Chromosome size and number polymorphisms in Leishmania infantum suggest amplification/deletion and possible genetic exchange.

We have studied the molecular karyotypes of 21 strains and 14 clones of Leishmania infantum using pulsed field gel electrophoresis (PFGE). We detected a high degree of polymorphism within this species, with 'strain-specific' patterns for most isolates, even within a restricted endemic area. Variations relate to both the size of chromosomes (270-2600 kb) and their number, which can vary from 24 to 31 between closely related isolates. This polymorphism does not correlate with isoenzyme analysis. Small size variations between homologous chromosomes of different strains are suggestive of DNA amplification/deletion events. Strains are also shown to be multiclonal, with slight differences between most clones, but with a predominant clone concealing the others in PFGE analysis. The analysis of these data leads to the hypothesis of occasional genetic exchange by nuclear fusion in Leishmania, as recently shown in the related protozoan Trypanosoma brucei.

Human plasmatic apolipoprotein H binds human immunodeficiency virus type 1 and type 2 proteins.

Apolipoprotein H (apo H), isolated from human plasma albumin solution, was shown to capture HIV-1-related antigens from antigen-positive sera (HIV-1 AG+) of AIDS patients, by using HIV-1-specific polyclonal antibodies. In an enzyme-linked immunosorbent assay and ligand blot and dot assays, apo H was able to bind recombinant retroviral HIV antigens, especially Gag proteins p18 of HIV-1, p26 of HIV-2, and Env gp160 of HIV-1. Binding was shown to be pH and NaCl dependent, with an optimum at acidic pH and low ionic strength. Specificity was demonstrated by saturation of this binding and inhibition either by homologous competition or by specific antisera. Binding was also observed in cell line-harvested viral proteins. The mechanism of this apo H-polyspecific binding is discussed in relation to conformational changes due to the influence of lipids or detergents.

A highly sensitive and rapid procedure for direct PCR detection of Leishmania infantum within human peripheral blood mononuclear cells.

We have developed a highly sensitive, simple and rapid procedure to detect Leishmania infantum within human macrophages. It only requires ficoll preparation of peripheral blood mononuclear cells from the patient, and their direct use for Leishmania kDNA amplification by polymerase chain reaction. Under these conditions, about one parasite can be detected in a one million human cell environment. Results, including those of a hybridization step to confirm the diagnosis specificity, are obtained with 24 h, a very short period as compared to current diagnostic methods. This procedure is of particular interest for early detection and early drug treatment of leishmaniasis, especially in the case of HIV coinfection. Furthermore, the method could be useful for monitoring the efficiency of new leishmaniasis treatments in infected patients.

Malaria: use of restriction endonuclease digestion and mutation-specific PCR for antifolate resistance isolate detection.

Antifolate resistance isolates of Plasmodium falciparum in the blood of 56 patients was investigated by using PCR technology. DNA was extracted with three different methods from parasite lysate by phenol-chloroform, or from whole blood and from blood collected onto dry filter paper, by chelex-100. The expected 727-bp PCR product was obtained in all samples extracted by chelex-100, while three samples prepared by phenol-chloroform failed to show any amplified product. The crucial point mutation within the dhfr gene leading to pyrimethamine and cycloguanil resistance is localised in an Alul recognition site. Thus, the 727-bp PCR product was submitted to endonuclease digestion. Fifty out of the 56 blood samples analysed yielded the two expected restriction fragments and an undigested 727-bp band. These 50 samples likely represent mixed infection as also confirmed the specific mutation PCR. The six undigested samples amplify a 339-bp fragment using a nested PCR-specific for pyrimethamine resistance mutation. Our results show that, the rapid DNA extraction from blood using chelex-100 and the PCR endonuclease assay can be efficiently used for accurate chemosensitivity analysis in the field.

"Pocket blotting": a method for transferring nucleic acids onto nylon membranes.

We have developed a fast and efficient method for transferring nucleic acids onto nylon membranes. This method requires less DNA for transfer; no decrease in efficiency is observed after successive probing, and several gels can be processed simultaneously. We believe that this techniques is of general interest in routine analysis of multiple samples in population genetic studies or in diagnosis purposes.

General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction.

