Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

In situ hybridization of hepatitis C virus RNA in liver cells of an experimentally infected rhesus macaque

The liver tissue of a rhesus macaque inoculated with hepatitis C virus (HCV) has been analyzed for the presence of HCV RNA using the technique of in situ hybridization, both at light and electron microscopy levels. The animal was inoculated by the intrasplenic route using a HCV infected autogenic hepatocyte transplant. The serum sample used to infect the hepatocyte cells was characterized by polymerase chain reaction technique and shown to be positive for HCV RNA, genotype 3 with 10(7) RNA copies/ml. In situ hybridization was performed using a complementary negative strand probe made with the specific primer. We were able to detect and localize viral RNA in altered membranes of the rough endoplasmic reticulum of infected liver cells, showing evidence of virus replication in vivo.

Leishmania major-specific B cells are necessary for Th2 cell development and susceptibility to L. major LV39 in BALB/c mice.

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Effect of blood components, abdominal distension, and ecdysone therapy on the ultrastructural organization of posterior midgut epithelial cells and perimicrovillar membranes in Rhodnius prolixus

The effects of blood components, nerve-cord severance, and ecdysone therapy on the posterior midgut epithelial cells of 5th-instar Rhodnius prolixus nymphs 10 days after feeding were analyzed by transmission electron microscopy. Cutting the nerve-cord of the blood-fed insects partially reduced the development of microvilli and perimicrovillar membranes (PMM), and produced large vacuoles and small electrondense granules; insects fed on Ringer's saline diet exhibited well developed microvilli and low PMM production; swolled rough endoplasmatic reticulum and electrondense granules; Ringer's saline meal with ecdysone led to PMM development, glycogen particles, and several mitochondria in the cytoplasm; epithelial cells of the insects fed on Ringer's saline meal whose nerve-cord was severed showed heterogeneously distributed microvilli with reduced PMM production and a great quantity of mitochondria and glycogen in the cytoplasm; well developed microvilli and PMM were observed in nerve-cord severed insects fed on Ringer's saline meal with ecdysone; Ringer's saline diet containing hemoglobin recovered the release of PMM; and insects fed on human plasma showed slightly reduced PMM production, although the addition of ecdysone in the plasma led to a normal midgut ultrastructural organization. We suggest that the full development of microvilli and PMM in the epithelial cells depends on the abdominal distension in addition to ingestion of hemoglobin, and the release of ecdysone.

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Location of corneal epithelial stem cells Reply

The longstanding concept that corneal epithelial stem cells reside mainly in the limbus is supported by the absence of major corneal epithelial differentiation markers, that is, K3 and K12 keratins, in limbal basal cells (these markers are expressed, however, in corneal basal cells, thus distinguishing the mode of keratin expression in corneal epithelium from that of all other stratified epithelia), the centripetal migration of corneal epithelial cells, the exclusive location of slow-cycling cells in the limbal basal layer, the superior in vitro proliferative potential of limbal epithelial cells, and the transplanted limbal cells' ability to reconstitute corneal epithelium in vivo (reviewed in refs 1-4). Moreover, previous data indicate that corneal and conjunctival epithelia represent two separate cell lineages (reviewed in refs 1-4). Majo et al. suggested, however, that corneal and conjunctival epithelia are equipotent, and that identical oligopotent stem cells are present throughout the corneal, limbal and conjunctival epithelia. We point out here that these suggestions are inconsistent with many known growth, differentiation and cell migration properties of the anterior ocular epithelia.

Oligoclonal expansions in the CD8(+)CD28(-) T cells largely explain the shorter telomeres detected in this subset: analysis by flow FISH.

We have previously reported that CD8(+)CD28(-) T cells have relatively shorter telomeres compared with CD8(+)CD28(+) T cells. Oligoclonal expansion is a common feature of CD8(+) T cells in human peripheral blood, and these expansions predominantly occur in the CD57(+)/CD28(-) population. We studied the telomere length in subsets of CD8(+) T cells using quantitative fluorescence in situ hybridization and flow cytometry (flow FISH). Our results confirm that CD8(+)CD28(-) T cells have shorter telomeres as compared with their CD28(+) counterpart cells. In addition, the oligoclonally expanded cells within the CD8(+)CD28(-) T cell subset generally have even shorter telomeres than the CD28(-) subset as a whole. We conclude that the presence of clonal expansions in the CD8(+)CD28(-) T cell population largely explain the shorter telomeres in this subset. These clonally expanded CD8(+)CD28(-) T cells generally have characteristics of terminally differentiated effector cells. Nevertheless, there is considerable individual variation in the degree of telomere shortening in these cells, which may reflect host genetic factors as well as the type and timing of the antigenic exposure.

NKT cells: what's in a name?

Recent years have seen so-called natural killer T (NKT) cells emerge as important regulators of the immune response. The existence of NKT-cell subsets, and other types of T cell that resemble NKT cells, is an ongoing source of confusion in the literature. This perspective article seeks to clarify which cells fall under the NKT-cell umbrella, and which might be best considered as separate.

