The chromosomes of Leishmania.

Chromosome size polymorphisms occur in Leishmania such that each strain of a given species has a distinctive molecular karyotype. Despite this variability, the chromosomal similarities among closely related strains of Leishmania are sufficiently characteristic to permit classification of unidentified clinical isolates. Mechanisms generating chromosome size polymorphisms are related to chromosomal evolution. In this review, Geoffrey Lighthall and Suzanne Giannini explain that the chromosomal profiles of members of different species may be diverging from a conserved 'consensus' karyotype at different rates, and present a current understanding of the genomic organization of Leishmania with emphasis on chromosomal elements.

Effects of ultraviolet B irradiation on cutaneous leishmaniasis.

Protection against many infectious diseases is mediated by cellular immunity in the competent host. Ultraviolet (UV) radiation, a component of sunlight, is a potent suppressor of cell-mediated immune responses. Suzanne Holmes Giannini discusses the possible relevance of ambient levels of UVB to pathogenesis and immunity in infectious diseases, with special reference to cutaneous leishmaniasis.

Leishmaniasis: an undesirable import.

The impact of leishmaniasis on public health is often underestimated. Prompt diagnosis and appropriate therapy may minimize the development of serious sequelae.

Characterization of effector functions of v-raf/mil- and v-myc-transformed murine splenic macrophage cell lines.

We have characterized several of the cytocidal effector functions of a series of cell lines derived by recombinant retroviral transformation of individual clones of C3H/HeJ mouse splenic macrophages. The three cell lines described in this report (4.01, 4.07, 4.14) all expressed equivalent tumoricidal activity against P815 tumor target cells. However they differed in their high avidity binding of tumor cells (4.01 = 4.14 greater than 4.07), as well as in the killing of Leishmania major (4.01 = 4.07 greater than 4.14), the expression of antibody-dependent cellular cytotoxicity against chicken erythrocytes (4.14 greater than 4.01 greater than 4.07), and finally, in the tumor-stimulated release of tumor necrosis factor-alpha (4.01 = 4.14 greater than 4.07). The stable and restricted expression of distinct effector functions among these three cell lines makes them particularly valuable as models for establishing the precise mechanisms by which cytocidal functions are effected. In addition, they should also prove of value in understanding the basis for macrophage functional diversity.

Recurrent lesions in human Leishmania braziliensis infection--reactivation or reinfection?

Strains of Leishmania braziliensis subspecies isolated from initial and recurrent lesions in 24 patients from the Pacific coast of Colombia were examined for distinguishing polymorphisms by enzyme electrophoresis, restriction endonuclease analysis of kDNA, and molecular karyotyping of nuclear DNA. Recurrent strains from 12 patients (50%) were identical to the initially infecting strain by all methods of characterisation. Phenotypic and genotypic identity, together with clinical data, support endogenous reactivation as the mechanisms of recurrent disease in these 12 patients. 5 of the 24 (22%) recurrent strains differed from the initial strain by all methods. The remaining 7 strain pairs, not separated by enzyme polymorphisms, showed differing schizodeme and/or karyotype profiles. Patients whose recurrent lesions were caused by strains different from those causing the initial lesions had a significantly longer disease-free interval than patients whose lesions were caused by identical strains. Recurrent lesions occurred further from initial lesions in the former than in the latter group. Exogenous reinfection is the most plausible explanation for recurrences due to disparate organisms. These findings have important implications for both treatment evaluation and vaccination strategies for American tegumentary leishmaniasis.

Size-conserved chromosomes and stability of molecular karyotype in cloned stocks of Leishmania major.

Molecular karyotypes for 5 stocks of Leishmania major were derived by pulsed-field gradient gel electrophoresis and transverse alternating field electrophoresis. Chromosome sizes obtained by the two methods agreed within less than or equal to 50kb. A set of 10 size-concordant chromosome bands between approx. 350-1000 kb was found in all stocks, plus a variable number of polymorphic chromosomes. Cloned gene probes, and DNA purified from individual chromosomes, hybridized to individual size-concordant chromosomes in different stocks, indicating a high degree of sequence homology among bands of similar size. Since the stocks were isolated over a 25-year period in a wide geographic area, we interpret these size-conserved chromosomes to be characteristic of the L. major karyotype. We were unable to identify irreversible genomic rearrangements in Leishmania cloned from the midgut of sandflies or cultivated from the skins of infected mice, which might have explained the origin of the size-variable chromosomes. For stocks that are maintained in the laboratory, the molecular karyotype appears to be a stable characteristic of a cloned population of Leishmania.

Structure and expression of the hsp 70 gene family of Leishmania major.