Our strategy of probes designing for major clones of Trypanosoma cruzi was performed taking into account the: (i) clear identification of the major clones under multilocus study; (ii) hypothesis of a parallel evolution between the extranuclear and nuclear markers; (iii) structure of kDNA which allowed to amplify high variable regions of the minicircle (HVRm) by PCR. The large production of HVRm was very useful to test their ability to be used as probes for detection of DNA from a diversified genetic panel of T. cruzi. Our success in designing such probes has important implications on: the enhancement of 2 evolutive hypothesis about the clonal structure of T. cruzi and the parallel evolution of their extranuclear and nuclear genetics markers; direct diagnosis in patients and vectors. Studies on bio-clinical significance of major clones are discussed. This procedure could be used as a general strategy to generate DNA probes for Kinetoplastida.

Trichomonas vaginalis: repeated DNA target for highly sensitive and specific polymerase chain reaction diagnosis.

Trichomoniasis is recognised as a major sexually transmitted disease (STD) in the world and may act as an acquired immunodeficiency syndromes (AIDS) co-factor by enhancing the transmission of human immunodeficiency virus (HIV). Diagnosis of Trichomonas vaginalis can be achieved by several methods, but sensitive detection means are still lacking. In this study a 2000-bp repeated DNA fragment of T. vaginalis was cloned. Part of a conserved region of this insert was sequenced, two primers (TVK3 and TVK4) were chosen and a highly sensitive detection by polymerase chain reaction (PCR) was then developed for T. vaginalis. All strains of T. vaginalis analysed with these primers gave the expected 350-bp fragment and a 450-bp additional fragment. Sequence analysis of these PCR amplification products revealed that the 450-bp fragment contained the 350-bp with a 100-bp insertion characterised by a TGG microsatellite. A second primer set, namely TVK3 and TVK7 (determined at the border of the insertion), yielded PCR products of expected sizes. After amplification we were able to detect a single parasite. We also detected T. vaginalis in vaginal fluids of patients with STD. There was no reaction with human DNA or other infectious agents. It appears that the two set primers are highly specific of T. vaginalis and provide a useful tool for PCR diagnosis in asymptomatic and symptomatic patients especially among the HIV at risk individuals.

The SU glycoprotein 120 from HIV-1 penetrates into lipid monolayers mimicking plasma membranes.

Increasing evidence suggests that the HIV envelope binds through its surface (SU) gp120 not only to receptors and coreceptors, but also to other components of the cellular membrane where the glycolipids appear to be good candidates. To assess the ability of HIV-1 SU gp120 to penetrate into phospholipid membranes, we carried out a study of the interactions between a recombinant SU gp120 from HIV-1/HXB2 and artificial lipid monolayers mimicking the composition of the outer leaflet of the lymphocytes and which were spread at the air-water interface. We show that the protein, in its aggregated form, has amphipathic properties and that the insertion of this amphipathic species into lipids is favored by the presence of sphingomyelin. Furthermore, cholesterol enhances the penetration into mixed phosphatidylcholine-sphingomyelin monolayers. Coexistence of different physical states of the lipids and thus of domains appears to play a major role for protein penetration independently of the presence of receptors and coreceptors.

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure.

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.

Golgi vacuolization and immunotoxin enhancement by monensin and perhexiline depend on a serum protein. Implications for intracellular trafficking.

Vacuole formation around the Golgi and immunotoxin enhancement induced by low doses of the ionophore monensin were inhibited by 50% human plasma (final concentration), whereas the lysosomal pH increase remained unaffected. Immunotoxin enhancement by the Ca2+ antagonist perhexiline was also inhibited by plasma. The inhibiting factor was present in different species and highly concentration-dependent. After purification on DEAE- and CM-Sepharose it showed a heterogeneous distribution between 45 and 50 kDa, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an extreme isoelectric point near 3.5, and binding to wheat germ agglutinin-Sepharose. Maximum inhibition was found in the lower molecular mass fraction of 45 kDa. The 50-kDa fraction, although showing immunological identity reactions, remained almost inactive. The simultaneous inhibition of morphological alterations and the enhancement of immunotoxin activity by the highly enriched protein provides a first direct link between both events. Apart from a role of this serum glycoprotein on in vivo inhibition of immunotoxin enhancement, its ability to maintain normal intracellular trafficking in the presence of blocking agents, such as monensin and perhexiline, suggests a more fundamental role in the regulation of these mechanisms.

