCLC who were treated with at least one cytotoxic regimen at Kyoto University Hospital between 2003 and 2008. We investigated the following VEGF polymorphisms: –460T > C (rs833061), +405G > C (rs2010963), +936C > T (rs3025039), –1154G > A (rs1570360), and –2578C > A (rs699947). Analyses of genotype associations with survival outcomes were performed using Cox proportional models, Kaplan–Meier methods, and the log‐rank test. There were 126 patients and 80 deaths. On a Cox regression analysis of a current and former smoker (hazards ratio [HR], 1.422; 95% confidence interval [CI], 1.111–1.859; P = 0.0046), poor performance status (PS) (HR, 2.524; 95% CI, 1.483–3.827; P = 0.0019), the VEGF –460CC genotype (HR, 1.719; 95% CI, 1.166–2.390; P = 0.0084), VEGF –1154AA and AG genotypes (HR, 1.482; 95% CI, 1.144–1.897; P = 0.0034), and VEGF –2578AA genotype (HR, 1.797; 95% CI, 1.219–2.495; P = 0.0047) had a significant prognostic effect on survival based on univariate analysis. Based on multivariate analysis of a current and former smoker (HR, 1.407; 95% CI, 1.095–1.840; P = 0.0070), poor PS (HR, 2.249; 95% CI, 1.309–3.468; P = 0.0058), and the VEGF –1154AA and AG genotypes (HR, 1.419; 95% CI, 1.033–1.901; P = 0.0316) were significant independent prognostic factors for survival. In this study, polymorphisms in VEGF may affect survival in advanced NSCLC. (Cancer Sci 2009; 100: 1917–1922)
Protein tyrosine phosphatase SHP‐2: A proto‐oncogene product that promotes Ras activation
Authors: Matozaki Takashi , Murata Yoji , Saito Yasuyuki , Okazawa Hideki , Ohnishi Hiroshi
Published: June 2009

SHP‐2 is a cytoplasmic protein tyrosine phosphatase (PTP) that contains two Src homology 2 (SH2) domains. Although PTPs are generally considered to be negative regulators on the basis of their ability to oppose the effects of protein tyrosine kinases, SHP‐2 is unusual in that it promotes the activation of the Ras‐MAPK signaling pathway by receptors for various growth factors and cytokines. The molecular basis for the activation of SHP‐2 is also unique: In the basal state, the NH2‐terminal SH2 domain of SHP‐2 interacts with the PTP domain, resulting in autoinhibition of PTP activity; the binding of SHP‐2 via its SH2 domains to tyrosine‐phosphorylated growth factor receptors or docking proteins, however, results in disruption of this intramolecular interaction, leading to exposure of the PTP domain and catalytic activation. Indeed, SHP‐2 proteins with artificial mutations in the NH2‐terminal SH2 domain have been shown to act as dominant active mutants in vitro. Such activating mutations of PTPN11 (human SHP‐2 gene) were subsequently identified in individuals with Noonan syndrome, a human developmental disorder that is sometimes associated with juvenile myelomonocytic leukemia. Furthermore, somatic mutations of PTPN11 were found to be associated with pediatric leukemia. SHP‐2 is also thought to participate in the development of other malignant disorders, but in a manner independent of such activating mutations. Biochemical and functional studies of SHP‐2 and genetic analysis of PTPN11 in human disorders have thus converged to provide new insight into the pathogenesis of cancer as well as potential new targets for cancer treatment. (Cancer Sci 2009; 100: 1786–1793)
Characterization of brain cancer stem cells: a mathematical approach
Authors: Turner C. , Stinchcombe A. R. , Kohandel M. , Singh S. , Sivaloganathan S.
Published: June 2009

Objective:  In recent years, support has increased for the notion that a subpopulation of brain tumour cells in possession of properties typically characteristic of stem cells is responsible for initiating and maintaining the tumour. Unravelling details of the brain tumour stem cell (BTSC) hierarchy, as well as interactions of these cells with various therapies, will be essential in the design of optimal treatment strategies.

Materials and methods:  Motivated by this, we have developed a mathematical model of the BTSC hypothesis that may aid in characterization of brain tumours, as well as in prediction of effective therapeutic strategies, which can be further validated in experimental and clinical studies. At the level of a small number of cells, the model developed herein is stochastic. For larger populations of cancer cells, the model is handled from a deterministic approach.