Role of oxidative stress in plasticity of Dendritic cells

Dendritic cells (DCs) are antigen presenting cells with an unique ability to induce primary immune responses. Different DCs subsets with an intrinsic capacity to polarise Tcells have been described: myeloid (Th1) and lymphoid (Th2). Plasticity is defined as DCs capacity to polarise T cells independent of the DCs origin. We investigated the potential role played by oxidants such as superoxide anion (·O2-), in the plasticity of DCs, measured by the induction of a specific DCs subset, cytokine release and antigen presentation. Furthermore, we are interested in the amplification of immune response analysed by the exosomes production after oxidative stress and LPS stimulation. Recently, we have demonstrated that exposure of cells to superoxide anions resulted in the activation of DC2 profile. To analyse the role of oxidative stress in DCs subsets, we used BDCA-1 and BDCA-2 antibodies, which identify myeloid and plasmacytoid DCs respectively. Freshly isolated monocytes have shown to be BDCA-1-, but BDCA-2+ populations. During 6 days culture up-regulation of BDCA-1, but a down-regulation of BDCA-2 were observed, giving a clear myeloid population. When DC were stimulated with superoxide anions or LPS, we have observed that both down regulate the expression of BDCA-1 when compared to immature DC. Antigen presentation was markedly altered according to the periodicity used, and antigens and oxidants exposures. Using DCs trapped in collagen "matrix" after LPS activation we were able to quantify DCs-exosomes (small membrane vesicles ~50-100 nm in diameter) by reconstruction pictures in three dimensions. Using double vital staining we have found that exosomes from activated DCs can fuse with the membrane of resting DCs. Understanding the capacity of DCs to integrate external signals we will be able to unravel and control Tcells-polarisation triggering a specific immune response or tolerance. We will be able also to understand the amplification role of DCs-exosomes in remote not yet activated DCs.

On the presence of hepatic stellate cells in portal spaces

Previous studies in mice with hypervitaminosis A have demonstrated that fat-storing cells (hepatic stellate cells-HSCs) participate in schistosomal granuloma fibrogenesis. The origin of such cells in portal areas, away from the Disse spaces, was herein investigated. HSCs were identified in frozen sections of the liver by means of Sudan III staining. They appeared as red-stained cells disposed along the sinusoids of normal mice, but were never found within portal spaces. However, in the chronically inflamed portal spaces of Capillaria hepatica-infected mice, Sudan III-positive cells were frequently present among leukocytes and fibroblast-like cells. Thus, there are no resident HSCs in portal spaces, but their presence there in chronic inflammatory processes indicates that they are able to migrate from peri-sinusoidal areas in order to reach the portal areas.

Benznidazole-induced genotoxicity in diploid cells of Aspergillus nidulans

Genotoxic effects of benznidazole were studied by the induction of homozygosis of genes previously present in heterozygous. UT448//A757 diploid strain was used in the benznidazole's recombinagenesis test. Although toxic effects on growth of colonies were not observed, 75 and 100 µM benznidazole induced an increasing of mitotic recombination events in diploid strain. Results were related to the induction of chromosomal breaks by the antiparasitic drug.

Melan-A-specific cytotoxic T cells are associated with tumor regression and autoimmunity following treatment with anti-CTLA-4.

PURPOSE: Ipilimumab is a monoclonal antibody that blocks the immune-inhibitory interaction between CTL antigen 4 (CTLA-4) and its ligands on T cells. Clinical trials in cancer patients with ipilimumab have shown promising antitumor activity, particularly in patients with advanced melanoma. Often, tumor regressions in these patients are correlated with immune-related side effects such as dermatitis, enterocolitis, and hypophysitis. Although these reactions are believed to be immune-mediated, the antigenic targets for the cellular or humoral immune response are not known. EXPERIMENTAL DESIGN: We enrolled patients with advanced melanoma in a phase II study with ipilimumab. One of these patients experienced a complete remission of his tumor. The specificity and functional properties of CD8-positive T cells in his peripheral blood, in regressing tumor tissue, and at the site of an immune-mediated skin rash were investigated. RESULTS: Regressing tumor tissue was infiltrated with CD8-positive T cells, a high proportion of which were specific for Melan-A. The skin rash was similarly infiltrated with Melan-A-specific CD8-positive T cells, and a dramatic (>30-fold) increase in Melan-A-specific CD8-positive T cells was apparent in peripheral blood. These cells had an effector phenotype and lysed Melan-A-expressing tumor cells. CONCLUSIONS: Our results show that Melan-A may be a major target for both the autoimmune and antitumor reactions in patients treated with anti-CTLA-4, and describe for the first time the antigen specificity of CD8-positive T cells that mediate tumor rejection in a patient undergoing treatment with an anti-CTLA-4 antibody. These findings may allow a better integration of ipilimumab into other forms of immunotherapy.

Autophagy promotes survival of retinal ganglion cells after optic nerve axotomy in mice.

Autophagy is an essential recycling pathway implicated in neurodegeneration either as a pro-survival or a pro-death mechanism. Its role after axonal injury is still uncertain. Axotomy of the optic nerve is a classical model of neurodegeneration. It induces retinal ganglion cell death, a process also occurring in glaucoma and other optic neuropathies. We analyzed autophagy induction and cell survival following optic nerve transection (ONT) in mice. Our results demonstrate activation of autophagy shortly after axotomy with autophagosome formation, upregulation of the autophagy regulator Atg5 and apoptotic death of 50% of the retinal ganglion cells (RGCs) after 5 days. Genetic downregulation of autophagy using knockout mice for Atg4B (another regulator of autophagy) or with specific deletion of Atg5 in retinal ganglion cells, using the Atg5(flox/flox) mice reduces cell survival after ONT, whereas pharmacological induction of autophagy in vivo increases the number of surviving cells. In conclusion, our data support that autophagy has a cytoprotective role in RGCs after traumatic injury and may provide a new therapeutic strategy to ameliorate retinal diseases.