In Vitro Evaluation of a Soluble Leishmania Promastigote Surface Antigen as a Potential Vaccine Candidate against Human Leishmaniasis

International audiencePSA (Promastigote Surface Antigen) belongs to a family of membrane-bound and secreted proteins present in severalLeishmania (L.) species. PSA is recognized by human Th1 cells and provides a high degree of protection in vaccinated mice.We evaluated humoral and cellular immune responses induced by a L. amazonensis PSA protein (LaPSA-38S) produced in aL. tarentolae expression system. This was done in individuals cured of cutaneous leishmaniasis due to L. major (CCLm) or L.braziliensis (CCLb) or visceral leishmaniasis due to L. donovani (CVLd) and in healthy individuals. Healthy individuals weresubdivided into immune (HHR-Lm and HHR-Li: Healthy High Responders living in an endemic area for L. major or L. infantuminfection) or non immune/naive individuals (HLR: Healthy Low Responders), depending on whether they produce high orlow levels of IFN-c in response to Leishmania soluble antigen. Low levels of total IgG antibodies to LaPSA-38S were detectedin sera from the studied groups. Interestingly, LaPSA-38S induced specific and significant levels of IFN-c, granzyme B and IL-10 in CCLm, HHR-Lm and HHR-Li groups, with HHR-Li group producing TNF-a in more. No significant cytokine response wasobserved in individuals immune to L. braziliensis or L. donovani infection. Phenotypic analysis showed a significant increasein CD4+ T cells producing IFN-c after LaPSA-38S stimulation, in CCLm. A high positive correlation was observed between thepercentage of IFN-c-producing CD4+ T cells and the released IFN-c. We showed that the LaPSA-38S protein was able toinduce a mixed Th1 and Th2/Treg cytokine response in individuals with immunity to L. major or L. infantum infectionindicating that it may be exploited as a vaccine candidate. We also showed, to our knowledge for the first time, the capacityof Leishmania PSA protein to induce granzyme B production in humans with immunity to L. major and L. infantum infectio

Successful reversal of transgene silencing in Chlamydomonas reinhardtii

Abstract Chlamydomonas reinhardtii has been successfully engineered to produce compounds of interest following transgene integration and heterologous protein expression. The advantages of this model include the availability of validated tools for bioengineering, its photosynthetic ability, and its potential use as biofuel. Despite this, breakthroughs have been hindered by its ability to silence transgene expression through epigenetic changes. Histone deacetylases (HDAC) are main players in gene expression. We hypothesized that transgene silencing can be reverted with chemical treatments using HDAC inhibitors. To analyze this, we transformed C. reinhardtii, integrating into its genome the mVenus reporter gene under the HSP70-rbcs2 promoter. From 384 transformed clones, 88 (22.9%) displayed mVenus positive (mVenus+) cells upon flow-cytometry analysis. Five clones with different fluorescence intensities were selected. The number of integrated copies was measured by qPCR. Transgene expression levels were followed over the growth cycle and upon SAHA treatment, using a microplate reader, flow cytometry, RT-qPCR, and western blot analysis. First, we observed that expression varies with the cell cycle, reaching a maximum level just before the stationary phase in all clones. Second, we uncovered that supplementation with HDAC inhibitors of the hydroxamate family, such as vorinostat (suberoylanilide-hydroxamic-acid, SAHA) at the initiation of culture increases the frequency (% of mVenus+ cells) and the level of transgene expression per cell over the whole growth cycle, through histone deacetylase inhibition. Thus, we propose a new tool to successfully trigger the expression of heterologous proteins in the green algae C. reinhardtii, overcoming its main obstacle as an expression platform

The impact of clothing style on bone mineral density among post menopausal women in Morocco: a case-control study