Results and conclusions:  In the stochastic regime, importance of a relationship between the likelihoods of two distinct types of symmetric BTSC divisions in determining BTSC survival rates becomes apparent, consequently emphasizing the need for a set of biomarkers that are able to better characterize the BTSC hierarchy. At the large scale, we predict the importance of the aforementioned symmetric division rates in dictating brain tumour composition. Furthermore, we demonstrate possible therapeutic benefits of considering combination treatments of radiotherapy and putative BTSC inhibitors, such as bone morphogenetic proteins, while reinforcing the importance of developing novel treatment strategies that specifically target the BTSC subpopulation.
A practice framework to guide screening and assessment in the Australian Family Relationship Centres and Advice Line
Authors: Winkworth Gail , McArthur Morag
Published: June 2009

In recent years, there has been a growing recognition of the reactive way in which policy is translated into practice on the front lines of child and family welfare organizations, particularly in how risk is assessed and responded to by staff at all levels in these organizations. Major reforms to family law in Australia and programmes set up to complement the existing Commonwealth‐Funded Family Relationship Services Program provide an opportunity to rethink how staff can be provided with a systematic and research‐based set of guidelines for screening and assessment. This paper examines the role of practice frameworks in general and the Australian family relationships screening and assessment practice framework in particular to demonstrate not only how this broader evidence‐based approach encourages well‐informed, professional and ethical practice but also how it leaves room for creativity and local, place‐based responsiveness.
A low dose of Mycoplasma pneumoniae infection enhances an established allergic inflammation in mice: the role of the prostaglandin E2 pathway
Authors: Wu Q. , Martin R. J. , LaFasto S. , Chu H. W.
Published: June 2009

Background
Over 40% of chronic stable asthma patients have evidence of respiratory Mycoplasma pneumoniae (Mp) infection as detected by PCR, but not by serology and culture, suggesting that a low‐level Mp is involved in chronic asthma. However, the role of such a low‐level Mp infection in the regulation of allergic inflammation remains unknown.

Objective
To determine the impact of a low‐level Mp infection in mice with established airway allergic inflammation on allergic responses such as eosinophilia and chemokine eotaxin‐2, and the underlying mechanisms [i.e. the prostaglandin E2 (PGE2) pathway] since PGE2 inhalation before an allergen challenge suppressed the eosinophil infiltration in human airways.

Methods
BALB/c mouse models of ovalbumin (OVA)‐induced allergic asthma with an ensuing low‐ or high‐dose Mp were used to assess IL‐4 expression, bronchoalveolar lavage (BAL) eosinophil, eotaxin‐2 and PGE2 levels, and lung mRNA levels of microsomal prostaglandin E synthase‐1 (mPGES‐1). Primary alveolar macrophages (pAMs) from naïve BALB/c mice were cultured to determine whether Mp‐induced PGE2 or exogenous PGE2 down‐regulates IL‐4/IL‐13‐induced eotaxin‐2.

Results
Low‐dose Mp in allergic mice significantly enhanced IL‐4 and eotaxin‐2, and moderately promoted lung eosinophilia, whereas high‐dose Mp significantly reduced lung eosinophilia and tended to decrease IL‐4 and eotaxin‐2. Moreover, in both OVA‐naïve and allergic mice, lung mPGES‐1 mRNA and BAL PGE2 levels were elevated in mice infected with high‐dose, but not low‐dose Mp. In pAMs, IL‐4/IL‐13 significantly increased eotaxin‐2, which was reduced by Mp infection accompanied by dose‐dependent PGE2 induction. Exogenous PGE2 inhibited IL‐4/IL‐13‐induced eotaxin‐2 in a dose‐dependent manner.

Conclusions
This study highlights a novel concept on how different bacterial loads in the lung modify the established allergic airway inflammation and thus interact with an allergen to further induce Th2 responses. That is, unlike high‐level Mp, low‐level Mp fails to effectively induce PGE2 to down‐regulate allergic responses (e.g. eotaxin‐2), thus maintaining or even worsening allergic inflammation in asthmatic airways.
Glucocorticoid treatment restores the impaired suppressive function of regulatory T cells in patients with relapsing–remitting multiple sclerosis
Authors: Xu L , Xu Z , Xu M
Published: June 2009

Patients relapsing from multiple sclerosis (MS) are treated with high-dose, short-term intravenous injection of glucocorticoid (GC), although its mechanism of action remains only partly understood. We evaluated the ex vivo and in vitro effects of GC on regulatory T cell (Treg) function in 14 relapsing–remitting MS (RR-MS) patients in acute phase and 20 healthy controls (HC). Treg function was enhanced significantly after 5 days of GC treatment. Furthermore, there was a trend towards increasing proportions of CD4+CD25+forkhead box P3+ T cells and interleukin-10 secretion with GC treatment when compared with HC. In conclusion, GC treatment restores the impaired Treg function in patients with RR-MS in its acute phase.
Conserved T cell and natural killer cell function in treatment-experienced adults receiving tenofovir plus didanosine as nucleoside reverse transcription inhibitor backbone
Authors: Costa P , Bozzano F , Fenoglio D , Beltrame A , Cenderello G , Di Biagio A , Ferrea G , Pagano G , De Maria A
Published: June 2009