A CD4-independent interaction of human immunodeficiency virus-1 gp120 with CXCR4 induces their cointernalization, cell signaling, and T-cell chemotaxis.

The gp120 envelope glycoprotein of human immunodeficiency virus-1 (HIV-1) interacts with the CXCR4 chemokine receptor, but it is not known whether gp120 activates CXCR4-mediated signaling cascades in the same manner as its natural ligand, SDF1alpha. We assessed the effects of wild-type gp120 and a mutant gp120 that interacts with CXCR4 but not CD4 on CD4(-)/CXCR4(+) cells and CD4(+)/CXCR4(+) cells, respectively. Under both experimental conditions, the interaction of CXCR4 and gp120 resulted in their CD4-independent cointernalization. Both molecules were translocated into early endosomes, whereas neither protein could be detected in late endosomes. Binding of gp120 to CXCR4 resulted in a CD4-independent phosphorylation of Pyk2 and an induction of chemotactic activity, demonstrating that this interaction has functional consequences. Interestingly, however, whereas SDF1alpha activated the ERK/MAP kinase pathway, this cascade was not induced by gp120. Together, these results suggest that the pathology of HIV-1 infection may be modulated by the distinct signal transduction pathway mediated by gp120 upon its interaction with CXCR4.

Successful treatment of Trypanosoma cruzi encephalitis in a patient with hemophilia and AIDS.

Although the frequency of infection with the human immunodeficiency virus (HIV) is increasing dramatically in areas where Trypanosoma cruzi is endemic, trypanosomiasis has been rarely reported in persons with HIV infection or AIDS. Persons with hemophilia who receive multiple blood product transfusions from blood banks with little or no screening for infectious agents are at particularly high risk for infections with both HIV and T. cruzi. We describe the case of a person with hemophilia who was infected by blood transfusion with HIV and T. cruzi and in whom a multifocal, necrotic trypanosomal encephalitis was demonstrated by brain biopsy and electron microscopy. Treatment with benznidazole followed by that with itraconazole and fluconazole was associated with significant clinical and radiographic improvement.

HIV-1 glycoprotein 120 induces the MMP-9 cytopathogenic factor production that is abolished by inhibition of the p38 mitogen-activated protein kinase signaling pathway.

It has been previously shown that the HIV-1 envelope glycoprotein 120 (gp120) activates cell signaling by CXCR4, independently of CD4. The present study examines the involvement of different intracellular signaling pathways and their physiopathologic consequences following the CD4-independent interaction between CXCR4 or CCR5 and gp120 in different cell types: primary T cells, CD4(-)/CXCR4(+)/CCR5(+) T cells, or glioma cells. These interactions were compared with those obtained with natural ligands, stromal cell-derived factor 1 alpha (SDF-1alpha) (CXCL12) and macrophage inflammatory protein 1 beta (MIP-1beta) (CCL4) of their respective coreceptors. Thus, both p38 and SAPK/Jun N-terminal kinase mitogen-activated protein kinases (MAPKs) are activated on stimulation of these cells with either T- or M-tropic gp120, as well as with SDF-1alpha or MIP-1beta. In contrast, extracellular signal-related kinase 1 and 2 MAPKs are only activated by MIP-1beta but not by M-tropic gp120. Importantly, T- and M-tropic gp120 are able to induce the secretion of matrix metalloproteinase 9 (MMP-9), an extracellular metalloproteinase present in cerebrospinal fluid of patients with HIV-1 by T cells or glioma cells. Specific inhibition of MAPK p38 activation resulted in a complete abrogation of the induction of the MMP-9 pathogenic factor expression by gp120 or chemokines in both cell types. Because neurodegenerative features in acquired immune deficiency syndrome dementia may involve demyelinization by MMP-9, the specific targeting of p38 could provide a novel means to control HIV-induced cytopathogenic effects and cell homing to viral replication sites. (Blood. 2001;98:541-547)

Genetic diversity of simian immunodeficiency viruses from West African green monkeys: evidence of multiple genotypes within populations from the same geographical locale.

