In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

Citrobacter rodentium Requires Intestinal Neural Wiskott-Aldrich Syndrome Protein (N-WASp) for Actin Pedestal Formation, Colonization and Epithelial Barrier Disruption In Vivo

Background: Citrobacter rodentium is a natural mouse pathogen that is genetically closelyrelated to the human enteric pathogens enteropathogenic and enterohemorrhagic E. coli.Among the repertoire of conserved virulence factors that these pathogens deliver via typeIII secretion, Tir and EspF are responsible for the formation of characteristic actin-richpedestals and disruption of tight junction integrity, respectively. There is evidence In Vitrothese effectors accomplish this, at least in part, by subverting the normal host cellularfunctions of N-WASP, a critical regulator of branched chain actin assembly. Although NWASPhas been shown to be involved in pedestal formation In Vitro, the requirements ofN-WASP-mediated actin pedestals for intestinal colonization by attaching/effacing (A/E)pathogens In Vivo is not known. Furthermore, it is not known whether N-WASP is requiredfor EspF-mediated tight junction disruption. Methods: To investigate the role of N-WASPin the gut epithelium, we generated mice with intestine-specific deletion of N-WASP(iNWKO), by mating mice homozygous for a floxed N-WASP allele (N-WASPL2L/L2L) tomice expressing Cre recombinase under the villin promoter. Separately housed groups ofWT and iNWKO mice were inoculated with 5x108 GFP-expressing C. rodentium by intragastriclavage. Stool was collected 2, 4, 7, and 12 days after infection, and recoverablecolony forming units (CFUs) of C. rodentium were quantified by plating serial dilutions ofhomogenized stool on MacConkey's agar. GFP+ colonies were counted after 24 hoursincubation at 37°C. The presence of actin pedestals was investigated by electron microscopy(EM), and tight junction morphology was assessed by immunofluorescence staining ofoccludin, ZO-1 and claudin-2. Results: C. rodentium infection did not result in mortalityin WT or iNWKO mice. Compared to controls, iNWKO mice exhibited higher levels ofbacterial shedding during the first 4 days of infection (day 4 average: WT 5.2x104 CFU/gvs. iNWKO 4.7x105 CFU/g, p=0.08), followed by a more rapid clearance of C. rodentium, (day7-12 average: WT 2x106 CFU/g vs. iNWKO 2.7x105, p=0.01). EM and immunofluorescencerevealed the complete lack of actin pedestals in iNWKO mice and no mucosa-associatedGFP+ C. rodentium by day 7. WT controls exhibited tight junction disruption, reflected byaltered distribution of ZO-1, whereas iNWKO mice had no change in the pattern of ZO-1.Conclusion: Intestinal N-WASP is required for actin pedestal formation by C. rodentium InVivo, and ablation of N-WASP is associated with more rapid bacterial clearance and decreasedability of C. rodentium to disrupt intercellular junctions.

Long-term in vivo regeneration of peripheral nerves through bioengineered nerve grafts.

Although autologous nerve graft is still the first choice strategy in nerve reconstruction, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to improve nerve regeneration. Nerve fibrin conduits were seeded with various cell types: primary Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC). Two further control groups were fibrin conduits without cells and autografts. Conduits were used to bridge a 1 cm rat sciatic nerve gap in a long term experiment (16 weeks). Functional and morphological properties of regenerated nerves were investigated. A reduction in muscle atrophy was observed in the autograft and in all cell-seeded groups, when compared with the empty fibrin conduits. SC showed significant improvement in axon myelination and average fiber diameter of the regenerated nerves. dASC were the most effective cell population in terms of improvement of axonal and fiber diameter, evoked potentials at the level of the gastrocnemius muscle and regeneration of motoneurons, similar to the autografts. Given these results and other advantages of adipose derived stem cells such as ease of harvest and relative abundance, dASC could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

In vivo 13C NMR spectroscopy and metabolic modeling in the brain: a practical perspective.

In vivo 13C NMR spectroscopy has the unique capability to measure metabolic fluxes noninvasively in the brain. Quantitative measurements of metabolic fluxes require analysis of the 13C labeling time courses obtained experimentally with a metabolic model. The present work reviews the ingredients necessary for a dynamic metabolic modeling study, with particular emphasis on practical issues.