Anti-retroviral treatment (ART) usually results in efficient control of virus replication and in immune reconstitution. Among potential adverse effects, impairment of immune responses in terms of CD4+ T cell counts has been attributed to some ART regimens, as with didanosine–tenofovir. We studied the functional integrity of adaptive and innate immunity during didanosine–tenofovir-containing ART. Two groups of extensively pretreated patients completing at least 48 weeks of ART containing either lamivudine–didanosine (n = 21) or tenofovir–didanosine (n = 25) were identified. In addition to standard clinical immune and virological parameters, we performed a flow cytometric analysis of natural killer (NK) cells, of memory and naive CD4+ T cells and of T cell receptor αβ+ T cells co-expressing inhibitory NK receptors. Functional analysis consisted in specific and total interferon-γ production by NK cells and of recall antigen proliferation of peripheral blood mononuclear cells. Comparable clinical immunological reconstitution and virological control were confirmed in the two groups of patients in the absence of clinically relevant adverse effects. The proportion of CD4+CD45RA+ T cells and of functionally inhibited killer immunoglobulin-like receptor T cell receptor αβ+ cells, the proliferation to recall antigens as well as NK cell phenotype and function as determined by interferon-γ production in patients treated with tenofovir–didanosine were comparable to those treated with a different regimen. Thus, no differences in functional innate or adaptive immune reconstitution are detected in drug-experienced human immunodeficiency virus-infected patients on tenofovir–didanosine nucleoside reverse transcription inhibitor regimens.
Pegylated interferon alpha‐2b (Peg‐IFN α‐2b) affects early virologic response dose‐dependently in patients with chronic hepatitis C genotype 1 during treatment with Peg‐IFN α‐2b plus ribavirin
Authors: Oze T. , Hiramatsu N. , Yakushijin T. , Kurokawa M. , Igura T. , Mochizuki K. , Imanaka K. , Yamada A. , Oshita M. , Hagiwara H. , Mita E. , Ito T. , Inui Y. , Hijioka T. , Tamura S. , Yoshihara H. , Hayashi E. , Inoue A. , Imai Y. , Kato M. , Yoshida Y. , Tatsumi T. , Ohkawa K. , Kiso S. , Kanto T. , Kasahara A. , Takehara T. , Hayashi N.
Published: June 2009

Summary.  Chronic hepatitis C (CH‐C) genotype 1 patients who achieved early virologic response have a high probability of sustained virologic response (SVR) following pegylated interferon (Peg‐IFN) plus ribavirin therapy. This study was conducted to evaluate how reducing drug doses affects complete early virologic response (c‐EVR) defined as hepatitis C virus (HCV) RNA negativity at week 12. Nine hundred eighty‐four patients with CH‐C genotype 1 were enrolled. Drug doses were evaluated independently on a body weight base from doses actually taken. From multivariate analysis, the mean dose of Peg‐IFN α‐2b during the first 12 weeks was the independent factor for c‐EVR (P = 0.02), not ribavirin. The c‐EVR rate was 55% in patients receiving ≥1.2 μg/kg/week of Peg‐IFN, and declined to 38% at 0.9–1.2 μg/kg/week, and 22% in patients given <0.9 μg/kg/week (P < 0.0001). Even with stratified analysis according to ribavirin dose, the dose‐dependent effect of Peg‐IFN on c‐EVR was observed, and similar c‐EVR rates were obtained if the dose categories of Peg‐IFN were the same. Furthermore, the mean dose of Peg‐IFN during the first 12 weeks affected HCV RNA negativity at week 24 (P < 0.0001) and SVR (P < 0.0001) in a dose‐dependent manner. Our results suggest that Peg‐IFN was dose‐dependently correlated with c‐EVR, independently of ribavirin dose. Thus, maintaining the Peg‐IFN dose as high as possible during the first 12 weeks can yield HCV RNA negativity and higher c‐EVR rates, leading to better SVR rates in patients with CH‐C genotype 1.
Invasion or invisibility: using genetic and distributional data to investigate the alien or indigenous status of the Atlantic populations of the peracarid isopod, Stenosoma nadejda (Rezig 1989)
Authors: XAVIER R. , SANTOS A. M. , LIMA F. P. , BRANCO M.
Published: June 2009

