Apoptosis and apoptotic mimicry in Leishmania: an evolutionary perspective.

Apoptotic death and apoptotic mimicry are defined respectively as a non-accidental death and as the mimicking of an apoptotic-cell phenotype, usually by phosphatidylserine (PS) exposure. In the case of the murine infection by Leishmania spp, apoptotic death has been described in promastigotes and apoptotic mimicry in amastigotes. In both situations they are important events of the experimental murine infection by this parasite. In the present review we discuss what features we need to consider if we want to establish if a behavior shown by Leishmania is altruistic or not: does the behavior increases the fitness of organisms other than the one showing it? Does this behavior have a cost for the actor? If we manage to show that a given behavior is costly for the actor and beneficial for the recipient of the action, we will be able to establish it as altruistic. From this perspective, we can argue that apoptotic-like death and apoptotic mimicry are both altruistic with the latter representing a weaker altruistic behavior than the former.

Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles.

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

Apoptosis and apoptotic mimicry: the Leishmania connection.

Different death-styles have been described in unicellular organisms. In most cases they evolve with phenotypic features similar to apoptotic death of animal cells, such as phosphatidylserine (PS) exposure, oligonucleosomal DNA fragmentation, and loss of mitochondrial transmembrane potential, hinting that similar mechanisms operate in both situations. However, the biochemical pathways underlying death in unicellular organisms are still unclear. Host recognition of PS exposed on the surface of unicellular parasites is an important feature of the process of infection and progression of the disease. Here, we discuss data showing that entirely different mechanisms of PS exposure co-exist during the life-cycle of Leishmania amazonensis: in the case of promastigotes, a sub-population dies by apoptosis; in the case of amastigotes, the entire population exposes PS, not necessarily followed by apoptotic death. This phenomenon has been called apoptotic mimicry. The elusive caspase-like activities described in protozoa are also discussed.

Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner.

Exposure of phosphatidylserine (PS) on cellular membranes and membrane-derived microvesicles stimulates a number of anti-inflammatory responses involved in malignant processes. Herein we show that B16F10 cells, a highly metastatic melanoma cell line, produce large quantities of PS-containing microvesicles in vitro. Tumor microvesicles increased TGF-beta(1) production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, microvesicles induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Altogether, this is the first demonstration that tumor-derived microvesicles favor the establishment of melanoma metastasis in a PS-dependent manner, possibly by down-regulating the host's inflammatory and/or anti-tumoral immune responses.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma.

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P < 0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P < 0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells.

Mimicry of apoptotic cells by exposing phosphatidylserine participates in the establishment of amastigotes of Leishmania (L) amazonensis in mammalian hosts.

Signaling through exposed phosphatidylserine (PS) is fundamental for the TGFbeta1-dependent, noninflammatory phagocytosis of apoptotic cells. This same mechanism operates in the internalization of amastigotes of Leishmania (L) amazonensis (L(L)a) in a process quoted as apoptotic mimicry. Now we show that the host modulates PS exposure by the amastigotes and, as a consequence, BALB/c mice-derived amastigotes expose significantly more PS than those derived from C57BL/6 mice. Due to this difference in the density of surface PS molecules, the former are significantly more infective than the latter, both in vivo, in F1 (BALB/c x C57BL/6) mice, and in vitro, in thioglycollate-derived macrophages from this same mouse strain. PS exposure increases with progression of the lesion and reaches its maximum value in amastigotes obtained at the time point when the lesion in C57BL/6 mice begins to decrease in size and the lesions in BALB/c mice are still growing in size. Synthesis of active TGFbeta1, induction of IL-10 message, and inhibition of NO synthesis correlate with the amount of surface PS displayed by viable (propidium iodide-negative) infective amastigote. Furthermore, we also show that, similar to what happens with apoptotic cells, amastigotes of L(L)a are internalized by macropinocytosis. This mechanism of internalization is consistent with the large phagolysosomes characteristic of L(L)a infection. The intensity of macrophage macropinocytic activity is dependent on the amount of surface PS displayed by the infecting amastigote.

G-CSF-treated granulocytes inhibit acute graft-versus-host disease.