Experimental cutaneous Leishmaniasis: a powerful model to study in vivo the mechanisms underlying genetic differences in Th subset differentiation.

The murine model of infection with Leishmania major has allowed the demonstration in vivo of the importance CD4+ T cell subsets, distinguishable by the pattern of cytokines they produce, on the outcome of infectious diseases. Genetically determined resistance and susceptibility to infection with this parasite are the result of the development of Th1 and Th2 response, respectively. In this short paper, we present some results obtained in our group pertaining to the analysis of the mechanisms, operational during the early phase of this infection, responsible for the maturation of these functionally distinct CD4+ responses.

In vitro and in vivo evaluation of sunitinib eluting beads

Background: Chemoembolization is used to treat liver malignancies. However recurrence occurs frequently, possibly because of neoangiogenesis triggered by ischemia caused by the embolic agent. In this context, the combination of an embolic agent with an anti-angiogenic drug seems appealing. This study characterizes the in vitro loading and release profile of sunitinib eluting beads of different sizes and their pharmacokinetic profile in a rabbit model. Methods: 70-150 μm and 100-300 μm drug eluting beads (DC Bead, Biocompatibles UK) were loaded by incubation in a sunitinib hydrochloride solution. Drug was quantified by spectrophotometry at 430 nm. Drug release was measured over one-week periods and normalized using an internal standard in 30% ethanol in NaCl 0.9%. New-Zealand white rabbits were used. Eight animals received 0.2 ml of 100-300 μm DC Bead loaded with 6 mg of sunitinib in the hepatic artery (group 1) and 4 animals received 6 mg of sunitinib p.o. (group 2). Half of the animals were sacrificed after 6 hours and half after24 hours. Liver enzymes were measured at 0, 6 and 24 hours in both groups. Plasmatic sunitinib concentration was determined by tandem mass spectroscopy (LC MS/MS) at 0, 1, 2, 3, 4, 5, 6 and 24 hours. At sacrifice, the livers were harvested and sunitinib concentration in liver tissue was assessed by LC MS/MS. Results: High drug loading was obtained for both microsphere bead sizes. Particle shrinking was observed with adsorption of sunitinib. Almost complete release of sunitinib was detected under physiological conditions, with very similar release for 70-150 μm and 100-300 μm (t50%=1.2 h) DC Bead. Conclusions: Sunitinib eluting beads are well tolerated by rabbits when administered in the hepatic artery. No unexpected toxicity was observed. Very high drug concentration can be obtained at the site of embolization with minimal systemic passage.

Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice.

The bioavailability of nitric oxide (NO) within the vascular wall is limited by superoxide anions (O2.-). The relevance of extracellular superoxide dismutase (ecSOD) for the detoxification of vascular O2.- is unknown. We determined the involvement of ecSOD in the control of blood pressure and endothelium-dependent responses in angiotensin II-induced hypertension and renovascular hypertension induced by the two-kidney, one-clip model in wild-type mice and mice lacking the ecSOD gene. Blood pressure was identical in sham-operated ecSOD+/+ and ecSOD-/- mice. After 6 days of angiotensin II-treatment and 2 and 4 weeks after renal artery clipping, blood pressure was significantly higher in ecSOD-/- than ecSOD+/+ mice. Recombinant ecSOD selectively decreased blood pressure in hypertensive ecSOD-/- mice, whereas ecSOD had no effect in normotensive and hypertensive ecSOD+/+ mice. Compared with sham-operated ecSOD+/+ mice, sham-operated ecSOD-/- mice exhibited attenuated acetylcholine-induced relaxations. These responses were further depressed in vessels from clipped animals. Vascular O2.-, as measured by lucigenin chemiluminescence, was higher in ecSOD-/- compared with ecSOD+/+ mice and was increased by clipping. The antioxidant tiron normalized relaxations in vessels from sham-operated and clipped ecSOD-/-, as well as from clipped ecSOD+/+ mice. In contrast, in vivo application of ecSOD selectively enhanced endothelium-dependent relaxation in vessels from ecSOD-/- mice. These data reveal that endogenous ecSOD is a major antagonistic principle to vascular O2.-, controlling blood pressure and vascular function in angiotensin II-dependent models of hypertension. ecSOD is expressed in such an abundance that even in situations of high oxidative stress no relative lack of enzyme activity occurs.

