Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

Role of the chemokine receptor CX3CR1 in the mobilization of phagocytic retinal microglial cells.

We recently showed that subretinal CX3CR1-dependent microglial cell (MC) accumulation may lead to age-related macular degeneration. The fate of MC after engulfing retinal debris is poorly understood. Severe photoreceptor degeneration was observed 40days after exposure to bright light in CX3CR1-deficient but not control mice, and more MCs accumulated in the subretinal space of the former than the latter. To study the fate of subretinal MCs in CX3CR1 competent animals, we used a dystrophic rat model in which abundant subretinal MC accumulation is observed secondary to retinal degeneration. In dystrophic rats, MCs containing rhodopsin or rod outer segment (ROS) debris were found outside the outer retina at sites suggesting choroidal and ciliary egress. In conclusion, our data indicate that MC accumulation at injury sites is independent of CX3CR1 and precedes photoreceptor degeneration. The ectopic presence of rhodopsin-positive MCs suggests that CX3CR1 participates in MC egress from the outer retina.

Neuroprotective role of lactate after cerebral ischemia.

It is well established that lactate can be used as an energy substrate by the brain by conversion to pyruvate and a subsequent oxidation in the mitochondria. Knowing the need for readily metabolizable substrates directly after ischemia and the protective effect of lactate after excitotoxicity, the aim of this study was to investigate whether lactate administration directly after ischemia could be neuroprotective. In vitro, the addition of 4 mmol/L L-lactate to the medium of rat organotypic hippocampal slices, directly after oxygen and glucose deprivation (OGD), protected against neuronal death, whereas a higher dose of 20 mmol/L was toxic. In vivo, after middle cerebral artery occlusion in the mouse, an intracerebroventricular injection of 2 microL of 100 mmol/L L-lactate, immediately after reperfusion, led to a significant decrease in lesion size, which was more pronounced in the striatum, and an improvement in neurologic outcome. A later injection 1 h after reperfusion did not reduce lesion size, but significantly improved neurologic outcome, which is an important point in the context of a potential clinical application. Therefore, a moderate increase in lactate after ischemia may be a therapeutic tool.

Expression pattern of sirtuins in innate immune cells and influence of Sirtuin 1/2 inhibition on innate immune responses to Toll-like receptor ligands and to infection

Purpose/Objective: The family of histone deacetylases comprises 18 members in mammals, among which seven sirtuins (SIRT1-7). Sirtuins are NADP-dependent enzymes that have been involved in the control of cell metabolism, proliferation and survival. The expression pattern of sirtuins and their influence on host response to microbial infection remain largely unknown. The aim of the study was to analyze the expression of SIRT1-7 and to address the effects of SIRT1/2 inhibition on innate immune responses in vitro and in vivo.. Materials and methods: in vitro: Bone marrow (BM), BM-derived macrophages (BMDMs) and dendritic cells (BMDCs) and RAW 264.7 and J774.1 macrophage cell lines were stimulated for 0, 2, 6 and 18 h with LPS, Pam3CSK4 and CpG ODN. SIRT1-7 mRNA was quantified by real time-PCR. TNF was measured by ELISA. In vivo: BALB/c mice were challenged with LPS (350 lg i.p.) with or without a SIRT1/2 inhibitor. Blood and organs were collected after 0, 1, 4, 8 and 24 h to quantify SIRT1-7 and TNF. Mortality was assessed daily. Results: Bone marrow, macrophages and DCs express, in order of abundance, SIRT2 > > SIRT1, SIRT3 and SIRT6 > SIRT4, SIRT5 and SIRT7. Microbial products decrease the expression of all sirtuins except SIRT6 in a time dependent manner in BMDMs (0_24 h). SIRT2 is the most expressed sirtuin also in the liver, kidney (together with SIRT3) and spleen. Upon LPS challenge, SIRT1, SIRT3, SIRT4 and SIRT7 mRNA levels decrease in the liver (from 4 h to 24 h), whereas SIRT1-7 mRNA levels decrease within 1 h in both kidney and spleen. Pharmacological inhibition of SIRT1/2 decreases TNF production by macrophages stimulated with LPS, Pam3CSK4 and CpG ODN (n = 6; P < 0.001). In agreement, prophylactic treatment with a SIRT1/2 inhibitor decreases TNF production (n = 8; P = 0.04) and increases survival (n = 13, P = 0.03) of mice challenged with LPS. Conclusions: Sirtuins are expressed in innate immune cells. Inhibition of SIRT1/2 activity decreases cytokine production by macrophages and protects from endotoxemia, suggesting that sirtuin inhibitors may represent novel adjunctive therapy for treating inflammatory disorders such as sepsis.