It has been shown that in vivo and in vitro treatment with G-CSF induces the generation of low-density granulocytes (LDGs), which copurify with PBMCs and inhibit IFN-gamma production by human T cells. These results prompted us to postulate an immunomodulatory role for LDGs in acute graft-versus-host disease (aGVHD). Here it is shown that in the mouse experimental model, in vivo and in vitro G-CSF treatment generates LDGs capable of inhibiting 80% of T-cell IFN-gamma production. To assess the role of these LDGs in aGVHD, lethally irradiated (C57BL/6 x BALB/c) F1 hosts were reconstituted with T cell-depleted bone marrow cells plus nylon wool-purified spleen cells from G-CSF-treated (G-NWS) or -nontreated (NWS) C57BL/6 donors. Recipients of G-NWS had a 75% survival rate in contrast to a rate of 25% in the NWS recipients. The protective effect was completely abolished, and the mortality rate was 100% if donor-cell infusion was treated with anti-Gr1. Moreover, if LDGs were infused with NWS, full protection of aGVHD was observed, and no signs of disease were evidenced by mortality rate, weight loss, or histopathology of target organs. These results revealed the unexpected immunosuppressive capacity of G-CSF based on the generation of LDGs, leading to the possibility of using these cells as inhibitors of aGVHD.

Flow cytometric assessment of Leishmania spp metacyclic differentiation: validation by morphological features and specific markers.

Characterization of infective metacyclic promastigotes of Leishmania spp can be an essential step in several experimental protocols. Metacyclic forms of all Leishmania species display a typical morphology with short, narrow cell body, and an elongated flagellum. This feature suggests that metacyclics can be distinguished from procyclic forms by non-fluorimetric flow cytometric parameters thus enabling the follow-up of their appearance and acquisition of specific properties, during metacyclogenesis in in vitro cultures. Here we describe the flow cytometric parameters of stage-specific promastigotes of Leishmania major, Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis. Our findings were validated by optical microscopy morphology and specific procyclic labeling with FITC-peanut agglutinin. Furthermore, we show that parasite's distribution in the plot during differentiation in culture is not species specific and that the parasites displaying low forward-angle light scatter (FSC(low)) are three times more infective than the FSC(high) ones. The method here described can be applied to the identification of metacyclics of different Leishmania spp within the whole stationary population.

Normal hematopoiesis is maintained by activated bone marrow CD4+ T cells.

CD4(+) T cells produce hematopoietic-related cytokines and are essential for hematopoiesis stimulation during infection and hematologic recovery after bone marrow transplantation. However, it remains unclear if T cells are necessary to maintain normal hematopoiesis. We report here that, in T-cell-deficient mice, terminal differentiation of myeloid progenitors is defective, resulting in very low levels of granulocytes in the periphery. Hematopoiesis is restored after thymus graft or reconstitution with CD4(+) T cells but not CD8(+) T cells. Bone marrow CD4(+) T cells have an activated phenotype and produce cytokines, apparently, in the absence of exogenous stimulation. Transgenic mice carrying T-cell receptor specific for an ovalbumin peptide presented in the context of a specific class II molecule (I-A(d)) (DO11.10 RAG(-/-)) show the same hematopoietic deficiency as athymic mice. Their bone marrow CD4(+) T cells are not activated, suggesting that hematopoiesis maintenance requires the presence of cognate antigen in order to activate bone marrow T-helper cells. In fact, priming of transgenic mice with ovalbumin restores normal hematopoiesis. The data show that the current concept of "normal hematopoiesis" does not reflect a basal bone marrow activity, but it is an antigen-induced state maintained by constant activation of bone marrow CD4(+) T cells.

Antisense intronic non-coding RNA levels correlate to the degree of tumor differentiation in prostate cancer.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems.

Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS) phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.

Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS) phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.

