Role of astrocyte senescence regulated by the non- canonical autophagy in the neuroinflammation associated to cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is a fatal neuroinflammatory syndrome caused (in humans) by the protozoa Plasmodium (P.) falciparum. Glial cell activation is one of the mechanisms that contributes to neuroinflammation in CM. RESULT: By studying a mouse model of CM (caused by P. berghei ANKA), we describe that the induction of autophagy promoted p21-dependent senescence in astrocytes and that CXCL-10 was part of the senescence-associated secretory phenotype. Furthermore, p21 expression was observed in post-mortem brain and peripheral blood samples from patients with CM. Lastly, we found that the depletion of senescent astrocytes with senolytic drugs abrogated inflammation and protected mice from CM. CONCLUSION: Our data provide evidence for a novel mechanism through which astrocytes could be involved in the neuropathophysiology of CM. p21 gene expression in blood cell and an elevated plasma CXCL-10 concentration could be valuable biomarkers of CM in humans. In the end, we believe senolytic drugs shall open up new avenues to develop newer treatment options.

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana.

Cutaneous leishmaniasis (CL) caused by infection with the parasite Leishmania exhibits a large spectrum of clinical manifestations ranging from single healing to severe chronic lesions with the manifestation of resistance or not to treatment. Depending on the specie and multiple environmental parameters, the evolution of lesions is determined by a complex interaction between parasite factors and the early immune responses triggered, including innate and adaptive mechanisms. Moreover, lesion resolution requires parasite control as well as modulation of the pathologic local inflammation responses and the initiation of wound healing responses. Here, we have summarized recent advances in understanding the in situ immune response to cutaneous leishmaniasis: i) in North Africa caused by Leishmania (L.) major, L. tropica, and L. infantum, which caused in most cases localized autoresolutives forms, and ii) in French Guiana resulting from L. guyanensis and L. braziliensis, two of the most prevalent strains that may induce potentially mucosal forms of the disease. This review will allow a better understanding of local immune parameters, including cellular and cytokines release in the lesion, that controls infection and/or protect against the pathogenesis in new world compared to old world CL.

Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia.

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

rs401502 and rs11575934 Polymorphisms of the IL-12 Receptor Beta 1 Gene are Protective Against Colorectal Carcinogenesis.

Background: Colorectal cancer (CRC) is a major public health problem worldwide and in Tunisia. It ranks among the main cancers in terms of incidence and cancer-related cause of death. Its pathogenesis is currently considered to be multifactorial involving genetic and environmental factors. Recent studies have suggested that the gene encoding the ß1 subunit of the IL-12 receptor, an important pro-inflammatory cytokine of the anti-tumor response, could be involved in the susceptibility to inherited CRC. Hence, it would be interesting to study the role of single nucleotide polymorphisms (SNPs) within the IL-12RB1 gene (rs401502 and rs11575934) in CRC susceptibility. Aim: Our purpose was to assess whether genetic variants IL-12RB1 +1196G/C (rs401502) and IL-12RB1 +705A/G (rs11575934) within the IL-12RB1 gene are associated with the sporadic CRC risk. Methods: A total of 110 Tunisian patients with sporadic CRC and 141 healthy control subjects were included in this study. Genotyping was performed by high-resolution melting (HRM) analysis. All results were confirmed by direct DNA sequencing or PCR-RFLP methods. Later, the allele frequencies and genotype distribution were established and compared between the control group and CRC patients. Results: The obtained results showed that the two target SNPs were in Hardy-Weinberg equilibrium (HWE) in both patients and controls. Minor allele frequencies of rs401502 SNP were 16.4% in CRC cases and 23.8% in controls. Mutant allele of rs11575934 SNP was present with 21.4% in CRC patients and 29.8% in control group. An association study showed a significant association of two target polymorphisms with CRC, according to the dominant genetic model with OR = 0.577, 95% CI = [0.343 to 0.972], p = 0.038 and OR = 0.547, 95% CI = [0.328 to 0.911], p = 0.02, respectively. Conclusion: In this study, we found, for the first time, a potential protective effect of two SNPs in the IL-12RB1 gene, namely rs401502 and rs11575934, in sporadic colorectal cancer in Tunisians.

A noncanonical autophagy is involved in the transfer of Plasmodium-microvesicles to astrocytes.