The peracarid isopod, Stenosoma nadejda (Rezig 1989), until recently considered to be endemic of the Mediterranean region, was first reported in the Atlantic coast of southern Spain in 2001, and in 2006 abundant populations were discovered throughout the southwestern Portuguese coast. This fast expansion was intriguing because, as a direct brooder, this species has limited mechanisms for dispersal, such as rafting on seaweeds. Did S. nadejda recently extend its range into the Atlantic or was it overlooked in the past? We examined the patterns of genetic diversity and population differentiation accordingly by sequencing the cytochrome c oxidase subunit I mitochondrial gene from 75 individuals collected in five locations in Atlantic Iberia and one in the Mediterranean. Our results indicate that the newly discovered Atlantic populations of S. nadejda appear to be old and have long persisted on Atlantic shores rather than being a recent introduction. High levels of genetic diversity and geographic structure were uncovered in what was initially suspected to be an ‘invasive’ species. Recent changes in population dynamics may have made S. nadejda more conspicuous in the Atlantic shores, or a more comprehensive survey led to the recognition of this species where it was not expected.
In situpopulation structure andex siturepresentation of the endangered Amur tiger
Authors: HENRY P. , MIQUELLE D. , SUGIMOTO T. , McCULLOUGH D. R. , CACCONE A. , RUSSELLO M. A.
Published: June 2009

The Amur tiger (Panthera tigris altaica) is a critically endangered felid that suffered a severe demographic contraction in the 1940s. In this study, we sampled 95 individuals collected throughout their native range to investigate questions relative to population genetic structure and demographic history. Additionally, we sampled targeted individuals from the North Americanex situpopulation to assess the genetic representation found in captivity. Population genetic and Bayesian structure analyses clearly identified two populations separated by a development corridor in Russia. Despite their well‐documented 20th century decline, we failed to find evidence of a recent population bottleneck, although genetic signatures of a historical contraction were detected. This disparity in signal may be due to several reasons, including historical paucity in population genetic variation associated with postglacial colonization and potential gene flow from a now extirpated Chinese population. Despite conflicting signatures of a bottleneck, our estimates of effective population size (Ne = 27–35) andNe/Nratio (0.07–0.054) were substantially lower than the only other values reported for a wild tiger population. Lastly, the extent and distribution of genetic variation in captive and wild populations were similar, yet gene variants persistedex situthat were lostin situ. Overall, our results indicate the need to secure ecological connectivity between the two Russian populations to minimize loss of genetic diversity and overall susceptibility to stochastic events, and support a previous study suggesting that the captive population may be a reservoir of gene variants lostin situ.
The ethanolamine branch of the Kennedy pathway is essential in the bloodstream form of Trypanosoma brucei
Authors: Gibellini Federica , Hunter William N. , Smith Terry K.
Published: June 2009

Phosphatidylethanolamine (GPEtn), a major phospholipid component of trypanosome membranes, is synthesized de novo from ethanolamine through the Kennedy pathway. Here the composition of the GPEtn molecular species in the bloodstream form of Trypanosoma brucei is determined, along with new insights into phospholipid metabolism, by in vitro and in vivo characterization of a key enzyme of the Kennedy pathway, the cytosolic ethanolamine‐phosphate cytidylyltransferase (TbECT). Gene knockout indicates that TbECT is essential for growth and survival, thus highlighting the importance of the Kennedy pathway for the pathogenic stage of the African trypanosome. Phosphatiylserine decarboxylation, a potential salvage pathway, does not appear to be active in cultured bloodstream form T. brucei, and it is not upregulated even when the Kennedy pathway is disrupted. In vivo metabolic labelling and phospholipid composition analysis by ESI‐MS/MS of the knockout cells confirmed a significant decrease in GPEtn species, as well as changes in the relative abundance of other phospholipid species. Reduction in GPEtn levels had a profound influence on the morphology of the mutants and it compromised mitochondrial structure and function, as well as glycosylphosphatidylinositol anchor biosynthesis. TbECT is therefore genetically validated as a potential drug target against the African trypanosome.
The ObgE/CgtA GTPase influences the stringent response to amino acid starvation in Escherichia coli
Authors: Persky Nicole S. , Ferullo Daniel J. , Cooper Deani L. , Moore Hayley R. , Lovett Susan T.
Published: June 2009

The stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro, implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo. Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.
Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis
Authors: Baraquet Claudine , Théraulaz Laurence , Iobbi‐Nivol Chantal , Méjean Vincent , Jourlin‐Castelli Cécile
Published: June 2009

Shewanella oneidensis uses a wide range of terminal electron acceptors for respiration. In this study, we show that the chemotactic response of S. oneidensis to anaerobic electron acceptors requires functional electron transport systems. Deletion of the genes encoding dimethyl sulphoxide and trimethylamine N‐oxide reductases, or inactivation of these molybdoenzymes as well as nitrate reductase by addition of tungstate, abolished electron acceptor taxis. Moreover, addition of nigericin prevented taxis towards trimethylamine N‐oxide, dimethyl sulphoxide, nitrite, nitrate and fumarate, showing that this process depends on the ΔpH component of the proton motive force. These data, together with those concerning response to metals (Bencharit and Ward, 2005), support the idea that, in S. oneidensis, taxis towards electron acceptors is governed by an energy taxis mechanism. Surprisingly, energy taxis in S. oneidensis is not mediated by the PAS‐containing chemoreceptors but rather by a chemoreceptor (SO2240) containing a Cache domain. Four other chemoreceptors also play a minor role in this process. These results indicate that energy taxis can be mediated by new types of chemoreceptors.
Listeria monocytogenesl‐forms respond to cell wall deficiency by modifying gene expression and the mode of division
Authors: Dell'Era Simone , Buchrieser Carmen , Couvé Elisabeth , Schnell Barbara , Briers Yves , Schuppler Markus , Loessner Martin J.
Published: June 2009

