Anti-galactose-alpha(1,3)Galactose antibody production in alpha1, 3-galactosyltransferase gene knockout mice after xeno- and allotransplantation.
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Biomedical subjects
Publications and source records attributed to L S Diamond.
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Antibodies (Abs) that mediate the hyperacute rejection and acute vascular rejection/delayed xenograft rejection of pig organs in humans and Old World primates are predominantly directed at a single carbohydrate epitope, galactose-alpha1,3-galactose (alpha1,3Gal). The T-cell dependence of elicited anti-alpha1,3Gal Ab responses in humans and Old World primates is controversial. In this study we have characterized anti-alpha1,3Gal Ab production in mice with disrupted alpha1,3-galactosyltransferase genes (GT-Ko mice) and determined the T-cell dependence of anti-alpha1,3Gal Ab responses, following xenograft and allograft transplantation. GT-Ko mice produce natural anti-alpha1,3Gal IgM and IgG in an age-dependent manner, however, these Abs could not elicit hyperacute rejection nor affect the rate of cardiac xenograft (3-5 days) or allograft rejection (7-9 days). Transplantation of xenogeneic Lewis rats hearts elicited modest anti-alpha1,3Gal Ab, but vigorous xenoAb responses. The anti-alpha1,3Gal Ab response was restricted to the IgM and IgG3 subclass while the xenoAb response comprised IgM and all four IgG subclasses. Transplantation of allogeneic C3H hearts elicited weak anti-alpha1,3Gal Ab responses that were primarily IgM, but vigorous alloAb responses. Despite the restriction of elicited anti-alpha 1,3Gal Ab responses to the IgM and IgG3 isotypes, these responses are T-cell dependent. The ability of allografts to elicit weak anti-alpha1,3Gal but strong allo-Ab responses, can be explained by the dependence of alpha1,3Gal-specific B cells on cognate help from T cells.
We sequenced small-subunit ribosomal RNA genes (16S-like rDNAs) of 10 species belonging to the genera Entamoeba and Endolimax. This study was undertaken to (1) resolve the relationships among the major lineages of Entamoeba previously identified by riboprinting; (2) examine the validity of grouping the genera Entamoeba and Endolimax in the same family, the Entamoebidae; and (3) examine how different models of nucleotide evolution influence the position of Entamoeba in eukaryotic phylogenetic reconstructions. The results obtained with distance, parsimony, and maximum-likelihood analyses support monophyly of the genus Entamoeba and are largely in accord with riboprinting results. Species of Entamoeba producing cysts with the same number of nuclei from monophyletic groups. The most basal Entamoeba species are those that produce cysts with eight nuclei, while the group producing four-nucleated cysts is most derived. Most phylogenetic reconstructions support monophyly of the Entamoebidae. In maximum-likelihood and parsimony analyses, Endolimax is a sister taxon to Entamoeba, while in some distance analyses, it represents a separate lineage. The secondary loss of mitochondria and other organelles from these genera is confirmed by their relatively late divergence in eukaryotic 16S-like rDNA phylogenies. Finally, we show that the positions of some (fast-evolving) eukaryotic lineages are uncertain in trees constructed with models that make corrections for among-site rate variation.
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Entamoeba histolytica has been redescribed as the separate species E. histolytica and Entamoeba dispar. E. dispar is apparently never invasive in humans, while E. histolytica is the etiologic agent of amebic colitis and liver abscess. Virulence factors that may enable E. histolytica to invade include a galactose-specific adhesin, secreted proteases, extracellular matrix receptors, and a cell surface lipophosphoglycan. Progress in vaccine development includes the identification of the cysteine-rich domain of the adhesin and the serine-rich surface protein as protective antigens.
The ability to identify individual isolates of Entamoeba histolytica Schaudinn 1903 (Emend. Walker 1911) is necessary before several important epidemiological questions can be answered. We have developed such a method based on our discovery of extensive polymorphism in two E. histolytica genes--the serine-rich antigen gene and the "strain specific gene"--each of which has an internal tandemly repeated structure. Using the polymerase chain reaction we detected both size and restriction site polymorphisms in the repetitive regions. When the two genes were used in combination we obtained 16 distinct DNA patterns out of 18 isolates examined. Moreover, these patterns proved to be stable under a variety of conditions--long-term culture, axenization, cell cloning, and animal passage.
The reported conversion of "nonpathogenic" Entamoeba histolytica isolates to the "pathogenic" form during attempted axenization of the amebae is highly controversial. After failing to obtain conversions ourselves we concluded that the simplest explanation for the published observations would be contamination of nonpathogenic cultures with pathogenic amebae. To address this possibility we used a method based on analysis of stable DNA polymorphisms that allows the positive identification of individual pathogenic isolates. The DNA patterns obtained using the "converted" amebae proved to be identical to those of reference isolates present in the laboratories at the time of conversion. We also found that very few cells need be transferred for a pathogenic contaminant to become established in a nonpathogenic culture. Cross-contamination fully explains the conversion phenomenon and thus recognition of nonpathogenic and pathogenic amebae as the distinct species Entamoeba dispar Brumpt 1925 and E. histolytica Schaudinn 1903 (Emend. Walker 1911), respectively, is upheld.