Cerebral malaria is a neuroinflammatory disease induced by P. falciparum infection. In animal models, the neuro-pathophysiology of cerebral malaria results from the sequestration of infected red blood cells (iRBCs) in microvessels that promotes the activation of glial cells in the brain. This activation provokes an exacerbated inflammatory response characterized by the secretion of proinflammatory cytokines and chemokines, leading to brain infiltration by pathogenic CD8+ T lymphocytes. Astrocytes are a major subtype of brain glial cells that play an important role in maintaining the homeostasis of the central nervous system, the integrity of the brain-blood barrier and in mounting local innate immune responses. We have previously shown that parasitic microvesicles (PbA-MVs) are transferred from iRBCs to astrocytes. The present study shows that an unconventional LC3-mediated autophagy pathway independent of ULK1 is involved in the transfer and degradation of PbA-MVs inside the astrocytes. We further demonstrate that inhibition of the autophagy process by treatment with 3-methyladenine blocks the transfer of PbA-MVs, which remain localized in the astrocytic cell membrane and are not internalized. Moreover, bafilomycin A1, another drug against autophagy promotes the accumulation of PbA-MVs inside the astrocytes by inhibiting the fusion with lysosomes, and prevents ECM in mice infected with PbA. Finally, we establish that RUBCN/rubicon or ATG5 silencing impede astrocyte production in CCL2 and CXCL10 chemokines induced by PbA stimulation. Altogether, our data suggest that a non-canonical autophagy-lysosomal pathway may play a key role in cerebral malaria through regulation of brain neuro-inflammation by astrocytes.

Use of Immunomagnetic Separation tool in Leishmania promastigotes capture.

Immunomagnetic Separation (IMS) assay has been used for isolation of viable whole organisms. The objective of our work is to produce anti-Leishmania magnetic beads and to assess the efficiency of the IMS technique on Leishmania promastigote capture in culture media. Polyclonal anti-Leishmania antibodies were produced by intravenous injection of viable metacyclic promastigotes of Leishmania (L.) major to rabbit. Purified anti-Leishmania IgG was assessed for their reactivity against both L. major and L. infantum promastigotes then covalently conjugated to magnetic beads and used for IMS. This latter was applied on either L. major promastigote cultures of known concentrations or early stage (24h, 48h, 72h) Novy-MacNeal-Nicolle (NNN) cultures of tissue fluid obtained from cutaneous leishmaniasis (CL) lesions. Promastigotes capture was assessed by either microscopy or qPCR after sample boiling. Indirect immunofluorescence assay showed that polyclonal antibodies reacted against both L. major and L. infantum promastigotes. In 50 µL solution, immunomagnetic beads were able to capture 5 live promastigotes out of 20 and 1050 out of 2500, giving an estimated efficiency of 25-42%. The efficiency of the IMS was lower for a lower number of parasites but still repeatable. On the other hand, IMS-qPCR applied to 14 NNN cultures of confirmed Leishmania lesions showed a higher sensitivity to detect live parasites than routine microscopy observation of promastigotes growth (93% positivity at 72h versus 50% positivity within 2-4 weeks incubation). The estimated number of captured parasites at 72h ranged from 1 to more than 100 parasites / 50 µL liquid phase of culture. These preliminary results open the way for interesting perspectives in the use of cultures for leishmaniasis diagnosis and also for other applications such as Leishmania detection in cultures taken from reservoir animals or sandflies.

Association of CCR5Δ32 Deletion and Human Cytomegalovirus Infection With Colorectal Cancer in Tunisia.

Background and objectives: Human cytomegalovirus (HCMV) and genetic polymorphisms of the chemokine receptor 5 have been suggested as factors associated with the progression of colorectal cancer (CRC). The aim of the study was to evaluate the associations of both CCR5Δ32 genetic deletion and/or HCMV virus infection with CRC in Tunisia. MATERIALS AND METHODS: The association between HCMV and CRC was validated by Nested PCR technology performed for HCMV and HCMV-specific serum IgG and IgM antibodies were investigated by enzyme-linked immunosorbent assay. Experiments were carried out on 40 tumor and 35 peri-tumor tissues, 100 blood from CRC patients and on 140 blood samples from healthy subjects and finaly serum samples of 80 patients with CRC and 100 healthy individuals. A conventional PCR has been optimized for the detection of CCR5Δ32 in100 CRC patients and 100 healthy subjects. RESULTS: Our results show that HCMV is significantly active in 93% of patients compared to 60% in controls (p < 0.0001, OR = 8.85, 95% CI: 3.82 -20.50). Compared to the healthy controls, the titers of IgG and IgM antiCMV antibodies in CRC patients were significantly higher than in healthy subjects (p value < 0,0001 for IgG and IgM). Statistical analysis revealed a lack of association between CCR5Δ32 mutation and colorectal cancer (p = 0.788, OR = 1.265, 95% CI: 0.228-7.011). CONCLUSION: our data confirmed that the HCMV infection was related to the development of CRC and that CRC cells may be infected more favorably by HCMV. Given the importance of the CCR5 in inflammation and therefore CRC progression, further studies still needed to evaluate CCR5 role as a potential candidate gene for CRC susceptibility under other polymorphisms.