Immunological selection for Leishmania (Viannia) braziliensis antigens.

Comparative ELISA and selective immunoblotting procedures were used in attempts to identify differential serological indicators of infection with the Leishmania (Viannia) braziliensis complex, infection with the L. braziliensis species, and therapeutic cure of localized or mucocutaneous leishmaniasis (LCL or MCL). Although mean ELISA absorbance values were significantly higher for MCL sera than for LCL sera, absorbance could not be used as a reliable indicator of the clinical form of disease. Immunoblotting profiles were similar with sera from MCL and LCL. Pre-adsorption with heterologous trypanosomatid antigens indicated that recognition of antigens of about 56, 60, 66, 72, 88 and 110 kDa might be specific to the subgenus Viannia. In two-colour, sequential, dual ELISA-based immunoblotting, no antigens recognized only by sera from MCL patients were detected. After glucantime therapy, immunoblotting profiles with LCL sera were reduced both in intensity and in the range of antigens detected; a 104-kDa antigen was newly detected with post-treatment LCL sera. Overall, the results show the value of differential immunological detection strategies and support the close relationship between species of the subgenus Viannia but fail to indicate a prognostic antigen for MCL.

Genomic diversity in the Leishmania donovani complex.

The Leishmania donovani complex is considered to be composed of 3 species; L. donovani, L. infantum and L. chagasi, although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum/L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.

Gene delivery to rat enteric neurons using herpes simplex virus-based vectors.

Neurons of the enteric (gut) nervous system can be cultured in vitro and readily survive transplantation into the brain making close connections with host neurons. As such, they could potentially be used to deliver therapeutic gene products to the brain after transduction with appropriate genes in culture. Here the authors report the first example of gene delivery to such cultured neurons using herpes simplex virus based vectors. They show that viruses lacking the immediate early gene encoding ICP27 (which are unable to replicate lytically) can efficiently deliver a marker gene to enteric neurons without producing extensive cellular damage. In contrast, viruses lacking only the viral neurovirulence factor encoded by ICP34.5 are inefficient in gene delivery, and produce extensive cellular damage, although they cannot replicate lytically in enteric neurons. A virus lacking both ICP27 and ICP34.5, however, produces less cellular damage than one lacking only ICP27, and is as efficient in gene transfer, whereas inactivation of VMW65 reduces toxicity further. The identification of this virus as a safe and efficient gene delivery vector for enteric neurons paves the way for the eventual delivery of therapeutic genes and subsequent transplantation of engineered neurons into the CNS.

Gene delivery to the heart in vivo and to cardiac myocytes and vascular smooth muscle cells in vitro using herpes virus vectors.

Herpes simplex virus 1 (HSV1), while usually thought of as neurotrophic, can also efficiently infect a wide variety of non-neuronal cell types and so might be developed as a vector for gene delivery to non-neuronal as well as neuronal cells. Here we have tested three different disabled HSV vectors for their ability to deliver a lacZ gene to primary cardiac myocytes and vascular smooth muscle cells in vitro, and used the most efficient virus to transfect the rat heart in vivo. We also assessed the degree of cytopathic effect of the various viruses on the cardiac myocytes in vitro by testing the effects on the frequency of beating in synchronously beating myocyte cultures. While an HSV mutant in which the essential immediate-early gene IE2 had been deleted gave high efficiency gene transfer to the cardiac myocytes in vitro and the rat heart in vivo, viruses in which ICP34.5 or ICP34.5 and VMW65 were inactive (and which were also unable to replicate in these cells) gave a much lower efficiency of gene transfer, mirroring the degree of cytopathic effect observed in the beating myocyte cultures. Gene transfer to the vascular smooth muscle cells was considerably less efficient than to the myocytes in all cases. These results indicate that while HSV may be inappropriate for highly efficient gene transfer to the arterial wall, efficient gene transfer can be achieved in the myocardium, and thus that HSV vectors may be suitable for the alteration of cardiac cell physiology in vivo.

Altered dinucleotide content within the latently transcribed regions of the DNA of alpha herpes viruses--implications for latent RNA expression and DNA structure.