Brain Spectrins 240/235 and 240/235E: Differential Expression During Development of Chicken Dorsal Root Ganglia in vivo and in vitro.

Brain spectrin, a membrane-related cytoskeletal protein, exists as two isoforms. Brain spectrin 240/235 is localized preferentially in the perikaryon and axon of neuronal cells and brain spectrin 240/235E is found essentially in the neuronal soma and dendrites and in glia (Riederer et al., 1986, J. Cell Biol., 102, 2088 - 2097). The sensory neurons in dorsal root ganglia, devoid of any dendrites, make a good tool to investigate such differential expression of spectrin isoforms. In this study expression and localization of both brain spectrin isoforms were analysed during early chicken dorsal root ganglia development in vivo and in culture. Both isoforms appeared at embryonic day 6. Brain spectrin 240/235 exhibited a transient increase during embryonic development and was first expressed in ventrolateral neurons. In ganglion cells in situ and in culture this spectrin type showed a somato - axonal distribution pattern. In contrast, brain spectrin 240/235E slightly increased between E6 and E15 and remained practically unchanged. It was localized mainly in smaller neurons of the mediodorsal area as punctate staining in the cytoplasm, was restricted exclusively to the ganglion cell perikarya and was absent from axons both in situ and in culture. This study suggests that brain spectrin 240/235 may contribute towards outgrowth, elongation and maintenance of axonal processes and that brain spectrin 240/235E seems to be exclusively involved in the stabilization of the cytoarchitecture of cell bodies in a selected population of ganglion cells.

Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo.

Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal. Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo. While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs. To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge. Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

Gene expression profiling in human pathogenic dermatophytes in vitro and in vivo

Objectives: Dermatophytes are highly specialized fungi which are the most common agents of superficial mycoses in humans and animals. The particular ability of these microorganisms to invade and multiply within keratinized host structures is presumably linked to their secreted keratinolytic activity, which is therefore a major putative virulence attribute of these fungi. The overall adaptation and transcriptional response of dermatophytes during protein degradation and/or infection is largely unknown. Methods: A Trichophyton rubrum cDNA microarray was developed and used for the transcriptional analysis of T. rubrum and Arthroderma benhamiae cells during growth on protein substrates. Moreover, the gene expression profile in A. benhamiae cells was monitored during infection of guinea pigs. Results: T. rubrum and A. benhamiae cells activate a large set of genes encoding secreted endo- and exoproteases during growth on soy and keratin. In addition, other specifically induced factors with potential implication in protein utilization were identified, e.g. multiple transporters, metabolic enzymes, transcription factors and hypothetical proteins with unknown function. Notably however, the protease gene expression profile in the fungal cells during infection was significantly different from the pattern elicited during in vitro growth on keratin. Conclusions: Our results suggest specific functions of individual proteases during infection, which may not be restricted to the degradation of keratin. This first, broad in vivo transcriptional profiling approach in dermatophytes gives new molecular insights into pathogenicity associated adaptation mechanisms that make these microorganisms the most successful causitive agents of superficial mycoses.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Fungos endofíticos isolados de ápices caulinares de pupunheira cultivada in vivo e in vitro

A perda de plantas micropropagadas ocorre, principalmente, pela presença de microrganismos, responsáveis pela morte das plantas no início da cultura ou em seu estabelecimento no campo. O trabalho teve como objetivo a identificação, por taxonomia clássica, e por meio de técnicas moleculares, de fungos presentes nos ápices caulinares de pupunheiras sadias, cultivadas no campo, e a comparação com os fungos isolados, em plantas micropropagadas há dois anos. Os isolados da microbiota fúngica endofítica, das plantas cultivadas in vitro, foram: Fusarium oxysporum, Neotyphodium sp. e Epicoccum nigrum; e das plantas in vivo, foram: Fusarium sp., F. proliferatum, F. oxysporum, Colletotrichum sp., Alternaria gaisen, Neotyphodium sp. e Epicoccum nigrum. As sete espécies de fungos foram reintroduzidas in vitro na planta hospedeira, demonstrando diferentes comportamentos. Neotyphodium sp. e E. nigrum estabeleceram uma interação endofítica com a planta, e as demais comportaram-se como patógenos, diminuindo o desenvolvimento das plântulas em relação às plantas sem inoculação. As espécies endofíticas apresentam potencial para o uso no controle biológico de patógenos de pupunha.