The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.
Entamoeba histolytica isolates have been shown to fall into two groups based on isoenzyme analysis. These groupings ("pathogenic" and "nonpathogenic") correlate well with the clinical course of the infection. A controversy exists over whether isoenzyme patterns are stable or whether under certain circumstances an isolate can convert from one form to the other. Resolution of this uncertainty is of importance since the nonpathogenic pattern has never been observed in amebae isolated from cases of active disease. This implies that, if the patterns are stable, carriers of amebae with this nonpathogenic pattern may never develop invasive disease. Although we set out to study isoenzyme conversion, we have been unable to replicate the two published accounts of this phenomenon. We have examined all of the variables proposed to be involved in the triggering of conversion, both individually and in combination. In none of the experiments was an alteration in the isoenzyme pattern observed. We now believe that isoenzyme patterns are stable and that all available evidence, other than the reported conversions, points to pathogenic and nonpathogenic E. histolytica being distinct species.
Changes in the cell surface of Entamoeba histolytica, a human intestinal parasite and the causative agent of amebic dysentery, were examined with a monoclonal antibody, 2D7.10, which selectively recognizes carbohydrate epitopes in some axenic amebic strains. While high-level expression of this epitope was observed in axenic amebae, it was either absent or present only in small amounts in xenic amebae. Furthermore, reassociation of the axenic amebae with intestinal flora resulted in loss of the 2D7.10 epitope. Our data suggest that surface antigens of E. histolytica can be modulated in response to bacteria and may provide an explanation for the observed influence of bacteria on amebic virulence.
Pap smears occasionally reveal protozoa of the genus Entamoeba in the uterus of intrauterine device (IUD) users, but definitive identification of the species involved has not been possible. Using riboprinting, a technique that compares ribosomal RNA gene sequences, we present evidence that the organism is Entamoeba gingivalis, an inhabitant of the mouth. Colonization most likely occurs via orogenital contact and requires the presence of an IUD and a concomitant bacterial infection.
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Differentiation of the pathogen Entamoeba histolytica from the variety of other amebas that can infect the human intestinal tract is vital for accurate diagnosis and treatment. Morphology and serology alone are not adequate for positive identification to be achieved. We have developed methods using the polymerase chain reaction to amplify amebal ribosomal RNA genes that allow either specific detection of E. histolytica or species identification.
Most infections with Entamoeba histolytica are asymptomatic. Two forms of the organism can be distinguished biochemically, and this finding has been explained by two distinct hypotheses: (1) there are two morphologically indistinguishable species, one of which causes disease; (2) there is one species which exists in two interconvertible forms, one of which causes disease. Knowledge of which hypothesis is correct has major implications for evaluation and treatment of carriers. We have studied the ribosomal RNA genes of the two forms hypothesizing that, if E. histolytica is one species, there should be no differences between them. We have found that the ribosomal RNA genes of the two forms are quite distinct, which supports the hypothesis that E. histolytica is two species.
A small number of Entamoeba isolates from humans, the best known of which is the 'Laredo' strain, have the ability to grow at room temperature. This peculiarity, along with other characteristics, distinguishes the strains from the human pathogen E. histolytica despite their being morphologically inseparable. In contrast, these 'E. histolytica-like' strains share several features with E. moshkovskii, which is most frequently isolated from polluted water. To examine the taxonomic relationships among these morphologically similar organisms, we have used polymerase chain reaction amplification of the small subunit ribosomal RNA gene combined with restriction fragment length polymorphism analysis, 'riboprinting'. The results clearly show that the 'E. histolytica-like' amoebae are indeed strains of E. moshkovskii, and not closely related to E. histolytica.
The in vivo incorporation of radiolabeled amino acids into antigens of Entamoeba histolytica, HM-1:IMSS, is reported. Immunoprecipitation with sera from patients with invasive amebiasis revealed a 28-kDa antigen present in whole cell lysates of E. histolytica. This antigen was of cytoplasmic origin, as indicated by cell fractionation and Triton X-114 detergent-phase separation. Immunoprecipitation, using sera from patients with invasive amebiasis and symptomless cyst passers, revealed the 28-kDa antigen as the major antigen recognized by the sera tested. Immunoprecipitation analysis using radiolabeled-released proteins instead of whole cell lysates showed a number of bands, including the 28-kDa antigen. The data suggest that the 28-kDa antigen is of cytoplasmic origin or is released from the cytoplasmic compartment.