The alphaherpesviruses establish latent infections in sensory neuronal cells, during which a number of latency-associated RNA transcripts (LATs) are produced. The reasons for the production of the LATs, however, and the mechanism by which the LAT region of the genome remains active during latency are unknown. Here we have analysed alphaherpesvirus sequences in an attempt to assess any differences between latently active regions of the genomes and the genomes as a whole which might be necessary for this differential expression pattern or for the LAT function, whatever that might be. We show that the LAT regions of all of the four alphaerpesviruses examined exhibit a previously unidentified and marked increase in CC + GG dinucleotides within the region and that three of the four viruses examined also show a marked decrease in CG + GC dinucleotides. The CC + GG increase was shown to occur throughout the LAT regions of these viruses, but the GC + CG decrease was shown to be confined only to LATs which are thought to be expressed as introns, and also to the intron sequences of the IE1 genes (at least for herpes simplex viruses 1 and 2), which overlap the LAT region. Bovine herpesvirus, the virus which did not show the GC + CG reduction in its LAT region but did show the GG + CC increase, is thought not to express LATs by a mechanism involving splicing, again indicating that the CG + GC reduction may be an intron-related phenomenon and also further suggesting that the two effects of CG + GC decrease and GG + CC increase may be independent of one another. Possible reasons for these unusual dinucleotide frequencies within the latently active regions of the alphaherpesviruses relating to DNA and RNA structure and to LAT function are discussed.

Down regulation of the octamer binding protein Oct-1 during growth arrest and differentiation of a neuronal cell line.

The octamer binding transcription/DNA replication factor Oct-1 is present in virtually all cell types including proliferating cell lines of neuronal origin but is not detectable in mature non-dividing neurons. Cell cycle arrest in G0/G1 and morphological differentiation of a neuronal cell line is accompanied by a decline in the level of Oct-1 DNA binding, although the level of DNA binding by another octamer binding protein, Oct-2 is unaltered. This effect is paralled by a decline in the level of the Oct-1 mRNA in the non-dividing cells. The decrease in Oct-1 levels occurs only with the production of a mature, non-dividing neuronal phenotype and not when the cells are arrested in late G1 and do not undergo morphological differentiation. The potential role of Oct-1 and other octamer binding proteins in gene regulation in neuronal cells and in their differentiation is discussed.

Heat shock proteins hsp90 and hsp70 protect neuronal cells from thermal stress but not from programmed cell death.

Prior exposure to a mild thermal stress can protect neuronal cells from a subsequent more severe stress including high temperature, ischemia, glutamate toxicity, or stimuli inducing apoptosis. Although the protective effect of thermal stress correlates with the elevated expression of the heat shock proteins (hsps), the protective effect of individual hsps has never been directly demonstrated in neuronal cells. Here we show that the constitutive overexpression of either of the major hsps, hsp90 or hsp70, can protect neuronal cells from thermal stress but not from stimuli that induce apoptosis. The possible mechanisms by which thermal stress can protect neuronal cells from apoptosis are discussed.

Visceral leishmaniasis in Teresina, state of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections

A Leishmania donovani-complex specific DNA probe was usedto confirm the widespread dissemination of amastigotes in apparently normal skinof dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35 per cent) fliesbecame infected: four of 65 flies (6 per cent) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previouslyon a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infectivebite (inner ear): 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67 per cent) of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L.chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. the Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies

A common pathway mediates retinoic acid and PMA-dependent programmed cell death (apoptosis) of neuronal cells.

The extent of programmed cell death (apoptosis) which occurs upon transfer of a neuronal cell line (ND7) to serum-free medium can be greatly increased by addition of retinoic acid (RA) to the medium. Here we show that the degree of apoptosis can also be enhanced by agents which activate protein kinase C (PKC) and that such agents synergize with RA in inducing apoptosis. In contrast chronic down regulation of PKC dramatically reduces the ability of RA to induce apoptosis whilst ND7 cell lines selected for resistance to RA-induced apoptosis are also resistant to apoptosis induced by PKC activation. This indicates that a common death pathway mediates the induction of apoptosis by PKC activators and RA. The potential nature of this pathway and the role of PKC in neuronal cell apoptosis is discussed.

