Relationships between specific antibody responses and clinical signs of dogs living in Tunisian endemic areas of canine leishmaniasis caused by Leishmania infantum.

The first step of the diagnostic process of canine leishmaniasis (CanL) is initiated by veterinarians and relies on their assessment of a high number of clinical signs common to other infectious diseases. We investigated herein the relationship between the clinical profile of 64 domestic dogs living in Tunisian endemic areas and their serological immune status with the aim to identify leishmanial serological markers of diagnosis and disease staging. Seven clinical signs were examined and a total clinical score that describes the number (TCS1) and the number plus the intensity of the clinical signs (TCS2) were determined. Laboratory tests consisted of parasitological examination (PE) of Giemsa-stained popliteal lymph node smears, indirect fluorescent antibody test (IFAT), IgG-, IgG1-, IgG2-Enzyme-Linked-Immunosorbent-Assay (ELISA), and IgG1-, IgG2- Western blotting (WB). Dogs' categorization according to the results of routine diagnostic tests, the TCS1 and TCS2, and the relative IgG1 and IgG2 specific reactivity allowed us to show that active CanL is characterized by an increased reactivity of the IgG2 specific antibodies. Interestingly, the IgG1 levels increased in parallel with the TCS1 and especially with the TCS2, indicating that this isotype is a better marker of dogs' health deterioration. PE & IFAT positive dogs which presented the highest TCS2 and IgG1 reactivity demonstrated significantly more severe weight loss and paleness of the mucosal membranes, suggesting that these signs characterize the latest stages of the disease. WB analysis showed that threeleishmanial polypeptides merit attention and further investigations. The antigens with MWs 32kDa reacting with IgG1 and 37kDa reacting withIgG2 antibodies were found associated with the results of diagnostic tests and late CanL stages, whereas the 24kDa antigen reacting with the IgG2 isotype and associated with low TCS2 seems to be a marker of the early stages.

Comparative analysis of the Leishmania infantum-specific antibody repertoires and the autoantibody repertoires between asymptomatic and symptomatic dogs.

Leishmania (L.) infantum-infected dogs may present with a large range of clinical signs, from apparently healthy with no or few (asymptomatic dogs, AD) to several clinical signs indicators of active infection (symptomatic dogs, SD). The present study is justified by the conflicting reports describing that either L. infantum-specific IgG1 or IgG2 antibodies may be used as isotype marker of the asymptomatic infection status and by the lack, to our knowledge, of previous analysis of the IgG sub-classes autoantibody repertoires of Leishmania-infected dogs. On the basis of clinical evaluation and laboratory testing (IFAT, parasitological examination of Giemsa-stained lymph node smears, L. infantum antigens-ELISA of total (Tot) IgG), 131 dogs were categorized as SD, asymptomatic seronegative (AND) or seropositive dogs (APD) from surrounding areas, and as negative control dogs (CTD). ELISA based on leishmanial native antigens or recombinant LACK and LeIF proteins showed that SD produce higher levels of specific Tot IgG, IgG1 and IgG2 antibodies than APD, and that for both clinical stages, the antibody titers of IgG2 isotype were constantly higher than those of the IgG1. The seroprevalences of Tot IgG, IgG2 did not differ between APD and SD groups (97 and 97% in SD; 100 and 96% in APD, respectively) whereas that of IgG1 was slightly lower in SD (88% of APD versus 82% of SD). The autoantibody repertoires were analyzed by ELISA using HEp-2 extracts, ds-DNA, human albumin and transferrin as self-antigens and by Western blot using HEp-2 proteins. ELISA results' indicated that APD develop higher levels of IgG1 autoantibodies, and higher seroprevalence (50% and 26% in APD and SD, respectively), contrasting with lower levels and seroprevalences of Tot IgG and IgG2 (43 and 68% for APD; 100 and 74% for SD). Interestingly, SD showed a stronger IgG1 and particularly IgG2 reactivity with transferrin, an iron-binding protein, than APD and AND. Western blotting experiments produced heterogeneous IgG1 and IgG2 inter- and intra-groups reactivity profiles towards HEp-2 proteins, to identify a specific antigenic profile. Generated data from competitive HEp-2-ELISA using leishmanial antigens as inhibitors were in favor that IgG1 antibodies are predominantly autoantibodies to self-antigens in APD whereas they are mainly cross-reactive (Leishmania/self-antigens) in SD.

Notch Stimulates Both Self-Renewal and Lineage Plasticity in a Subset of Murine CD9High Committed Megakaryocytic Progenitors.

This study aimed at reinvestigating the controversial contribution of Notch signaling to megakaryocytic lineage development. For that purpose, we combined colony assays and single cells progeny analyses of purified megakaryocyte-erythroid progenitors (MEP) after short-term cultures on recombinant Notch ligand rDLL1. We showed that Notch activation stimulated the SCF-dependent and preferential amplification of Kit+ erythroid and bipotent progenitors while favoring commitment towards the erythroid at the expense of megakaryocytic lineage. Interestingly, we also identified a CD9High MEP subset that spontaneously generated almost exclusively megakaryocytic progeny mainly composed of single megakaryocytes. We showed that Notch activation decreased the extent of polyploidization and maturation of megakaryocytes, increased the size of megakaryocytic colonies and surprisingly restored the generation of erythroid and mixed colonies by this CD9High MEP subset. Importantly, the size increase of megakaryocytic colonies occurred at the expense of the production of single megakaryocytes and the restoration of colonies of alternative lineages occurred at the expense of the whole megakaryocytic progeny. Altogether, these results indicate that Notch activation is able to extend the number of divisions of MK-committed CD9High MEPs before terminal maturation while allowing a fraction of them to generate alternative lineages. This unexpected plasticity of MK-committed progenitors revealed upon Notch activation helps to better understand the functional promiscuity between megakaryocytic lineage and hematopoietic stem cells.