Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Human antibody responses to Schistosoma mansoni: does antigen directed, isotype restriction result in the production of blocking antibodies?

After treatment young Kenyan schoolchildren are highly susceptible to reinfection with Schistosoma mansoni. Older children and adults are resistant to reinfection. There is no evidence that this age related resistance is due to a slow development of protective immunological mechanisms, rather, it appears that young children are susceptible because of the presence of blocking antibodies which decline with age, thus allowing the expression of protective responses. Correlations between antibody responses to different stages of the parasite life-cycle suggest that, in young children, antigen directed, isotype restriction of the response against cross-reactive polysaccharide egg antigens results in an ineffectual, or even blocking antibody response to the schistosomulum.

Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Small ubiquitin-like modifier conjugating enzyme with active site mutation acts as dominant negative inhibitor of SUMO conjugation in arabidopsis(F).

Small ubiquitin-like modifier (SUMO) conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.

Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

Simultaneous CD8+ T cell responses to multiple tumor antigen epitopes in a multipeptide melanoma vaccine.

The recent identification and molecular characterization of tumor-associated antigens recognized by tumor-reactive CD8+ T lymphocytes has led to the development of antigen-specific immunotherapy of cancer. Among other approaches, clinical studies have been initiated to assess the in vivo immunogenicity of tumor antigen-derived peptides in cancer patients. In this study, we have analyzed the CD8+ T cell response of an ocular melanoma patient to a vaccine composed of four different tumor antigen-derived peptides administered simultaneously in incomplete Freund's adjuvant (IFA). Peptide NY-ESO-1(157-165) was remarkably immunogenic and induced a CD8+ T cell response detectable ex vivo at an early time point of the vaccination protocol. A CD8+ T cell response to the peptide analog Melan-A(26-35 A27L) was also detectable ex vivo at a later time point, whereas CD8+ T cells specific for peptide tyrosinase(368-376) were detected only after in vitro peptide stimulation. No detectable CD8+ T cell response to peptide gp100(457-466) was observed. Vaccine-induced CD8+ T cell responses declined rapidly after the initial response but increased again after further peptide injections. In addition, tumor antigen-specific CD8+ T cells were isolated from a vaccine injection site biopsy sample. Importantly, vaccine-induced CD8+ T cells specifically lysed tumor cells expressing the corresponding antigen. Together, these data demonstrate that simultaneous immunization with multiple tumor antigen-derived peptides can result in the elicitation of multiepitope-directed CD8+ T cell responses that are reactive against antigen-expressing tumors and able to infiltrate antigen-containing peripheral sites.

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.

Reasons why emergency department providers do not rely on the pneumonia severity index to determine the initial site of treatment for patients with pneumonia.

BACKGROUND: Many emergency department (ED) providers do not follow guideline recommendations for the use of the pneumonia severity index (PSI) to determine the initial site of treatment for patients with community-acquired pneumonia (CAP). We identified the reasons why ED providers hospitalize low-risk patients or manage higher-risk patients as outpatients. METHODS: As a part of a trial to implement a PSI-based guideline for the initial site of treatment of patients with CAP, we analyzed data for patients managed at 12 EDs allocated to a high-intensity guideline implementation strategy study arm. The guideline recommended outpatient care for low-risk patients (nonhypoxemic patients with a PSI risk classification of I, II, or III) and hospitalization for higher-risk patients (hypoxemic patients or patients with a PSI risk classification of IV or V). We asked providers who made guideline-discordant decisions on site of treatment to detail the reasons for nonadherence to guideline recommendations. RESULTS: There were 1,306 patients with CAP (689 low-risk patients and 617 higher-risk patients). Among these patients, physicians admitted 258 (37.4%) of 689 low-risk patients and treated 20 (3.2%) of 617 higher-risk patients as outpatients. The most commonly reported reasons for admitting low-risk patients were the presence of a comorbid illness (178 [71.5%] of 249 patients); a laboratory value, vital sign, or symptom that precluded ED discharge (73 patients [29.3%]); or a recommendation from a primary care or a consulting physician (48 patients [19.3%]). Higher-risk patients were most often treated as outpatients because of a recommendation by a primary care or consulting physician (6 [40.0%] of 15 patients). CONCLUSION: ED providers hospitalize many low-risk patients with CAP, most frequently for a comorbid illness. Although higher-risk patients are infrequently treated as outpatients, this decision is often based on the request of an involved physician.