Visceral leishmaniasis in Teresina, State of Piauí, Brazil: preliminary observations on the detection and transmissibility of canine and sandfly infections.

A Leishmania donovani-complex specific DNA probe was used to confirm the widespread dissemination of amastigotes in apparently normal skin of dogs with canine visceral leishmaniasis. When Lutzomyia longipalpis were fed on abnormal skin of five naturally infected dogs 57 of 163 (35%) flies became infected: four of 65 flies (6%) became infected when fed on apparently normal skin. The bite of a single sandfly that had fed seven days previously on a naturally infected dog transmitted the infection to a young dog from a non-endemic area. Within 22 days a lesion had developed at the site of the infective bite (inner ear): 98 days after infection organisms had not disseminated throughout the skin, bone marrow, spleen or liver and the animal was still serologically negative by indirect immunofluorescence and dot-enzyme-linked immunosorbent assay. When fed Lu. longipalpis were captured from a kennel with a sick dog known to be infected, 33 out of 49 (67%) of flies contained promastigotes. In contrast only two infections were detected among more than 200 sandflies captured in houses. These observations confirm the ease of transmissibility of L. chagasi from dog to sandfly to dog in Teresina. It is likely that canine VL is the major source of human VL by the transmission route dog-sandfly-human. The Lmet2 DNA probe was a useful epidemiological tool for detecting L. chagasi in sandflies.

Inhibition of herpes simplex virus infection by ectopic expression of neuronal splice variants of the Oct-2 transcription factor.

Herpes simplex virus (HSV) is capable of lytic replication in most cells, such replication in epithelial cells resulting in the mucocutaneous lesions observed following in vivo infection. In addition however, the virus also establishes asymptomatic latent infections in sensory neurons which serve as a reservoir for further cycles of peripheral lytic infections. These latent infections are dependent upon the inhibition of viral immediate-early (IE) gene expression via the octamer-related TAATGARAT motif in the IE promoters resulting in the failure of the viral lytic cycle. Here we show that the ectopic expression of neuronal isoforms of the octamer/TAATGARAT-binding transcription factor Oct-2 in permissive BHK cells represses IE gene expression following HSV infection and inhibits the viral lytic cycle whereas the B lymphocyte isoform of Oct-2 does not have this effect. These results suggest that the neuronal isoforms of Oct-2 play a critical role in rendering neuronal cells non-permissive for the viral lytic cycle thereby allowing the establishment of latent infection. Moreover, this is the first time that the ectopic expression of a cellular transcription factor has been shown to inhibit infection with any virus, raising the possibility of therapeutically inhibiting lytic viral infections by inducing such ectopic expression.

Heat shock protects neuronal cells from programmed cell death by apoptosis.

The programmed cell death (apoptosis) of a proportion of the neurons which form plays a critical role in the development of the nervous system and ensures that the correct number of mature neurons are ultimately present. We show that the prior exposure of neuronal cells to an elevated temperature sufficient to induce the heat-shock response partially protects the cells from apoptotic cell death following subsequent transfer to serum-free medium. The degree of protection observed in experiments using different heat-shock or recovery times correlates with the extent of heat-shock protein synthesis. Similarly activation of heat-shock protein synthesis by inducers other than elevated temperature also results in protection from apoptosis. The mechanism by which the heat-shock proteins may protect neuronal cells from apoptosis is discussed.

Transactivation by the herpes simplex virus virion protein Vmw65 and viral permissivity in a neuronal cell line with reduced levels of the cellular transcription factor Oct-1.