Isolation and molecular identification of Leishmania chagasi from a bat (Carollia perspicillata) in northeastern Venezuela

This report describes the isolation of a Leishmania chagasi strain from a bat (Carollia perspicillata), and its identification using biological methods and molecular characterization. The parasites were isolated in an artificial culture medium from a blood sample extracted from a bat heart. The isolate was then inoculated into the footpads of Balb/c mice, which subsequently developed a typical nodular leishmanial lesion; the parasites were confirmed as Leishmania by smear and histopathology. Molecular characterization of the parasites was performed by polymerase chain reaction with species-specific primers, kDNA restriction pattern following Hae III endonuclease digestion and dot blot hybridization using a kDNA probe. This report demonstrates that bats can be hosts for L. chagasi species and suggests the need for studies to determine whether they may be involved in foci of visceral leishmaniasis.

Active antiviral T-lymphocyte response can be redirected against tumor cells by antitumor antibody x MHC/viral peptide conjugates.

PURPOSE: To redirect an ongoing antiviral T-cell response against tumor cells in vivo, we evaluated conjugates consisting of antitumor antibody fragments coupled to class I MHC molecules loaded with immunodominant viral peptides. EXPERIMENTAL DESIGN: First, lymphochoriomeningitis virus (LCMV)-infected C57BL/6 mice were s.c. grafted on the right flank with carcinoembryonic antigen (CEA)-transfected MC38 colon carcinoma cells precoated with anti-CEA x H-2D(b)/GP33 LCMV peptide conjugate and on the left flank with the same cells precoated with control anti-CEA F(ab')(2) fragments. Second, influenza virus-infected mice were injected i.v., to induce lung metastases, with HER2-transfected B16F10 cells, coated with either anti-HER2 x H-2D(b)/NP366 influenza peptide conjugates, or anti-HER2 F(ab')(2) fragments alone, or intact anti-HER2 monoclonal antibody. Third, systemic injections of anti-CEA x H-2D(b) conjugates with covalently cross-linked GP33 peptides were tested for the growth inhibition of MC38-CEA(+) cells, s.c. grafted in LCMV-infected mice. RESULTS: In the LCMV-infected mice, five of the six grafts with conjugate-precoated MC38-CEA(+) cells did not develop into tumors, whereas all grafts with F(ab')(2)-precoated MC38-CEA(+) cells did so (P = 0.0022). In influenza virus-infected mice, the group injected with cells precoated with specific conjugate had seven times less lung metastases than control groups (P = 0.0022 and P = 0.013). Most importantly, systemic injection in LCMV-infected mice of anti-CEA x H-2D(b)/cross-linked GP33 conjugates completely abolished tumor growth in four of five mice, whereas the same tumor grew in all five control mice (P = 0.016). CONCLUSION: The results show that a physiologic T-cell antiviral response in immunocompetent mice can be redirected against tumor cells by the use of antitumor antibody x MHC/viral peptide conjugates.

Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts

Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

Resistance of melanized yeast cells of Paracoccidioides brasiliensis to antimicrobial oxidants and inhibition of phagocytosis using carbohydrates and monoclonal antibody to CD18

Paracoccidioides brasiliensis, a thermal dimorphic fungal pathogen, produces a melanin-like pigment in vitro and in vivo. We investigated the involvement of carbohydrates and monoclonal antibody to CD18, on phagocytosis inhibition, involving macrophage receptors and the resistance of melanized fungal cells to chemically generated nitric oxide (NO), reactive oxygen species (ROS), hypochlorite and H2O2. Our results demonstrate that melanized yeast cells were more resistant than nonmelanized yeast cells to chemically generated NO, ROS, hypochlorite and H2O2, in vitro. Phagocytosis of melanized yeast cells was virtually abolished when mannan, N-acetyl glucosamine and anti-CD18 antibody were added together in this system. Intratracheal infection of BALB/c mice, with melanized yeast cells, resulted in higher lung colony forming units, when compared to nonmelanized yeast cells. Therefore, melanin is a virulence factor of P. brasiliensis.