Anatomy of contaminated aquifers of an industrial site: insights from the stable isotope compositions of waters and dissolved inorganic carbon

The hydrogen and oxygen isotopes of water and the carbon isotope composition of dissolved inorganic carbon (DIC) from different aquifers at an industrial site, highly contaminated by organic pollutants representing residues of the former gas production, have been used as natural tracers to characterize the hydrologic system. On the basis of their stable isotope compositions as well as the seasonal variations, different groups of waters (precipitation, surface waters, groundwaters and mineral waters) as well as seasonably variable processes of mixing between these waters can clearly be distinguished. In addition, reservoir effects and infiltration rates can be estimated. In the northern part of the site an influence of uprising mineral waters within the Quaternary aquifers, presumably along a fault zone, can be recognized. Marginal infiltration from the Neckar River in the cast and surface water infiltration adjacent to a steep hill on the western edge of the site with an infiltration rate of about one month can also be resolved through the seasonal variation. Quaternary aquifers closer to the centre of the site show no seasonal variations, except for one borehole close to a former mill channel and another borehole adjacent to a rain water channel. Distinct carbon isotope compositions and concentrations of DIC for these different groups of waters reflect variable influence of different components of the natural carbon cycle: dissolution of marine carbonates in the mineral waters, biogenic, soil-derived CO2 in ground- and surface waters, as well as additional influence of atmospheric CO2 for the surface waters. Many Quaternary aquifer waters have, however, distinctly lower delta(13)C(DIC) values and higher DIC concentrations compared to those expected for natural waters. Given the location of contaminated groundwaters at this site but also in the industrially well-developed valley outside of this site, the most likely source for the low C-13(DIC) values is a biodegradation of anthropogenic organic substances, in particular the tar oils at the site.

HCF-1 is cleaved in the active site of O-GlcNAc transferase.

Host cell factor-1 (HCF-1), a transcriptional co-regulator of human cell-cycle progression, undergoes proteolytic maturation in which any of six repeated sequences is cleaved by the nutrient-responsive glycosyltransferase, O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). We report that the tetratricopeptide-repeat domain of O-GlcNAc transferase binds the carboxyl-terminal portion of an HCF-1 proteolytic repeat such that the cleavage region lies in the glycosyltransferase active site above uridine diphosphate-GlcNAc. The conformation is similar to that of a glycosylation-competent peptide substrate. Cleavage occurs between cysteine and glutamate residues and results in a pyroglutamate product. Conversion of the cleavage site glutamate into serine converts an HCF-1 proteolytic repeat into a glycosylation substrate. Thus, protein glycosylation and HCF-1 cleavage occur in the same active site.

Directed Search over the Life Cycle

We develop a life-cycle model of the labor market in which different worker-firm matches have different quality and the assignment of the right workers to the right firms is time consuming because of search and learning frictions. The rate at which workers move between unemployment, employment and across different firms is endogenous because search is directed and, hence, workers can choose whether to seek low-wage jobs that are easy to find or high-wage jobs that are hard to find. We calibrate our theory using data on labor market transitions aggregated across workers of different ages. We validate our theory by showing that it predicts quite well the pattern of labor market transitions for workers of different ages. Finally, we use our theory to decompose the age profiles of transition rates, wages and productivity into the effects of age variation in work-life expectancy, human capital and match quality.

Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Lack of directed V alpha 14-J alpha 281 rearrangements in NK1+ T cells.

NK1.1+ T cells are an unusual subset of TCR alpha beta cells distinguished by their highly restricted V beta repertoire and predominant usage of an invariant V alpha 14-J alpha 281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant alpha chain, we have analyzed V alpha 14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1+ T cells. As expected a high proportion (17/20) of the hybrids had rearranged V alpha 14 to J alpha 281. However, V alpha 14-J alpha 281 rearrangements always occurred on only one chromosome and were accompanied by other V alpha-J alpha rearrangements (not involving V alpha 14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of V alpha 14-J alpha 281 rearrangements in NK1.1+ T cells.