Cell wall‐deficient bacteria referred to as l‐forms have lost the ability to maintain or build a rigid peptidoglycan envelope. We have generated stable, non‐reverting l‐form variants of the Gram‐positive pathogen Listeria monocytogenes, and studied the cellular and molecular changes associated with this transition. Stable l‐form cells can occur as small protoplast‐like vesicles and as multinucleated, large bodies. They have lost the thick, multilayered murein sacculus and are surrounded by a cytoplasmic membrane only, although peptidoglycan precursors are still produced. While they lack murein‐associated molecules including Internalin A, membrane‐anchored proteins such as Internalin B are retained. Surprisingly, l‐forms were found to be able to divide and propagate indefinitely without a wall. Time‐lapse microscopy of fluorescently labelled l‐forms indicated a switch to a novel form of cell division, where genome‐containing membrane vesicles are first formed within enlarged l‐forms, and subsequently released by collapse of the mother cell. Array‐based transcriptomics of parent and l‐form cells revealed manifold differences in expression of genes associated with morphological and physiological functions. The l‐forms feature downregulated metabolic functions correlating with the dramatic shift in surface to volume ratio, whereas upregulation of stress genes reflects the difficulties in adapting to this unusual, cell wall‐deficient lifestyle.
Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase
Authors: Payne Kimberly , Sun Qingan , Sacchettini James , Hatfull Graham F.
Published: June 2009

Mycobacteriophages encounter a unique problem among phages of Gram‐positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid‐rich outer membrane covalently attached to the arabinogalactan–peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an α/β hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four‐helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles ΔlysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan–peptidoglycan layer.
In vivo localizations of membrane stress controllers PspA and PspG in Escherichia coli
Authors: Engl Christoph , Jovanovic Goran , Lloyd Louise J. , Murray Heath , Spitaler Martin , Ying Liming , Errington Jeff , Buck Martin
Published: June 2009

The phage shock protein (Psp) response in Gram‐negative bacteria counteracts membrane stress. Transcription of the PspF regulon (pspABCDE and pspG) in Escherichia coli is induced upon stresses that dissipate the proton motive force (pmf). Using GFP fusions we have visualized the subcellular localizations of PspA (a negative regulator and effector of Psp) and PspG (an effector of Psp). It has previously been proposed that PspA evenly coates the inner membrane of the cell. We now demonstrate that instead of uniformly covering the entire cell, PspA (and PspG) is highly organized into what appear to be distinct functional classes (complexes at the cell pole and the lateral cell wall). Real‐time observations revealed lateral PspA and PspG complexes are highly mobile, but absent in cells lacking MreB. Without the MreB cytoskeleton, induction of the Psp response is still observed, yet these cells fail to maintain pmf under stress conditions. The two spatial subspecies therefore appear to be dynamically and functionally distinct with the polar clusters being associated with sensory function and the mobile complexes with maintenance of pmf.
Identification of small RNAs in Mycobacterium tuberculosis
Authors: Arnvig Kristine B. , Young Douglas B.
Published: June 2009

In spite of being one of our most prominent bacterial pathogens, the presence of small regulatory RNAs (sRNAs) has not previously been investigated in Mycobacterium tuberculosis. Post‐transcriptional regulation of gene expression by sRNA molecules has been demonstrated in a wide range of pathogenic bacteria and has been shown to play a significant role in the control of virulence. By screening cDNA libraries prepared from low‐molecular weight RNA from M. tuberculosis we have identified nine putative sRNA molecules, including cis‐encoded antisense transcripts from within open reading frames and trans‐encoded transcripts from intergenic regions. sRNAs displayed differential expression between exponential and stationary phase, and during a variety of stress conditions. Two of the cis‐encoded sRNAs were associated with genes encoding enzymes involved in lipid metabolism, desA1 and pks12. These sRNAs showed complementarity to multiple M. tuberculosis genes, suggesting the potential to act as both cis‐encoded and trans‐encoded sRNAs. Overexpression of selected trans‐encoded sRNAs had profound impact on growth of M. tuberculosis and M. smegmatis. This is the first experimental evidence of sRNAs in M. tuberculosis and it will be important to consider the potential influence of sRNA regulation when studying the transcriptome and the proteome of M. tuberculosis during infection.
Transient lower oesophageal sphincter relaxation in achalasia: everything but LOS relaxation
Authors: Kwiatek M. A. , Post J. , Pandolfino J. E. , Kahrilas P. J.
Published: June 2009