High simian immunodeficiency virus (SIV) seroprevalence rates have been reported in the different African green monkey (AGM) subspecies. Genetic diversity of these viruses far exceeds the diversity observed in the other lentivirus-infected human and nonhuman primates and is thought to reflect ancient introduction of SIV in the AGM population. We investigate here genetic diversity of SIVagm in wild-living AGM populations from the same geographical locale (i.e., sympatric population) in Senegal. For 11 new strains, we PCR amplified and sequenced two regions of the genome spanning the first tat exon and part of the transmembrane glycoprotein. Phylogenetic analysis of these sequences shows that viruses found in sympatric populations cluster into distinct lineages, with at least two distinct genotypes in each troop. These data strongly suggest an ancient introduction of these divergent viruses in the AGM population.

Regulatory genes of simian immunodeficiency viruses from west African green monkeys (Cercopithecus aethiops sabaeus).

The high seroprevalence of simian immunodeficiency viruses (SIVs) in African green monkeys (AGMs) without immunological defects in their natural hosts has prompted consideration of SIV-infected AGMs as a model of apathogenic SIV infection. Study of the molecular mechanisms of SIVagm asymptomatic infection could thus provide clues for understanding the pathogenesis of human immunodeficiency viruses. Regulatory genes could be candidates for genetic control of SIVagm apathogenicity. We have characterized Vpr, Tat, Rev, and Nef genes of two SIVagm strains isolated from naturally infected sabaeus monkeys captured in Senegal. The results provide further evidence that SIVagm from West African green monkeys is the most divergent class of AGM viruses, with structural features in long terminal repeat sequences and Vpr and Tat genes that distinguish them from viruses isolated from other AGM species (vervet, grivet, and tantalus monkeys).

Simian immunodeficiency virus infection in a patas monkey (Erythrocebus patas): evidence for cross-species transmission from African green monkeys (Cercopithecus aethiops sabaeus) in the wild.

Socio-ethological studies on troops of African green monkeys (AGMs) (Cercopithecus aethiops sabaeus) and patas monkeys (Erythrocebus patas) in Senegal have documented physical contacts between these two species. Elevated simian immunodeficiency virus (SIV) seroprevalence rates have been reported for the different AGM subspecies. We report here the extent to which patas monkeys are infected and compare the relatedness of the viruses isolated from theses two different species. Among the 85 AGMs and 54 patas monkeys studied, 47% of 7.5%, respectively, had antibodies that cross-reacted with HIV-2 envelope proteins. From two AGMs a virus was isolated. From the patas monkeys, virus isolation was generally not possible, but from one animal that was ill a virus designated pamG31 was amplified by PCR. In addition, for the two SIVagm isolates, an 830 bp region spanning the env and nef genes was amplified and sequenced. Comparisons of sequences from the env/nef region revealed 80% identity between pam G31 and SIVagm isolates from AGMs of the sabaeus subspecies, and 94% identity between the two SIVagm isolates. Phylogenetic analysis showed that pamG31 belongs to the SIVagm sabaeus subgroup. This is the first report of a lentiviral infection in a patas monkey. The close genetic relatedness between pamG31 and SIVagm sabaeus viruses is a strong argument in favour of cross-species transmission of SIV between AGMs and patas monkeys in the wild. For these reasons, we propose to refer to this patas virus as SIVagm-pamG31.

Hepatitis B virus Dane particles bind to human plasma apolipoprotein H.

Human apolipoprotein H (apo H) was found to bind specifically to hepatitis B surface antigen (HBsAg) from hepatitis B virus (HBV)-infected individuals. We used recombinant HBsAg proteins to analyze HBV domains recognized by apo H. We showed that the myristylated pre-S1 domain of HBsAg strongly interacted with apo H. This binding involved phospholipid components of the HBV envelope because their removal by detergent prevented apo H-HBsAg interaction. The opposite effects of iron and zinc metal ions on binding suggest that the oxidation of phospholipids also affects apo H-HBsAg interaction. After fractionation of viral particles on a sucrose gradient, and their addition to microtiter plates coated with apo H or anti-HBsAg, we observed that the maximal anti-HBsAg capture activity corresponded to a sucrose concentration of 36%, whereas the maximal apo H capture activity corresponded to a concentration of 39%. Electron microscopy and polymerase chain reaction (PCR) Southern blot studies of these fractions showed that the fraction with maximal apo H binding predominantly contained full Dane particles. Finally, we studied apo H-HBsAg binding relative to the presence of hepatitis B virus markers and observed that apo H binding activity for HBsAg was higher in sera from patients in the active virus replication phase.