Absence of Fas-L aggravates renal injury in acute Trypanosoma cruzi infection

Trypanosoma cruzi infection induces diverse alterations in immunocompetent cells and organs, myocarditis and congestive heart failure. However, the physiological network of disturbances imposed by the infection has not been addressed thoroughly. Regarding myocarditis induced by the infection, we observed in our previous work that Fas-L-/- mice (gld/gld) have very mild inflammatory infiltration when compared to BALB/c mice. However, all mice from both lineages die in the early acute phase. Therefore, in this work we studied the physiological connection relating arterial pressure, renal function/damage and cardiac insufficiency as causes of death. Our results show that a broader set of dysfunctions that could be classified as a cardio/anaemic/renal syndrome is more likely responsible for cardiac failure and death in both lineages. However, gld/gld mice had very early glomerular deposition of IgM and a more intense renal inflammatory response with reduced renal filtration, which is probably responsible for the premature death in the absence of significant myocarditis in gld/gld.

Genetic investigation of the functions of c-Myc and N-Myc in Hematopoietic stem cells

Résumé : c-Myc, le premier facteur de transcription de la famille Myc a été découvert il y a maintenant trente ans. Il reste à l'heure actuelle parmi les plus puissants proto-oncogènes connus. c-Myc est dérégulé dans plus de 50% des cancers, où il promeut la prolifération, la croissance cellulaire, et la néoangiogenèse. Myc peut aussi influencer de nombreuses autres fonctions de par sa capacité à activer ou à réprimer la transcription de nombreux gènes, et à agir globalement sur le génome à travers des modifications épigénétiques de la chromatine. La famille d'oncogènes Myc comprend, chez les mammifères, trois protéines structurellement proches: c-Myc, N-Myc et L-Myc. Ces protéines ont les mêmes proprietés biochimiques, exercent les mêmes fonctions mais sont le plus souvent exprimées de façon mutuellement exclusive. Myc a été récemment identifié comme un facteur clef dans la maintenance des cellules souches embryonnaires et adultes ainsi que dans la réacquisition des proprietés des cellules souches. Nous avons précédemment démontré que l'élimination de c-Myc provoque une accumulation de cellules souches hématopoïétiques (CSH) suite à un défaut de différenciation lié à la niche. Les CSH sont responsables de la production de tous les éléments cellulaires du sang pour toute la vie de l'individu et sont définies par leur capacité à s'auto-renouveler tout en produisant des précurseurs hématopoïétiques. Afin de mieux comprendre la fonction de Myc dans les CSH, nous avons choisi de combiner l'utilisation de modèles de souris génétiquement modifiées à une caractérisation systématique des schémas d'expression de c-Myc, N-Myc et L-Myc dans tout le système hématopoïétique. Nous avons ainsi découvert que les CSH les plus immatures expriment des quantités équivalentes de transcrits de c-myc et N-myc. Si les CSH déficientes en N-myc seulement ont une capacité d'auto-renouvellement à long-terme réduite, l'invalidation combinée des gènes c-myc et N-myc conduit à une pan-cytopénie suivie d'une mort rapide de l'animal, pour cause d'apoptose de tous les types cellulaires hématopoïétiques. En particulier, les CSH en cours d'auto-renouvelemment, mais pas les CSH quiescentes, accumulent du Granzyme B (GrB), une molécule fortement cytotoxique qui provoque une mort cellulaire rapide. Ces données ont ainsi mis au jour un nouveau mécanisme dont dépend la survie des CSH, à savoir la répression du GrB, une enzyme typiquement utilisée par le système immunitaire inné pour éliminer les tumeurs et les cellules infectées par des virus. Dans le but d'évaluer l'étendue de la redondance entre c-Myc et N-Myc dans les CSH, nous avons d'une part examiné des souris dans lesquelles les séquences codantes de c-myc sont remplacées par celles de N-myc (NCR) et d'autre part nous avons géneré une série allèlique de myc en éliminant de façon combinatoire un ou plusieurs allèles de c-myc et/ou de N-myc. Alors que l'analyse des souris NCR suggère que c-Myc et N-Myc sont qualitativement redondants, la série allélique indique que les efficiences avec lesquelles ces deux protéines influencent des procédés essentiels à la maintenance des CSH sont différentes. En