Abstract  In conducting clinical high‐resolution oesophageal pressure topography (HROPT) studies we observed that after subjects sat upright between series of supine and upright test swallows, they frequently had a transient lower oesophageal sphincter relaxation (TLOSR). When achalasia patients were studied in the same protocol, they exhibited a similar HROPT event leading to the hypothesis that achalasics had incomplete TLOSRs. We reviewed clinical HROPT studies of 94 consecutive non‐achalasics and 25 achalasics. Studies were analyzed for a TLOSR‐like event during the study and, when observed, that TLOSR‐like event was characterized for the degree and duration of distal oesophageal shortening, the degree of LOS relaxation, associated crural diaphragm (CD) inhibition, oesophageal pressurization and upper oesophageal sphincter (UOS) relaxation. About 64/94 (68%) non‐achalasics and 15/24 (63%) of achalasics had a pressure topography event after the posture change characterized by a prolonged period of distal oesophageal shortening and/or LOS relaxation. Events among the non‐achalasics and achalasics were similar in terms of magnitude and duration of shortening and all were associated with CD inhibition. Similar proportions had associated non‐deglutitive UOS relaxations. The only consistent differences were the absence of associated LOS relaxation and the absence of HROPT evidence of reflux among the achalasics leading us to conclude that their events were incomplete TLOSRs. Achalasic patients exhibit a selective defect in the TLOSR response suggesting preservation of all sensory, central and efferent aspects of the requisite neural substrate with the notable exception of LOS relaxation, a function of inhibitory (nitrergic) myenteric plexus neurons.
Mycobacterium tuberculosis Entry into Mast Cells Through Cholesterol‐rich Membrane Microdomains
Authors: Muñoz S. , Rivas‐Santiago B. , Enciso J. A.
Published: June 2009

Cholesterol‐enriched membrane microdomains (lipid rafts) play a role in the uptake of many pathogens. Mycobacteria are one of the intracellular pathogens that utilize lipid rafts in order to invade both phagocytic and non‐phagocytic cells. However, the mechanism of Mycobacterium tuberculosis uptake by mast cell is not known. To address this issue, we investigated the interaction of M. tuberculosis (H37Rv strain) with mast cells. Confocal microscopy showed that interaction of mycobacterium with mast cell resulted in changes in the mast cell surface, with formation of pseudopod‐like structure and activation with visibly extruded granules. Moreover, infection of mast cells with Mycobacteria induced cholesterol accumulation at the site of bacterial entry and around intracellular mycobacteria. Disruption of mast cells lipid rafts by cholesterol depletion markedly inhibited the mycobacterium entry. Intracellular multiplication of M. tuberculosis within mast cells was also observed. Overall, our results indicate that M. tuberculosis employs a cholesterol‐dependent pathway to infect mast cells, which leads to degranulation and mast cell morphological changes. These results suggest that although mast cells are capable to respond to M. tuberculosis infection, entry of mycobacterium through lipid rafts may allow replication within mast cells.
A Systematic Review and Meta‐analysis of the Efficacy and Safety of Etanercept for Treating Rheumatoid Arthritis
Authors: Wiens Astrid , Correr Cassyano Januario , Pontarolo Roberto , Venson Rafael , Quinalha Juliana Vasconcelos , Otuki Michel Fleith
Published: June 2009

The aim of this study was to evaluate the efficacy and safety of etanercept (ETA) for treating rheumatoid arthritis. A systematic review was performed to search for randomized clinical trials comparing subcutaneous doses of ETA at 25 mg twice a week or 50 mg weekly to a placebo group, with or without methotrexate. Studies of low quality (less than 3 points on Jadad’s scale) were excluded. The efficacy was assessed by using the criteria of the American College of Rheumatology (ACR). Safety data were evaluated based on serious adverse events, serious infections, malignancy and deaths. Withdrawals as a result of adverse events or lack of efficacy were also evaluated. Eight studies met the inclusion criteria, comprising 2385 patients. In the efficacy meta‐analysis, a greater number of ETA‐treated patients achieved the efficacy criteria within 6 months of treatment, where the relative risk (RR) was 2.94 [2.27, 3.81] for achieving ACR20, 5.28 [3.12, 8.92] for ACR50 and 4.83 [1.74, 13.47] for ACR70. After 1 year, the RR for achieving ACR20, ACR50 and ACR70 were 1.14 [1.07, 1.23], 1.36 [1.21, 1.53] and 1.56 [1.30, 1.88], respectively. This response rates were higher for ETA‐treated patients in comparison with control group patients. For safety, there were no statistically significant differences between treated patients and controls. This was also confirmed by withdrawals as a result of adverse events, which were not statistically different between the two groups. However, more patients withdrew from control groups because of a lack of efficacy as compared with ETA groups (RR = 0.48 [0.30, 0.78]).
Reduced Apoptosis of Mouse Macrophages Induced by yscW Mutant of Yersinia pestis Results from the Reduced Secretion of YopJ and Relates to Caspase‐3 Signal Pathway
Authors: Bi Y. , Du Z. , Yang H. , Guo Z. , Tan Y. , Zhu Z. , Yang Ruifu
Published: June 2009

