Kinetoplast minicircle DNA of Trypanosoma evansi encode guide RNA genes.
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Animals↗
Biomedical subjects
R Hamers
Publications and source records attributed to R Hamers.
Dynamics and size polymorphisms of minichromosomes in Leishmania major LV-561 cloned lines.
Various lines and cloned lines of Leishmania major of varying degrees of virulence in BALB/c mice possessed size polymorphic multicopy minichromosomes related to previously described LD1/CD1 and 715-class DNAs of Leishmania. The minichromosomes were not necessary for virulence. Two of these DNAs (M180 and M210), coexisting in a single cloned line, showed remarkable dynamics in terms of loss or gain when followed through multiple transfers during in vitro culture and in vivo passage in BALB/c mice. Although there was significant sequence heterogeneity among minichromosomes, M180 sequences were present within large (megabase) and in intermediate (550-760 kb) chromosomes in the L. major lines analysed. M180 related small DNAs were also detected in Leishmania mexicana and Leishmania donovani isolates, suggesting that the generation of these molecules involves a common, probably functional basic mechanism widespread in Leishmania.
Animals↗
Structure of a Plasmodium chabaudi acidic phosphoprotein that is associated with the host erythrocyte membrane.
We have characterized by molecular cloning and sequencing a Plasmodium chabaudi antigen that is associated with the membrane of the infected erythrocyte throughout the entire intraerythrocytic cycle. The protein (PcEMA1) has a predicted size of 50 kDa and contains a major tandem repeat array of 16 octapeptides that constitutes almost 30% of the protein. At its amino-terminus, PcEMA1 has a string of hydrophobic residues characteristic of a secreted protein, but does not contain a hydrophobic membrane-spanning segment. The antigen appears to reside on the cytoplasmic face of the erythrocytic membrane. PcEMA1 has a predicted pI of 4.4 and is a potential phosphoprotein.
Amino Acid Sequence↗
DNA recombination associated with short direct repeats in Leishmania mexicana M379.
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Animals↗
Only the serum-resistant bloodstream forms of Trypanosoma brucei rhodesiense express the serum resistance associated (SRA) protein.
The Trypanosoma brucei species consists of three subspecies. T. b. rhodesiense and T. b. gambiense are human infective forms, while T. b. brucei is lysed upon exposure to the cytotoxic factor in normal human serum. T. b. rhodesiense can however occur as a serum-resistant (R) and as a serum-sensitive form (S). In a study of the molecular basis of serum resistance in T. b. rhodesiense it was shown that in the cloned ETaR1-repertoire only the serum-resistant variants express specific transcripts, encoding a protein with VSG-characteristics. When a serial of freshly isolated T. b. rhodesiense isolates was tested, the presence of the R-transcripts in the serum-resistant populations was confirmed. The absence of the serum resistance associated (SRA) mRNAs in several isolates from T. b. brucei and especially T. b. gambiense (including an a-typical one), indicates that more than one mechanism might be involved in the phenomenon of serum resistance in the T. brucei group. Neither T. evansi, nor T. equiperdum expresses the SRA-transcripts. Interestingly, the transcription of the R-specific transcripts is not maintained during the life cycle of T. b. rhodesiense; in the procyclic forms the SRA-mRNAs are no longer present.
Animals↗
Mechanisms underlying trypanosome-elicited immunosuppression.
T-cell proliferative responses of lymph node cells are profoundly suppressed during experimental infections of mice with Trypanosoma brucei. The active suppression of lymph node T-cell proliferative responses is attributed to the coexistence of at least two unlinked suppressive mechanisms that block different T-cell regulatory steps and operate through different effector mechanisms. The generation of prostaglandin-producing macrophages is entirely responsible for the suppression of IL-2 production whereas the induction of a prostaglandin-independent suppressive mechanism accounts for the suppression of the expression of IL-2 receptors (IL-2R). Both mechanisms are mediated by the cells that co-purify which macrophages. Despite an impairment at the level of T-cell proliferation, lymph node cells from T. brucei infected animals produce substantial amounts of interferon-gamma (IFN-gamma) and this lymphokine participates in the down-regulation of IL-2R expression. T-brucei-pulsed macrophage cell lines acquire concomitantly the potential to suppress T-cell proliferative responses and to stimulate CD8+ T-cells to secrete IFN-gamma. The sensibilization of CD8+ T cells by T. brucei-pulsed macrophages might be mediated by TNF-alpha. Collectively, these results indicate that the uptake of T. brucei by macrophages, either in vivo or in vitro, results in the generation of suppressive cells that annihilate T-cell proliferative responses. Furthermore, at least two cytokines (i.e., TNF-alpha and IFN-gamma) are released during these interactions. Besides playing a role in the pathway of T-cell immunosuppression, TNF-alpha and IFN-gamma could also contribute to immunopathological features that occur during trypanosome infections.