conclusion, nos données génétiques montrent que l'activité générale de MYC, fournie par c-Myc et N-Myc, contrôle plusieurs aspects cruciaux de la fonction des CSH, notamment l'auto-renouvellement, la survie et la différenciation. Abstract : c-Myc, the first Myc transcription factor was discovered 30 years ago and is to date one of the most potent proto-oncogenes described. It is found to be misregulated in over 50% of all cancers, where it drives proliferation, cell growth and neo-angiogenesis. Myc can also influence a variety of other functions, owing to its ability to activate and repress transcription of many target genes and to globally regulate the genome via epigenetic modifications of the chromatin. The Myc family of oncogenes consists of three closely related proteins in mammals: c-Myc, N-Myc and L-Myc. These proteins share the same biochemical properties, exert mostly the same functions, but are most often expressed in mutually exclusive patterns. Myc is now emerging as a key factor in maintenance of embryonic and adult stem cells as well as in reacquisition of stem cell properties, including induced reprogramming. We previously showed that c-Myc deficiency can cause the accumulation of hematopoietic stem cells (HSCs) due to a niche dependent differentiation defect. HSCs are responsible for life-long replenishment of all blood cell types, and are defined by their ability to self-renew while concomitantly giving rise to more commited progenitors. To gain further insight into the function of Myc in HSCs, in this study we combine the use of genetically-modified mouse models with the systematic characterization of c-myc, N-myc and L-myc transcription patterns throughout the hematopoietic system. Interestingly, the most immature HSCs express not only c-myc, but also about equal amounts of N-myc transcripts. Although conditional deletion of N-myc alone in the bone marrow does not affect steady-state hematopoiesis, N-myc null HSCs show impaired long-term self-renewal capacity. Strikingly, combined deficiency of c-Myc and N-Myc results in pan-cytopenia and rapid lethality, due to the apoptosis of most hematopoietic cell types. In particular, self-renewing HSCs, but not quiescent HSCs or progenitor cell types rapidly up-regulate and accumulate the potent cytotoxic molecule GranzymeB (GrB), causing their rapid cell death. These data uncover a novel pathway on which HSC survival depends on, namely repression of GrB, a molecule typically used by the innate immune system to eliminate tumor and virus infected cells. To evaluate the extent of redundancy between c-Myc and N-Myc in HSCs, we examined mice in which c-myc coding sequences are replaced by that of N-myc (NCR) and also generated an allelic series of myc, by combinatorially deleting one or several c-myc and/or N-myc alleles. While the analysis of NCR mice suggests that c-Myc and N-Myc are qualitatively functionally redundant, our allelic series indicates that the efficiencies with which these two proteins affect crucial HSC maintenance processes are likely to be distinct. Collectively, our genetic data show that general "MYC" activity delivered by c-Myc and N-Myc controls crucial aspects of HSC function, including self-renewal, survival and niche dependent differentiation.

Chemistry, cytotoxicity and antileishmanial activity of the essential oil from Piper auritum

Leishmaniasis is one of the most important parasitic infections, but current treatments are unsatisfactory due to their toxicity, cost and resistance. Therefore, the development of new antileishmanial compounds is imperative. Many people who live in endemic areas use plants as an alternative to treat the disease. In this paper, we characterised the essential oil from Piper auritum, evaluated its cytotoxicity and determined its antileishmanial activity. The chromatogram obtained by gas chromatography revealed 60 peaks and we found that safrole was the most abundant compound, composing 87% of the oil. The oil was active against the promastigotes of Leishmania major, Leishmania mexicana, Leishmania braziliensis and Leishmania donovani with a favourable selectivity index against peritoneal macrophages from BALB/c mice. The Piper-oil inhibited the growing of intracellular amastigotes of L. donovani with an IC50 value of 22.3 ± 1.8 μg/mL. This study demonstrates the usefulness of the essential oils as a promising alternative to treat leishmaniasis.