The virulence of the pathogenic Yersinia species depends on a plasmid‐encoded type III secretion system (T3SS) that injects six Yersinia outer protein (Yop) effector proteins into the cytosol of macrophages, leading to disruption of host defence mechanisms. Here, we report that a T3SS structural protein YscW of Yersinia pestis contributed to the induction of apoptosis of murine macrophages. The apoptotic percentage of macrophages, from both mouse peritoneal cavity and spleen, and of RAW264.7 cell line, caused by the yscW mutant strain was significantly lower than that by wild type (WT) Y. pestis and yscW complemented strain. Meanwhile, detection of caspase‐3 activity in macrophages, a key apoptosis‐inducing protein, showed coincident results with the changes of macrophage apoptosis induced by WT, yscW mutant and complemented strains, indicating that macrophage apoptosis was related to caspase‐3 signal pathways. However, ectopic expression of YscW in RAW264.7 cells cannot increase the macrophage apoptosis and death, suggesting that YscW itself could not induce macrophage apoptosis directly. To get insight into the mechanism of this phenomenon, we investigated the secretion of YopJ, which has been thought to be the only Yop effector related to apoptosis, in WT, mutant and complemented strains, respectively. Results showed that in yscW mutant strain, secretion of YopJ was decreased significantly in the supernatant than that in WT or complemented strain. This means although YscW does not induce apoptosis directly, it can indirectly affect apoptosis through reducing the secretion of YopJ.
Studies on the Mechanism by Which Antigen‐Specific IgG Suppresses Primary Antibody Responses: Evidence for Epitope Masking and Decreased Localization of Antigen in the Spleen
Authors: Getahun A. , Heyman B.
Published: June 2009

Immunoglobulin (IgG) has the ability to suppress the Ab response against the Ag to which it binds. Although the mechanism remains unclear, this phenomenon has physiological relevance and is used clinically in Rh prophylaxis. As suppression works well in mice lacking the inhibitory FcγRIIB, the two most likely explanations are that IgG masks epitopes and/or that IgG increases the clearance of Ag. In the present study, mice were immunized with sheep red blood cells (SRBC) to which the hapten 5‐iodo‐4‐hydroxyl‐3‐nitrophenacetyl (NIP) was conjugated at high or low density and the ability of IgG anti‐NIP to suppress the Ab response to NIP and SRBC was assayed. Only the NIP‐specific response was suppressed when mice were immunized with SRBC‐NIPlow, whereas both NIP‐ and SRBC‐specific responses were suppressed when SRBC‐NIPhigh was used. This is best explained by epitope masking; at high epitope density, IgG also blocks neighbouring epitopes from recognition by B cells. We also examined the effects of IgG‐mediated suppression on T‐cell responses directly in vivo. While IgG anti‐SRBC administered with sheep red blood cells ovalbumin (SRBC‐OVA) almost completely suppressed the anti‐SRBC and anti‐OVA Ab responses, the OVA‐specific T‐cell response was still 50% of that observed in control mice. This is probably the result of decreased Ag exposure as IgG‐bound SRBC were cleared faster from the bloodstream and were found at lower concentration in the spleen than unbound SRBC. These results suggest that both Ag clearance and epitope masking occurs during IgG‐mediated suppression, but that under physiological circumstances epitope masking is the predominant mechanism.
HIV Induces both a Down‐Regulation of IRAK‐4 that Impairs TLR Signalling and an Up‐regulation of the Antibiotic Peptide Dermcidin in Monocytic Cells
Authors: Pathak S. , De Souza G. A. , Salte T. , Wiker H. G. , Åsjö B.
Published: June 2009