Comprehensive analysis of proteins secreted by dermatophytes in a protein medium which to some extent mimic "in vivo" growth parameters

RESUME : Les dermatophytes sont les agents infectieux les plus fréquents responsables de la plupart des mycoses superficielles chez les humains et chez les animaux. Ces infections, dermatophytoses, également appelées tineas ou teignes, sont fréquentes et causent des problèmes de santé publique au niveau mondial. La capacité d'envahir et de progresser au sein des structures kératinisées est probablement liée à la sécrétion de différentes enzymes kératinolytiques, qui sont considérées comme la principale caractéristique liée à la pathogénicité de ces champignons. L'objectif de ma thèse a été premièrement de progresser dans l'identification et la caractérisation des nouvelles protéines sécrétées, afin de mieux comprendre a) la capacité globale des dermatophytes à envahir les structures kératinisées, et b) les différences dans la virulence et la spécificité d'hôte que présentent les espèces étudiées .Pour progresser dans l'identification et la caractérisation de ces nouvelles protéines, les secretomes de six espèces de dermatophytes (Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii et Trichophyton tonsurans) ont été étudiés. Bien qu'il y ait un niveau globalement élevé de similitude entre les protéases sécrétées, les différentes espèces de dermatophytes sécrètent des profiles protéiques distincts lorsqu'elles sont cultivées dans les mêmes conditions de culture, et donc une signature spécifique a pu être associé à chaque espèce. Ces profiles ont été un outil avantageux pour identifier et cartographier les protéines orthologues aux six espèces et ont aussi permit la discrimination d'espèces très proches comme T. tonsurans et T. equinum qui ne peuvent pas être différenciées par l'ADN ribosomal. Ce travail également présente ce que l'on croit être la première identification global des protéines sécrétées par les dermatophytes dans des conditions de culture que incitent l'activité protéolytique extracellulaire. Ce catalogue de protéines, comprenant des endo- and exo- proteases, autres hydrolases, oxydoreductases et des protéines avec fonction inconnue, représente probablement le spectre d'enzymes qui permettent la dégradation des tissus kératinisés en composés qui peuvent être assimilés par le champignon. Les résultats suggèrent qu'un changement écologique pourrait être associé à une expression différentielle des gènes codant les protéines sécrétées, en particulier, les protéases, plutôt qu'à des divergences génétiques au niveau des gènes codant les protéines orthologues. Une sécrétion différentielle des protéines par les dermatophytes pourrait également être responsable de la variabilité inflammatoire qui causent ces agents infectieux chez les différents hôtes. Par conséquent, les protéines identifiées ici sont également importantes pour faire la lumière sur la réponse immunitaire de l'hôte au cours du processus infectieux. SUMMARY : Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Dermatophytoses, also called tineas or ringworm, are frequent and cause public health problems worldwide. The secretion of different keratinolytic enzymes is believed to be a key pathogenicity-related characteristic of these fungi. The aim of this work was first to progress in the identification and characterization of novel secreted proteins, in order to better understand a) the overall capability of dermatophytes to invade keratinised structures, and b) differences in virulence and host-specificity of the investigated species. To progress in the identification and characterization of novel proteins, the secretomes from Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii and Trichophyton tonsurans were studied. Although there is a high global level of similarity among the secreted proteases, different dermatophyte species produce distinct patterns of proteins when grown in the same culture medium, and so a specific signature could be associated to each species. These patterns were useful to identify and map orthologous proteins among the six species, as well as to discriminate the closely related species T. tonsurans and T. equinum, which cannot be differentiated by ribosomal DNA. This work also presents the first in-depth identification of the major proteins secreted by dermatophytes growing under conditions promoting extracellular proteolytic activity. This catalogue of proteins, which include several endo- and exo- proteases, other hydrolases, oxydoreductases, and proteins of unknown function, probably represents the spectrum of enzymes that allow the degradation of keratinized tissues into compounds which can be assimilated by the fungus. The results suggest that ecological switching could be related to a differential expression of genes encoding secreted proteins, particularly, proteases, rather than genetic divergences of the genes encoding orthologous proteins. Differential secretion of proteins by Dermatophyte species could also be responsible for the variable inflammation caused by the infectious agent within the host. Therefore, the proteins here identified are also important to shed light into the immune response of the host during the infection process.