Transactivation of the herpes simplex virus (HSV) immediate-early (IE) genes is dependent upon the formation of a complex between the viral protein Vmw65 and the cellular transactivation factor Oct-1. Differentiation of the proliferating ND7 neuronal cell to a nondividing phenotype results in a large fall in the amount of Oct-1 to a level characteristic of nondividing neuronal cells but does not dramatically affect the level of IE gene expression following infection or the ability of Vmw65 to transactivate the IE promoter in transfection experiments. This suggests that the low levels of Oct-1 in nonproliferating neuronal cells do not play a key role in the failure of IE gene expression following initial infection of these cells, which is an essential step in the establishment of latent infections with HSV.

Visceral leishmaniasis in Teresina, n. e. Brazil: towards a DNA probe kit and its adaptation to processing blood-contaminated samples.

The Lmet2 chemiluminescent DNA probe is a valuable tool for identifying parasites of the Leishmania donovani -complex in sand flies, dogs and human samples. Recent blood meals in sand flies or blood contamination of tissue samples inhibited probe sensitivity, whether radiolabelled or chemiluminescent detection systems were used. Treatment of membranes with protease before hybridisation restored positive signal. Alternatively samples could be lysed with protease and applied to membranes with a vacuum blotting apparatus. The Lmet2 protocol provides the basis for a DNA probe kit that is adaptable for use with a wide range of other probes.

Cell cycle arrest of proliferating neuronal cells by serum deprivation can result in either apoptosis or differentiation.

Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.

Human urine stimulates growth of Leishmania in vitro.

Enhancement of trypanosomatid metacyclogenesis by insect urine components led us to test the effect of human urine as a culture additive. The addition of 1-5% urine to Schneider's Drosophila medium containing 10% foetal calf serum enhanced the growth of 11 Leishmania strains representing 8 different taxonomic groups. Cell division was stimulated in cultures with non-dividing organisms. Peak cell density was increased, as was the efficiency with which L. donovani could be isolated from infected hamsters. Preliminary work suggested that the modified medium would be useful for field isolation of L. donovani and L. braziliensis. The active nutrients or growth factors are not known.

A sensitive repetitive DNA probe that is specific to the Leishmania donovani complex and its use as an epidemiological and diagnostic reagent.

Leishmania donovani (HU3 strain) metacyclic promastigotes generated in vitro were used to construct a cDNA library in the bacteriophage vector lambda gt10. A cDNA clone (Lmet 2), was isolated by differential screening with metacyclic-derived or log-phase promastigote-derived cDNA. The clone insert was comprised predominantly of four copies of an imperfect 60-bp repeat motif, which was represented in the genome by multiple tandem repeats distributed among at least six chromosomes. The corresponding mRNA transcript was a developmentally regulated 12-kb doublet. The Lmet 2 sequence was entirely specific to L. donovani donovani, L. donovani (East Africa), L. donovani infantum and L. donovani chagasi, even when genomic Southern blots and slot blots of other Leishmania species were washed at low stringencies. Twenty-two strains of L. donovani were clearly detected by radiolabelled Lmet 2 cDNA probe, with signals of approximately equal intensity, irrespective of geographical origin, which encompassed widely dispersed endemic regions. The probe could detect DNA from fewer than 100 organisms and identified small numbers of promastigotes in infected sand flies. Amastigotes were also detected in impression smears of organs from infected hamsters. The Lmet 2 probe is likely to be a valuable reagent for clinical diagnosis and epidemiological investigations.

Trypanosoma cruzi infection in the Mexican state of Guerrero: a seroepidemiological (ELISA) survey of 20 communities.

The enzyme linked immunosorbent assay (ELISA) was used to analyse 4372 blood samples from residents of 978 households in 20 representative communities in the Mexican state of Guerrero. Seventy-five individuals had very high titres of antibodies against Trypanosoma cruzi. Samples with intermediate optical density values, despite overlapping values with several control positives on a single-well test, did not sustain their positivity at high dilutions. 'Intermediate positives' had a different distribution among the 20 communities to samples sustaining reactivity at high dilutions, indicating possible cross-reactivity with another infectious agent. The finding of seropositive children under the age of 10 years in the Costa Chica, Acapulco and the Tierra Caliente regions, with family clustering of putative cases, indicates that recent transmission must be considered. Very few people interviewed in the 20 communities knew the triatomine bug could transmit a disease.