HIV‐infected individuals have an increased risk of invasive bacterial infections, even at early clinical stages with relatively normal CD4+ T‐cell counts. The pathogenic mechanisms behind this are not fully understood. However, an increasing number of studies indicate that HIV may impair the innate immunity to bacteria by infecting key cells of the monocyte/macrophage lineage. In this study, the effects of HIV infection on the protein profile of undifferentiated monocyte‐like THP‐1 cells were examined by a mass spectrometric approach based on stable isotope labelling with amino acid in cell culture (SILAC). We identified 651 proteins, of which nine proteins were down‐regulated and 17 proteins were up‐regulated in HIV‐infected THP‐1 cells as compared to uninfected controls. Most remarkably, the IL‐1 receptor associated kinase 4 (IRAK‐4), which is essential for virtually all TLR signalling, was suppressed, whereas the precursor for the antibiotic peptide Dermcidin was up‐regulated in HIV‐infected cells. Upon stimulation of either TLR2 or TLR4, the HIV‐infected THP‐1 cells displayed reduced TNF‐α secretion. The HIV‐induced down‐regulation of IRAK‐4 was reconfirmed in monocyte‐derived macrophage cell cultures. These data suggests that HIV may impair the TLR signalling cascade for pathogen recognition in cells of the monocyte/macrophage lineage and thus, may reduce the ability of the innate immune system to sense invading pathogens and initiate appropriate responses.
MHC II and the Endocytic Pathway: Regulation by Invariant Chain
Authors: Landsverk O. J. B. , Bakke O. , Gregers T. F.
Published: June 2009

The major histocompatibility complex (MHC) class I and II molecules perform vital functions in innate and adaptive immune responses towards invading pathogens. MHC class I molecules load peptides in the endoplasmatic reticulum (ER) and display them to the T cell receptors (TcR) on CD8+ T lymphocytes. MHC class II molecules (MHC II) acquire their peptides in endosomes and present these to the TcR on CD4+ T lymphocytes. They are vital for the generation of humoral immune responses. MHC II assembly in the ER and trafficking to endosomes is guided by a specialized MHC II chaperone termed the invariant chain (Ii). Ii self‐associates into a trimer in the ER, this provides a scaffold for the assembly of three MHC II heterodimers and blocks their peptide binding grooves, thereby avoiding premature peptide binding. Ii then transports the nascent MHC II to more or less specialized compartment where they can load peptides derived from internalized pathogens.
Increased Expression of Activation‐Induced Cytidine Deaminase is Associated with Anti‐CCP and Rheumatoid Factor in Rheumatoid Arthritis
Authors: Xu X. , Hsu H.‐C. , Chen J. , Grizzle W. E. , Chatham W. W. , Stockard C. R. , Wu Q. , Yang P. A. , Holers V. M. , Mountz J. D.
Published: June 2009

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL‐17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation‐induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL‐17. The results of immunohistochemical staining showed that organized AID+ germinal centres were observed in six of the 12 RA synovial samples, and AID+ cells were found almost exclusively in the B‐cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID+ cells. Significantly higher levels of AID mRNA (Aicda) detected by RT‐PCR were found in the PBMCs from RA patients than PBMCs from normal controls (P < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10+IgM+CD20+ B‐cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls (P < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) (P ≤ 0.0001) and anti‐cyclic citrullinated peptide (CCP) (P = 0.0005). Serum levels of IFN‐γ (P = 0.0005) and IL‐17 (P = 0.007), but not IL‐4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN‐γ and IL‐17, leading to the development of anti‐CCP and RF.
Diagnosis of antiretroviral therapy failure in Malawi: poor performance of clinical and immunological WHO criteria
Authors: Van Oosterhout Joep J.G. , Brown Lillian , Weigel Ralf , Kumwenda Johnstone J. , Mzinganjira Dalitso , Saukila Nasinuku , Mhango Brian , Hartung Thomas , Phiri Sam , Hosseinipour Mina C.
Published: June 2009

Objectives  In antiretroviral therapy (ART) scale‐up programmes in sub‐Saharan Africa viral load monitoring is not recommended. We wanted to study the impact of only using clinical and immunological monitoring on the diagnosis of virological ART failure under routine circumstances.

Methods  Clinicians in two urban ART clinics in Malawi used clinical and immunological monitoring to identify adult patients for switching to second‐line ART. If patients met clinical and/or immunological failure criteria of WHO guidelines and had a viral load <400 copies/ml there was misclassification of virological ART failure.

Results  Between January 2006 and July 2007, we identified 155 patients with WHO criteria for immunological and/or clinical failure. Virological ART failure had been misclassified in 66 (43%) patients. Misclassification was significantly higher in patients meeting clinical failure criteria (57%) than in those with immunological criteria (30%). On multivariate analysis, misclassification was associated with being on ART <2 years [OR = 7.42 (2.63, 20.95)] and CD4 > 200 cells/μl [OR = 5.03 (2.05, 12.34)]. Active tuberculosis and Kaposi’s sarcoma were the most common conditions causing misclassification of virological ART failure.

Conclusion  Misclassification of virological ART failure occurs frequently using WHO clinical and immunological criteria of ART failure for poor settings. A viral load test confirming virological ART failure is therefore advised to avoid unnecessary switching to second‐line regimens.