BACKGROUND: The clothing style is an important factor that influences vitamin D production and thus bone mineral density. We performed a case-control study in order to evaluate the effect of veil wearing (concealing clothing) on bone mineral density in Moroccan post menopausal women. METHODS: The cases were osteoporotic women whose disease was assessed by bone mineral density measurement. Each patient was matched with a non osteoporotic woman for age, and body mass index. All our patients were without secondary causes or medications that might affect bone density. The veil was defined as a concealing clothing which covered most of the body including the arms, the legs and the head. This definition is this of the usual Moroccan traditional clothing style. RESULTS: 178 post menopausal osteoporotic patients and 178 controls were studied. The mean age of the cases and the controls was 63.2 years (SD 7) and the mean body mass index was 32.1 (SD 8). The results of crude Odds Ratios analyses indicated that wearing a veil was associated with a high risk of osteoporosis: OR 2.29 (95% CI, 1.38–3.82). Multiparity or a history of familial peripheral osteoporotic fractures had also a significant effect on increasing the osteoporosis risk (ORs: 1.87 (95% CI, 1.05–3.49) and 2.01 (95% CI, 1.20–3.38)). After a multiple regression analysis, wearing the veil and a history of familial osteoporotic fractures remained the both independent factors that increased the osteoporosis risk (ORs: 2.20 (95% CI, 1.22–3.9) and 2.19 (95% CI, 1.12–4.29) respectively). CONCLUSION: our study suggested that in Moroccan post menopausal women, wearing a traditional concealing clothing covering arms, legs and head increased the risk of osteoporosis. Further studies are required to evaluate the clinical impact of the above findings and to clarify the status of vitamin D among veiled women in Morocco

Gain of chromosome arm 17q is associated with unfavourable prognosis in neuroblastoma, but does not involve mutations in the somatostatin receptor 2 (SSTR2) gene at 17q24

Deletion of chromosome arm 1p and amplification of the MYCN oncogene are well-recognized genetic alterations in neuroblastoma cells. Recently, another alteration has been reported; gain of the distal part of chromosome arm 17q. In this study 48 neuroblastoma tumours were successfully analysed for 17q status in relation to known genetic alterations. Chromosome 17 status was detected by fluorescence in situ hybridization (FISH). Thirty-one of the 48 neuroblastomas (65%) showed 17q gain, and this was significantly associated with poor prognosis. As previously reported, 17q gain was significantly associated with metastatic stage 4 neuroblastoma and more frequently detected than both deletion of chromosome arm 1p and MYCN amplification in tumours of all stages. 17q gain also showed a strong correlation to survival probability (P = 0.0009). However, the most significant correlation between 17q gain and survival probability was observed in children with low-stage tumours (stage 1, 2, 3 and 4S), with a survival probability of 100% at 5 years from diagnosis for children with tumours showing no 17q gain compared to 52.5% for those showing 17q gain (P = 0.0021). This suggests that 17q gain as a prognostic factor plays a more crucial role in low-stage tumours. Expression of the somatostatin receptor 2 (SSTR2), localized in chromosome region 17q24, has in previous studies been shown to be positively related to survival in neuroblastoma. A point mutation in the SSTR2 gene has earlier been reported in a human small-cell lung cancer. In this study, mutation screening of the SSTR2 gene in 43 neuroblastoma tumours was carried out with polymerase chain reaction-based single-stranded conformation polymorphism/heteroduplex (SSCP/HD) and DNA sequencing, and none of the tumours showed any aberrations in the SSTR2 gene. These data suggest that mutations in the SSTR2 gene are uncommon in neuroblastoma tumours and do not correlate with either the 17q gain often seen or the reason some tumours do not express SSTR2 receptors. Overall, this study indicates that gain of chromosome arm 17q is the most frequently occurring genetic alteration, and that it is associated with established prognostic factors. © 1999 Cancer Research Campaig

Suppression of LPS-induced inflammatory responses in macrophages infected with Leishmania