Animals↗
In vitro study on the impact of fish sera on the survival and fine structure of the eel-pathogenic acanthocephalan Paratenuisentis ambiguus.
The effects of fish sera on the growth and fine structure of infective larvae of the eel-pathogenic acanthocephalan Paratenuisentis ambiguus (Eoacanthocephala: Tenuisentidae) were studied under in vitro conditions using sera from the final host Anguilla anguilla and from two accidental fish hosts as well as fetal calf serum. As controls larvae were also kept in medium in the absence of serum and in experimentally infected eels. Sera from the accidental fish hosts carp and rainbow trout exerted toxic effects on the acanthocephalans. Worms maintained in medium containing sera from these two fish were contracted and displayed inverted probosces. Moreover, the tegument exhibited vacuolization and the formation of necrotic areas, including lysis of the mitochondria. Due to these effects, the parasites died at 21 (rainbow trout) or 21-50 days (carp) post-incubation. Eel sera had no toxic effect on the infective larvae. The growth of the larvae in medium depended on the composition of the latter, but was reduced as compared with that in the natural final host. Based on these results, we conclude that components of the hosts' blood sera play a role in the determination of the host specificity of P. ambiguus.
Acanthocephala↗
Analysis of MSA-1 diversity in Plasmodium chabaudi chabaudi strains.
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Amino Acid Sequence↗
Chromosome rearrangement in Leishmania mexicana M379.
Circular extrachromosomal elements were observed in a variety of Leishmania species. We show here that two lines originating from the same isolate have been found to contain a circular DNA molecule of 26.6 kb and a linear chromosome of about 250 kb, respectively, which share a homology of more than 20 kb. The circular DNA molecule and its related region on the linear chromosome were cloned and their restriction maps compared. This investigation reveals information about chromosome rearrangement in L. mexicana M379. Further examination will enable us to understand the nature of chromosome rearrangement such as circularization or linearization.
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Evidence for widespread asymptomatic Trypanosoma rhodesiense human infection in the Luangwa Valley (Zambia).
The RoTat 1/2 CATT test developed for Trypanosoma evansi was used in comparison with other diagnostic tests for the detection of T. rhodesiense infection in the northern part of the Luangwa Valley. The human population, the domestic and a large number of game animals were positive with the RoTat 1/2 CATT, the Ag-ELISA, the IFAT and the radioimmunoprecipitation tests. Human sera from these areas precipitated the same trypanosome-antigen components 35S-methionine labelled whereas few differences in band patterns were found between individual game animals. Surprisingly, however, T. rhodesiense could not be isolated from the "Ag-ELISA and radioimmunoprecipitation" positive patients from the Musenga and Kasyasya localities. The fact that the CATT positive humans were positive in antigen detection tests, does indicate that in all probability they carry or had been carrying a subpatent infection. These results suggest that the reservoir for T. rhodesiense in that region is considerable, comprising the game animals and probably to an even greater extent, the human population.
Agglutination Tests↗
Infectivity of Trypanosoma (Trypanozoon) brucei gambiense for baboons (Papio hamadryas, Papio papio).
In order to study sensitivity or resistance of T.b. gambiense to baboon serum, two species of baboons, P. hamadryas and P. papio were inoculated with T.b. gambiense clone LiTat 1.1. Both species were receptive to infection but, parasitological and immunological parameters showed that P. papio was more trypanotolerant than P. hamadryas. The VAT-specific trypanolysis test and the ELISA, using MoAb for circulating antigen detection may be appropriate for the diagnosis of human trypanosomiasis due to T.b. gambiense.
