Cellular and humoral responses induced by Leishmania histone H2B and its divergent and conserved parts in cutaneous and visceral leishmaniasis patients, respectively.

Leishmania histone H2B has been reported to be a promising candidate for both vaccination and serodiagnosis. We evaluated the cellular immune responses induced by H2B and its divergent amino-terminal (H2B-N) and conserved carboxy-terminal (H2B-C) regions in individuals with a history of Localized Cutaneous Leishmaniasis (LCL) due to Leishmania (L.) major. H2B induced significantly high PBMC proliferation and IFNgamma levels in LCL individuals whereas significantly lower proliferation and IFNgamma levels were observed with the divergent part of the protein. All proteins induced IL10 in LCL and healthy individuals. We also evaluated the humoral responses induced by these proteins in patients with Mediterranean Visceral Leishmaniasis (MVL) due to L. infantum. H2B and H2B-N were highly recognized by MVL sera. Our results show that the entire H2B protein is more efficient than its amino- and carboxy-terminal regions in inducing a dominant Th1 profile in cured LCL subjects and suggest that this protein may constitute a potential vaccine against leishmaniasis. Furthermore, H2B and H2B-N are interesting antigens for serodiagnosis of MVL.

Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites.

Leishmania parasites are able to survive in host macrophages despite the harsh phagolysosomal vacuoles conditions. This could reflect, in part, their capacity to secrete proteins that may play an essential role in the establishment of infection and serve as targets for cellular immune responses. To characterize Leishmania major proteins excreted/secreted early after promastigote entry into the host macrophage, we have generated antibodies against culture supernatants of stationary-phase promastigotes collected 6 h after incubation in conditions that partially reproduce those prevailing in the parasitophorous vacuole. The screening of an L. major cDNA library with these antibodies led us to isolate 33 different cDNA clones that we report here. Sequence analysis revealed that the corresponding proteins could be classified in 3 groups: 9 proteins have been previously described as excreted/secreted in Leishmania and/or other species; 11 correspond to known proteins already characterized in Leishmania and/or other species although it is unknown whether they are excreted/secreted and 13 code for unknown proteins. Interestingly, the latter are transcribed as shown by RT-PCR and some of them are stage regulated. The L. major excreted/secreted proteins may constitute putative virulence factors, vaccine candidates and/or new drug targets.

Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major.

To identify approaches for vaccination against leishmaniasis, we analyzed the protective effect of different constructions using recombinant peptides from the protein Leishmania (L.) major histone H2B. H2B sequence displays two distinct regions: an amino-terminal region divergent from mammalian H2B (27% identity) and a carboxy-terminal region highly conserved with mammalian H2B (55% identity). We tested the ability of the entire H2B protein, its divergent or conserved regions to provide protection against virulent L. major challenge. While the recombinant H2B protein adjuved with CpG induces potent cellular and antibody responses when injected to BALB/c mice, only the divergent amino-terminal region of H2B is able to confer potent protection against a virulent challenge. These findings indicate that different portions of the same parasite protein may express contrasting protective effects likely through the induction of different effector mechanisms. Due to its potent protective properties in the BALB/c mouse model, the amino-terminal region of Leishmania H2B could constitute a good vaccine candidate.

Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor.

Several approaches have been previously used to elucidate the genetic basis of Leishmania virulence. In general, they were based on laboratory Leishmania clones genetically modified or grown in the presence of selecting agents. In a previous study, we demonstrated that Leishmania major freshly isolated from human cutaneous lesions showed significant differences in the severity of the experimental disease induced in BALB/c mice. Here, using the mRNA differential display technique, we analyzed gene expression in L. major promastigotes showing different levels of virulence. We have identified a novel Leishmania gene encoding a 477-amino-acid protein exhibiting two distinct regions that are identical to the putative active-site sequence (CGHC) of the eukaryotic protein disulfide isomerase (PDI). The recombinant protein displayed a specific PDI enzymatic activity. This L. major disulfide isomerase protein (LmPDI) is predominantly expressed, at both the mRNA and protein levels, in highly virulent strains. Specific PDI inhibitors abolished the enzymatic activity of the recombinant protein and profoundly affected parasite growth. These findings suggest that LmPDI may play an important role in Leishmania natural pathogenicity and may constitute a new target for anti-Leishmania chemotherapy.

Heterogeneity of wild Leishmania major isolates in experimental murine pathogenicity and specific immune response.

Virulence variability was investigated by analyzing the experimental pathogenicity of 19 Leishmania major strains in susceptible BALB/c mice. Twelve strains were isolated from Tunisian patients with zoonotic cutaneous leishmaniasis; seven strains were isolated in Syria (n = 1), Saudi Arabia (n = 2), Jordan (n = 2), or Israel (n = 2). BALB/c mice were injected in the hind footpad with 2 x 10(6) amastigotes of the various isolates, and lesion progression was recorded weekly for 9 weeks. Interleukin-4 (IL-4) and gamma interferon (IFN-gamma) production of lymph node mononuclear cells activated in vitro with parasite antigens were evaluated 5 weeks after infection. We show that disease progression induced by different L. major isolates was largely heterogeneous although reproducible results were obtained when using the same isolate. Interestingly, isolates from the Middle East induced a more severe disease than did the majority of Tunisian isolates. Strains with the highest virulence tend to generate more IL-4 and less IFN-gamma in vitro at week 5 postinfection as well as higher levels of early IL-4 mRNA in the lymph node draining the inoculation site at 16 h postinfection. These results suggest that L. major isolates from the field may differ in virulence, which influences the course of the disease induced in mice and the type of immune response elicited by the infected host.

Mapping the interacting domains between the rabies virus polymerase and phosphoprotein.

The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein (P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.

Ethical considerations: educational programme for assisting staff.

With the advancement of dialysis technique, it has become possible to treat older patients with more complicated conditions. More and more often, staff have to face difficult treatment decisions. This has led to many questions, concerning for example the indications for life support and the risk of "over-treatment". Patients, relatives, doctors, nurses and social workers all have different ways of looking at these problems.

Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism.

The phosphoprotein of rabies virus is a 297-amino-acid polypeptide encoded by the longest open reading frame of the P gene. Immunoprecipitation experiments using a monoclonal antiserum directed against the P protein detected the P protein and at least four additional shorter products in infected cells, cells transfected with a plasmid encoding the wild-type P protein, and purified virus (CVS strain). By means of deletion analyses, these proteins were shown to be translated from secondary downstream in-frame AUG initiation codons. Immunofluorescence experiments indicated that all these P products were found in the cytoplasm of transfected cells; however, the proteins initiated from the third, fourth, and fifth AUG codons were found mostly in the nucleus. Changes in the 5'-terminal region of the P mRNA (including site-specific mutations, deletions, and insertions) demonstrated that a leaky scanning mechanism is responsible for translation initiation of the P gene at several sites.

In vivo interaction of rabies virus phosphoprotein (P) and nucleoprotein (N): existence of two N-binding sites on P protein.

The rabies virus phosphoprotein (P) and nucleoprotein (N) are involved in transcription and replication of the viral genome. Interaction between N and P was studied in vivo in transfected cells expressing both proteins. Co-immunoprecipitation assays revealed that the N-P complex is present in cells expressing both proteins as well as in infected cells. Furthermore, immunostaining showed that coexpression of N and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in infected cells. In addition, deletion mutant analysis of P was performed to identify the regions of P interacting with N. The results indicate that at least two independent N-binding sites exist on P protein: one is located in the carboxy-terminal part of the protein and another between amino acids 69 and 177. The formation of cytoplasmic inclusions seems to require the presence of both N-binding sites on P protein.