<p>Abstract</p> <p>Background</p> <p>Chronic inflammation activated by macrophage innate pathogen recognition receptors such as TLR4 can lead to a range of inflammatory diseases, including atherosclerosis, Crohn's disease, arthritis and cancer. Unlike many microbes, the kinetoplastid protozoan pathogen <it>Leishmania </it>has been shown to avoid and even actively suppress host inflammatory cytokine responses, such as LPS-induced IL-12 production. The nature and scope of <it>Leishmania</it>-mediated inflammatory cytokine suppression, however, is not well characterized. Advancing our knowledge of such microbe-mediated cytokine suppression may provide new avenues for therapeutic intervention in inflammatory disease.</p> <p>Methods</p> <p>We explored the kinetics of a range of cytokine and chemokine responses in primary murine macrophages stimulated with LPS in the presence versus absence of two clinically distinct species of <it>Leishmania </it>using sensitive multiplex cytokine analyses. To confirm that these effects were parasite-specific, we compared the effects of <it>Leishmania </it>uptake on LPS-induced cytokine expression with uptake of inert latex beads.</p> <p>Results</p> <p>Whilst <it>Leishmania </it>uptake alone did not induce significant levels of any cytokine analysed in this study, <it>Leishmania </it>uptake in the presence of LPS caused parasite-specific suppression of certain LPS-induced pro-inflammatory cytokines, including IL-12, IL-17 and IL-6. Interestingly, <it>L. amazonensis </it>was generally more suppressive than <it>L. major</it>. We also found that other LPS-induced proinflammatory cytokines, such as IL-1α, TNF-α and the chemokines MIP-1α and MCP-1 and also the anti-inflammatory cytokine IL-10, were augmented during <it>Leishmania </it>uptake, in a parasite-specific manner.</p> <p>Conclusions</p> <p>During uptake by macrophages, <it>Leishmania </it>evades the activation of a broad range of cytokines and chemokines. Further, in the presence of a strong inflammatory stimulus, <it>Leishmania </it>suppresses certain proinflammatory cytokine responses in a parasite-specific manner, however it augments the production of other proinflammatory cytokines. Our findings highlight the complexity of inflammatory cytokine signalling regulation in the context of the macrophage and <it>Leishmania </it>interaction and confirm the utility of the <it>Leishmania</it>/macrophage infection model as an experimental system for further studies of inflammatory regulation. Such studies may advance the development of therapies against inflammatory disease.</p

Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases

Impact of Continuous Axenic Cultivation in Leishmania infantum Virulence

Experimental infections with visceral Leishmania spp. are frequently performed referring to stationary parasite cultures that are comprised of a mixture of metacyclic and non-metacyclic parasites often with little regard to time of culture and metacyclic purification. This may lead to misleading or irreproducible experimental data. It is known that the maintenance of Leishmania spp. in vitro results in a progressive loss of virulence that can be reverted by passage in a mammalian host. In the present study, we aimed to characterize the loss of virulence in culture comparing the in vitro and in vivo infection and immunological profile of L. infantum stationary promastigotes submitted to successive periods of in vitro cultivation. To evaluate the effect of axenic in vitro culture in parasite virulence, we submitted L. infantum promastigotes to 4, 21 or 31 successive in vitro passages. Our results demonstrated a rapid and significant loss of parasite virulence when parasites are sustained in axenic culture. Strikingly, the parasite capacity to modulate macrophage activation decreased significantly with the augmentation of the number of in vitro passages. We validated these in vitro observations using an experimental murine model of infection. A significant correlation was found between higher parasite burdens and lower number of in vitro passages in infected Balb/c mice. Furthermore, we have demonstrated that the virulence deficit caused by successive in vitro passages results from an inadequate capacity to differentiate into amastigote forms. In conclusion, our data demonstrated that the use of parasites with distinct periods of axenic in vitro culture induce distinct infection rates and immunological responses and correlated this phenotype with a rapid loss of promastigote differentiation capacity. These results highlight the need for a standard operating protocol (SOP) when studying Leishmania species

Youth as Actors of Change? The Cases of Morocco and Tunisia

In the last decades, ‘youth’ has increasingly become a fashionable category in academic and development literature and a key development (or security) priority. However, beyond its biological attributes, youth is a socially constructed category and also one that tends to be featured in times of drastic social change. As the history of the category shows in both Morocco and Tunisia, youth can represent the wished-for model of future citizenry and a symbol of renovation, or its ‘not-yet-adult’ status which still requires guidance and protection can be used as a justification for increased social control and repression of broader social mobilisation. Furthermore, when used as a homogeneous and undifferentiated category, the reference to youth can divert attention away from other social divides such as class in highly unequal societies