The parasitic protozoan Leishmania major differentiates in vitro, from the insect-adapted promastigote to the mammalian infective amastigote, in response to a temperature shift from 25 degrees C to 37 degrees C. We studied the genes encoding 70 kilodalton heat shock proteins (hsp 70 genes) in Leishmania substocks, which vary in their capability to differentiate. In total, four hsp 70 genes are arranged in tandem with intergenic regions of about 380 bp. These hsp 70 genes are 89% conserved at the aminoacid level when compared to the T. brucei hsp 70 genes. The expression of these four hsp 70 genes is increased, in vitro and in vivo, in response to a temperature shift from 25 degrees C to 37 degrees C. The parasite thus indeed responds to the transfer between hosts like it responds to a heat shock. In contrast, the high rate of transcription of a fifth identical hsp 70 gene, located at a separate locus, is unaffected by temperature shifts. The hsp 70 mRNAs have mini-exons trans-spliced onto their 5' ends and share unusually long (1000 nt) 3' untranslated extensions containing repetitive sequences. It is unclear whether or not the intergenic regions of the L. major hsp 70 genes function in transcription initiation and/or whether transcription results in the generation of polycistronic pre-mRNAs. Since each of the hsp 70 genes that we identified is expressed normally in an L. major substock that lost the capability to differentiate in response to an in vitro temperature shift, the inability to differentiate does not result from a general defect in the temperature-dependent control of transcription.

Karyotype analysis of Leishmania species and its use in classification and clinical diagnosis.

Chromosomes of four species of Leishmania represented by ten different geographic isolates were analyzed by pulsed field gradient gel electrophoresis (PFG) to assess chromosome stability in these parasitic protozoans. Among different geographic isolates of the same subspecies, more than two-thirds of chromosomes had similar sizes, ethidium bromide staining intensities, and locations of alpha,beta-tubulin genes. However, among New World Leishmania, members of different species or subspecies have fewer than one-third of their chromosomes in common. Therefore, PFG karyotypes of Leishmania exhibit intraspecific variability similar to that reported for other parasitic protozoans. The greater similarities of the karyotypes of members of the same Leishmania subspecies may indicate that they represent valid taxa. These similarities also allowed the use of PFG in clinical diagnosis for rapid and accurate typing of patient isolates.

Heat shock genes: regulatory role for differentiation in parasitic protozoa.

The parasitic protozoa Trypanosoma brucei and Leishmania major are transmitted by insect vectors to their mammalian hosts. The temperature difference between the hosts (25 degrees and 37 degrees C) may induce a heat shock response in the parasite. Transcripts of heat shock genes (homologous to Hsp70 and Hsp83) were 25 to 100 times more abundant in Trypanosoma brucei bloodstream forms (trypomastigotes) than in insect (procyclic) stages. In Leishmania major the patterns of heat shock gene expression in promastigotes (insect-adapted) and amastigotes (mammal-adapted) were different. A temperature shift in vitro induced differentiation of Leishmania major from promastigotes to amastigotes. Therefore, heat shock genes may be responsible for differentiation of these vector-borne parasites.

Induction and detection of leishmanial infections in Rattus norvegicus.

Weanling Sprague-Dawley rats were injected intradermally with Leishmania major or with L. donovani promastigotes. Parasites could be cultivated from the skin at times from 2 to 28 days after infection. At necropsy, no parasites were observed in spleen or liver impression smears, nor could they be cultivated from heart blood or spleen, even when skin cultures were positive. Rats were not highly susceptible to infection with L. donovani, since parasites could be cultured from skin only at 2 days after infection. L. major could establish an infection in rat skin, elicit antibodies, and in some cases, metastasize from the inoculation site. At necropsy, anti-leishmanial antibodies were detected in rats with parasites in their skin, although cutaneous lesions were not observed. These findings suggest that commonly used survey techniques are relatively insensitive, and might indicate possible involvement of Rattus norvegicus in the transmission cycle of cutaneous leishmaniasis.

Isolation of protective antigens from Trypanosoma lewisi by using trypanostatic (ablastic) immunoglobulin G from the surface coat.

Antigens were purified from extracts of Trypanosoma lewisi on immunoadsorbent columns of trypanostatic immunoglobulin G eluted from parasite surface coats at 8 days postinfection. Eight absorbed proteins, with molecular weights between 15,000 and 70,000, were identified. These surface coat antigens (SCAgs) were then used to immunize rats. After immunization, sera were assayed in vitro for levels of circulating trypanostatic and trypanocidal antibodies. Approximately half of the immune sera had higher levels of trypanostatic antibody, compared with control sera; no trypanocidal antibodies (agglutinins) were detected in any of the sera. The rats were then challenged intraperitonally, and the parasitemias and division rates of the parasites were monitored. Parasitemias of all immunized rats were significantly (P less than 0.01) lower and of shorter duration than those of the controls. Division rates of trypanosomes were also significantly (P less than 0.01) lower in all immunized rats at all times before total cessation of division compared with control rats. A clear dose-response effect was observed, with greater amounts of SCAg eliciting higher levels of protection. Purified SCAgs were also more effective immunogens than were the crude trypanosome extracts from which they had been purified, and in which other proteins in addition to the SCAgs were present. These data provide conclusive evidence that the immunoglobulin G in the surface coats of T. lewisi, adsorbed during the course of infection, is specific antibody, in that it can be used to isolate parasite antigens that elicit a trypanostatic response in rats immunized with them.

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi.

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.