Molecular aspects of prostate cancer: implications for future directions

Many studies have been developed trying to understand the complex molecular mechanisms involved in oncogenesis and progression of prostate cancer (PCa). Current biotechnological methodologies, especially genomic studies, are adding important aspects to this area. The construction of extensive DNA sequence data and gene expression profiles have been intensively explored to search for candidate biomarkers to evaluate PCa. The use of DNA micro-array robotic systems constitutes a powerful approach to simultaneously monitor the expression of a great number of genes. The resulting gene expressing profiles can be used to specifically describe tumor staging and response to cancer therapies. Also, it is possible to follow PCa pathological properties and to identify genes that anticipate the behavior of clinical disease. The molecular pathogenesis of PCa involves many contributing factors, such as alterations in signal transduction pathways, angiogenesis, adhesion molecules expression and cell cycle control. Also, molecular studies are making clear that many genes, scattered through several different chromosomal regions probably cause predisposition to PCa. The discovery of new molecular markers for PCa is another relevant advance resulting from molecular biology studies of prostate tumors. Interesting tissue and serum markers have been reported, resulting in many cases in useful novelties to diagnostic and prognostic approaches to follow-up PCa. Finally, gene therapy comes as an important approach for therapeutic intervention in PCa. Clinical trials for PCa have been demonstrating that gene therapy is relatively safe and well tolerated, although some improvements are yet to be developed.

Molecular aspects of prostate cancer: implications for future directions.

Many studies have been developed trying to understand the complex molecular mechanisms involved in oncogenesis and progression of prostate cancer (PCa). Current biotechnological methodologies, especially genomic studies, are adding important aspects to this area. The construction of extensive DNA sequence data and gene expression profiles have been intensively explored to search for candidate biomarkers to evaluate PCa. The use of DNA micro-array robotic systems constitutes a powerful approach to simultaneously monitor the expression of a great number of genes. The resulting gene expressing profiles can be used to specifically describe tumor staging and response to cancer therapies. Also, it is possible to follow PCa pathological properties and to identify genes that anticipate the behavior of clinical disease. The molecular pathogenesis of PCa involves many contributing factors, such as alterations in signal transduction pathways, angiogenesis, adhesion molecules expression and cell cycle control. Also, molecular studies are making clear that many genes, scattered through several different chromosomal regions probably cause predisposition to PCa. The discovery of new molecular markers for PCa is another relevant advance resulting from molecular biology studies of prostate tumors. Interesting tissue and serum markers have been reported, resulting in many cases in useful novelties to diagnostic and prognostic approaches to follow-up PCa. Finally, gene therapy comes as an important approach for therapeutic intervention in PCa. Clinical trials for PCa have been demonstrating that gene therapy is relatively safe and well tolerated, although some improvements are yet to be developed.

NFATC2 transcription factor regulates cell cycle progression during lymphocyte activation: evidence of its involvement in the control of cyclin gene expression.

Upon antigen stimulation, lymphocytes enter in cell cycle and proliferate, and most of the activated T cells die by apoptosis. Many of the proteins that regulate lymphocyte activation are Under the control of transcription factors belonging to the NFAT family. As previously demonstrated, NFATC2-/- mice consistently showed a marked increase in lymphocyte proliferation. Here, we evaluate the role of NFATC2 in regulating lymphocyte proliferation and its involvement in the control of cell cycle progression during lymphocyte activation. NFATC2-/- lymphocytes, including CD4+ T cells and B cells, hyperproliferated upon stimulation when compared with NFATC2+/+ cells. Analysis of cell death demonstrated that NFATC2-/- lymphocytes displayed an increased rate of apoptosis after antigen stimulation in addition to the hyperproliferation. Cell cycle analysis after antigen stimulation showed that NFATC2-/- cultures contained more cycling cells when compared with NFATC2+/+ cultures, which is related to a shortening in time of cell division upon activation. Furthermore, hyperproliferation of NFATC2-/- lymphocytes is correlated to an overexpression of cyclins A2, B1, E, and F. Taken together, our results suggest that the NFATC2 transcription factor plays an important role in the control of cell cycle during lymphocyte activation and may act as an inhibitor of cell proliferation in normal cells.

Parasite signalling by host-derived molecules: facts and hypotheses

A series of descriptions in the literature points to the fact that an interaction between host-derived cytokines and protozoan parasites can play a role in the natural history of a disease. In this paper those examples are reviewed and discussed. In most cases, the host-derived molecules act as growth factors for the parasites, and in one case, protect the infective form from heatinduced death. Since the molecules described act as growth factores or as protectors from death when acting on cells from the host, it is suggested that their mechanism of action is the same, when targeting mammalian cells or protozoan parasites. A hypothesis is formulated that protozoan parasites might adapt to their mammalian host by "mimicking" a host cytokine-dependent system of cell growth and differentiation.