Animals↗
Evidence for kinetoplast and nuclear DNA homogeneity in Trypanosoma evansi isolates.
The kinetoplast DNA minicircles from 13 stocks of trypanosomes designated as Trypanosoma evansi were digested with various restriction enzymes. We also examined the distribution of restriction site polymorphisms in the nuclear DNA of 9 of these stocks, using 7 different variable surface glycoprotein (VSG) and non-VSG probes. Restricted kinetoplast DNA (kDNA) fragments of some of these strains were cloned into M13 or PUC 18 vectors and sequenced. The restriction and sequence mapping showed that most of T. evansi isolates belonged to the A1 and A2 types of Borst and to two new closely related types A3 and A4. A notable exception was RoTat 4/1 derived from a Sudanese stock which was found to display a characteristic brucei-like minicircle heterogeneity. The T. evansi minicircles analysed are not only homogeneous in sequence but also the region similar to the conserved region in Trypanosoma brucei and Trypanosoma equiperdum is flanked on its 5' end by a palindromic repeat of part of the conserved region. The highly conserved sequence GGGCGGT which appears to correspond to the initiation of synthesis of one of the Okazaki fragments contains an additional G and is located as in T. brucei and T. equiperdum about 73 bp 5' from the ORI. The nuclear DNA analysis confirms the kDNA study in that all the T. evansi stocks are members of a very homogeneous group in terms of sequence divergence. Moreover, our analysis also confirms that T. evansi is more closely related to the West African T. b. brucei and T. b. gambiense than to other African trypanosomes.
Animals↗
Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chabaudi IP-PC1.
The complete nucleotide sequence of the gene encoding the precursor to the major merozoite surface antigens of Plasmodium chabaudi chabaudi strain IP-PC1 has been determined. A single open reading frame was detected, that coded for a protein of 199 kDa. The encoded protein (p199) contains putative signal and membrane anchor sequences and shows a clustering of Cys residues in the last 120 amino acids. Incompletely conserved tandem repeat oligopeptides are present at different positions in the molecule. P199 shows 69% overall homology to the analogous antigen in Plasmodium yoelii yoelii strain YM. The divergence between these antigens is largely confined to 4 areas where a number of insertions and/or deletions have occurred. All repeats occur in these divergent regions. The overall homology with both alleles of Plasmodium falciparum PMMSA is 33%.
Amino Acid Sequence↗
Different mechanisms account for the suppression of interleukin 2 production and the suppression of interleukin 2 receptor expression in Trypanosoma brucei-infected mice.
Lymph node cell populations derived from Trypanosoma brucei-infected mice failed to produce interleukin 2 (IL 2) in response to a potent mitogenic trigger and suppress the potential of normal lymph node cells to secrete IL 2 in co-culture assays. This suppression is promptly restored by the addition of indomethacin, which blocks prostaglandin synthesis, but is not markedly affected by the addition of catalase, which degrades H2O2. The suppression of the IL 2 receptor expression, on the other hand, is not restored by the addition of indomethacin, nor by the simultaneous supply of both indomethacin and catalase. This discrepancy is not caused by an extreme susceptibility of the receptor expression to low prostaglandin (PG) concentrations, but rather by the presence of suppressive cells that operate through a PG-independent mechanism. This suppressive mechanism accounts for the loss of the IL 2 receptors on both the Ly-2+ and the L3T4+ T cell compartment. The indomethacin-treated co-cultures, which manifest a normal IL 2 production but lack the IL 2 receptors, manifest an impaired DNA synthesis and contain a decreased number of T cell blasts.
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Dual role of macrophages in the suppression of interleukin 2 production and interleukin 2 receptor expression in trypanosome-infected mice.