Embryonic development of NMRI mice: relationship between the weight, age and ossification of embryos.

The time and the order of the appearance of the ossification centres were found to be similar in C3H and NMRI mice. Bodyweight comparisons confirmed these results. Location in the right as opposed to the left uterine horn, or in the upper, middle or lower part, was not found to influence the weight of the embryo. Significant differences in the weight of embryos within the same litter were used in investigating the sequence of ossification in embryos. This should prove useful in comparative morphology and teratology.

Acute oxygen sensing in heme oxygenase-2 null mice.

Hemeoxygenase-2 (HO-2) is an antioxidant enzyme that can modulate recombinant maxi-K(+) channels and has been proposed to be the acute O(2) sensor in the carotid body (CB). We have tested the physiological contribution of this enzyme to O(2) sensing using HO-2 null mice. HO-2 deficiency leads to a CB phenotype characterized by organ growth and alteration in the expression of stress-dependent genes, including the maxi-K(+) channel alpha-subunit. However, sensitivity to hypoxia of CB is remarkably similar in HO-2 null animals and their control littermates. Moreover, the response to hypoxia in mouse and rat CB cells was maintained after blockade of maxi-K(+) channels with iberiotoxin. Hypoxia responsiveness of the adrenal medulla (AM) (another acutely responding O(2)-sensitive organ) was also unaltered by HO-2 deficiency. Our data suggest that redox disregulation resulting from HO-2 deficiency affects maxi-K(+) channel gene expression but it does not alter the intrinsic O(2) sensitivity of CB or AM cells. Therefore, HO-2 is not a universally used acute O(2) sensor.

Differential tissue tropism of Trypanosoma cruzi strains: an in vitro study

We have previously demonstrated selection favoring the JG strain of Trypanosoma cruziin hearts of BALB/c mice that were chronically infected with an equal mixture of the monoclonal JG strain and a clone of the Colombian strain, Col1.7G2. To evaluate whether cell invasion efficiency drives this selection, we infected primary cultures of BALB/c cardiomyocytes using these same T. cruzi populations. Contrary to expectation, Col1.7G2 parasites invaded heart cell cultures in higher numbers than JG parasites; however, intracellular multiplication of JG parasites was more efficient than that of Col1.7G2 parasites. This phenomenon was only observed for cardiomyocytes and not for cultured Vero cells. Double infections (Col1.7G2 + JG) showed similar results. Even though invasion might influence tissue selection, our data strongly suggest that intracellular development is important to determine parasite tissue tropism.

Cryptococcus neoformans var. grubii - Pathogenicity of environmental isolates correlated to virulence factors, susceptibility to fluconazole and molecular profile

The pathogenicity of Cryptococcus neoformans is heterogeneous and is associated with the expression of virulence factors. This study aimed to correlate the pathogenicity of C. neoformans var. grubii in BALB/c mice with in vitro virulence factors, fluconazole minimal inhibitory concentrations (MICs) and molecular profiles, before and after animal passage. Ten environmental isolates and one ATCC strain of C. neoformans var. grubii mating type α were evaluated. Most isolates (91%) killed 50% or more of the infected animals by day 24 postinfection and were recovered from the lungs and brains of surviving animals on days 7 and 14 postinfection. The burden of yeast in the lungs was more variable than that in the brain. The differences in the expression of virulence factors (growth at 37ºC, presence and size of the capsule and production of melanin, urease, proteinase and phospholipase) by most isolates pre and postpassage in animals were not statistically significant. The fluconazole MICs in postpassaged lines differed by a one-dilution from the MIC of the corresponding prepassaged line for six isolates. Using molecular typing [polymerase chain reaction-fingerprinting with (GACA)4 and M13], eight isolates were identified as VNI and three as VNII. We concluded that different isolates with the same molecular and phenotypic profiles, including isolates that are markedly hypervirulent, span a wide range of virulence and there were no changes in virulence factors in the postpassaged lines when compared with the corresponding nonpassaged lines.