Lymph node cells derived from T. brucei-infected mice fail to produce interleukin 2-(IL2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL2 in co-culture experiments. The depletion of Thy-1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell-enriched nylon wool-nonadherent fraction, on the other hand, is not suppressive. The suppression of IL2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin-producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather directly interfere with the normal lymph node cells. In contrast to the essential role of prostaglandins in the impairment of IL2 production, these mediators are not involved in the suppression of IL2 receptor expression. Lymph node cells derived from Trypanosoma brucei-infected mice fail to produce interleukin 2 (IL2) subsequent to a potent mitogenic trigger and actively suppress the capacity of normal cells to produce IL2 in co-culture experiments. The depletion of Thy-1+ cells does not decrease but rather increases the suppressive potential of the LNC derived from infected mice. A T cell-enriched nylon wool-nonadherent fraction, on the other hand, is not suppressive. The suppression of IL2 production is promptly restored by the addition of prostaglandin synthesis inhibitors suggesting a key role of the prostaglandin-producing macrophages. Our data indicate that such macrophages do not act indirectly through the induction of suppressor T cells, but rather interfere directly with the normal lymph node cells.
Animals↗
Identification of virus-like particles in Eimeria stiedae.
When nucleic acid samples purified from sporozoites of Eimeria stiedae were analyzed by agarose gel electrophoresis, an ethidium-stainable band with an apparent electrophoretic mobility of 6.5 kb was consistently observed. The band was readily degradable upon RNAse treatment, and its susceptibility towards ribonuclease A on a decreasing ionic strength was suggestive of double-stranded RNA (dsRNA). Electron microscopy revealed spherical, probably icosahedral, virus-like particles (VLP) with a diameter of 35 nm in sporozoite lysates. The VLP were purified by CsCl buoyant density gradient centrifugation. Upon extraction, these particles yielded dsRNA molecules of a uniform length of 1.63 microns. The presence of the VLP was investigated in different Eimeria strains. All E. stiedae isolates contained the RNA virus, whereas the Eimeria intestinalis and Eimeria magna isolates tested did not. RNA/RNA hybridization experiments where the E. stiedae VLP dsRNA was probed to the genomes of the dsRNA viruses of Trichomonas vaginalis and Giardia intestinalis revealed a strong relatedness of the E. stiedae virus to the G. intestinalis virus, in contrast with the T. vaginalis virus, where no homology could be detected.
Animals↗
A gene expressed only in serum-resistant variants of Trypanosoma brucei rhodesiense.
The human infective African trypanosomes are host range variants of Trypanosoma brucei which are resistant to a lytic component in primate serum. T. b. rhodesiense occurs both as a form sensitive to lysis by normal human serum and as a form resistant to this lysis. Switching from one phenotype to the other has been observed in both directions. In the cloned T. b. rhodesiense ETAR1-repertoire we have detected 1.5-kb mRNAs only present in the resistant forms. In T. b. gambiense, which always occurs as a normal human serum-resistant form, no such transcript could be detected, indicating that another mechanism of resistance is involved here. Starting from an independent non-cloned T. b. rhodesiense population isolated from an infected patient, both resistant and sensitive trypanosomes have been prepared. Northern blot analysis of the total RNA prepared from these populations has revealed again the differential occurrence of the resistance-specific transcript, indicating that we are dealing with a general phenomenon associated with serum resistance in T. b. rhodesiense. As expected, Southern blot analyses have demonstrated that both serum-resistant and serum-sensitive forms of T. b. rhodesiense contain the gene coding for this transcript.
Amino Acid Sequence↗
Detection of circulating trypanosomal antigens in Trypanosoma evansi-infected animals using a T. brucei group-specific monoclonal antibody.
An antigen-detection enzyme immunoassay based on a T. brucei group-specific monoclonal antibody was used for the detection of circulating antigens in several animal species experimentally infected with T. evansi stocks from Sudan, Indonesia, Thailand and South America. Circulating antigens were detected as early as 6 days after infection, and they persisted throughout the observation period of up to 60 days postinfection. In an analysis of sera from naturally infected water buffaloes from Thailand, the test identified all the animals with positive parasitological findings, and 3 additional cases that had not been diagnosed by parasitological techniques. In an analysis of sera from pigs on a farm in Thailand suspected of a T. evansi outbreak, the assay detected "antigenaemia" in 66.7% of the animals, with antigen titres ranging from 1:2 to 1.512.
Animals↗