Biogeography of the marmosets and tamarins (Callitrichidae).

The marmosets and tamarins, Family Callitrichidae, are Neotropical primates with over 60 species and subspecies that inhabit much of South America. Although callitrichids exhibit a remarkable widespread distribution, attempts to unravel their biogeographic history have been limited by taxonomic confusion and the lack of an appropriate statistical biogeographic framework. Here, we construct a time-calibrated multi-locus phylogeny from GenBank data and the callitrichid literature for 38 taxa. We use this framework to conduct statistical biogeographic analyses of callitrichids using BioGeoBEARS. The DIVAj model is the best supported reconstruction of biogeographic history among our analyses and suggests that the most recent common ancestor to the callitrichids was widespread across forested regions c. 14 Ma. There is also support for multiple colonizations of the Atlantic forest region from the Amazon basin, first by Leontopithecus c. 11 Ma and later by Callithrix c. 5 Ma. Our results show support for a 9 million year old split between a small-bodied group and large-bodied group of tamarins. These phylogenetic data, in concert with the consistent difference in body size between the two groups and geographical patterns (small-bodied tamarins and large-bodied tamarins have an unusually high degree of geographic overlap for congeners) lend support to our suggestion to split Saguinus into two genera, and we propose the use of distinct generic names; Leontocebus and Saguinus, respectively.

Análise do espaçador intergênico do gene de calmodulina em parasitas da família trypanosomatidae: potencial como marcador molecular / Intergenic spacer analysis of calmodulin gene in the family Trypanosomatidae parasites: potential as a molecular marker

Caracterização e descrição de espécie na família Trypanosomatidae ainda depende da morfologia e identidade de hospedeiros dos tripanossomatídeos. Embora vários marcadores moleculaes tenham sido descritos nas ultimas décadas nenhum deles tem sido proposto como uma ferramenta molecular definitiva para complementar a informação ministrada pelas analises morfológicas. Com objetivo de avaliar o potencial do espaçador intergenico do gene de calmodulina como marcador molecular e estudar a composição desta região nos tripanossomatídeos, foram amplificados clonados e seqüenciados os espaçadores intergênicos de Crithidia fasciculata, C. deanei, C. guilhermei, C. acanthocephali, C. luciliae, C. desouzai, Trypanosoma rangeli e comparados com as seqüências que estão publicamente disponíveis de T. brucei, T. cruzi, Leishmania mexicana, L. major, L. infantum, L. arentolae e Phytomonas serpens. Características como organização genética do gene de calmodulina, conteúdo de G+C e presença de repeto~ções de simples seqüência também foram avaliados. O gene de calmodulina mostrou estar organizado in tandem de duas a quatro copias separados por espaçadores intergenicos os quais podem apresentar variação no tamanho. Estes espaçadores mostraram pouca variação intraespecífica no seu conteúdo de G+C sem importar qualquer diferença de tamanho. Nos tripanossomatídeos monoxênicos a tendência aponta que os organismos com um espaçador intergênico maior apresentam um conteúdo maior de G+C. evidenciou-se que a presença de repetições de seqüência simples nos espaçadores, elementos vinculados com a regulação da expressão genética dos tripanossomatideos. Embora o espaçador de calmoduline mostre variabilidade entre as copias do gene, nos demonstramos que seqüências na região 5UTR localizadas imediatamente antes do códon de inicio são espécie-específicas. Uma vez que este segmento genético é de fácil amplificação e esta limitado por uma seqüência conservada (ORF), resulta factível desenvolver uma ferramenta molecular que auxilie a morfologia tradicional na identificação de espécies na família Trypanosomatidae.

IgM/IgA nephropathy in callitrichids: antigen studies.

Renal tissues of callitrichids with IgM nephropathy were immunohistochemically examined for the participation of IgA in pathogenesis. In 58 histopathologically nephropathy-positive kidneys, IgM predominated in 20 cases and IgA in 7 cases, and in 31 cases both immunoglobulins were rated to be approximately equally involved. The disease, therefore, might be described as IgM/IgA nephropathy. The renal tissues and sera were also tested for nutritional antigens or antinutritional antigen antibodies, using immunohistochemistry and Western blots (tissues) and enzyme-linked immunosorbent assay (sera). Evidences of nutritional antigens in the renal tissues were inconclusive, although circulating IgG class antibodies against cereals, milk, and egg proteins were present in quite a number of sera. Particular consideration was paid to IgA-antigliadin antibodies, which were statistically significantly associated with nephropathy as were IgA rheumatoid factors. The findings are discussed in relation to human IgA and IgM nephropathies.

Mr 40,000 human colonic epithelial protein expression in colonic mucosa and presence of circulating anti-Mr 40,000 antibodies in cotton top tamarins with spontaneous colitis.

Saguinus oedipus, Callithrix jacchus, and Saguinus fuscicollis are three species of New World monkeys which develop a form of colitis that is similar to human ulcerative colitis. Only S oedipus, however, develop colon cancer. We examined intestinal tissues from these animals for the presence of an antigen cross reacting to the Mr 40,000 human colonic epithelial protein that acts as an autoantigen in ulcerative colitis. Using an anti-Mr 40,000 monoclonal antibody (7E12H12, IgM isotype), by an immunoperoxidase assay we showed that all colon specimens from S oedipus reacted with 7E12H12; however, the colonic tissue from C jacchus and S fuscicollis did not. In immunotransblot analysis eluted IgG antibody bound to human ulcerative colitis colon (CCA-IgG) reacted with Mr 40,000 protein(s) present in the extracts of colon from S oedipus animals and humans. Small intestinal tissue reacted neither with 7E12H12 nor with CCA-IgG. In S oedipus, the Mr 40,000 protein was localised exclusively to colonic epithelial cells. Preincubation of seven S oedipus colon specimens with eight of 10 sera from animals with acute or chronic colitis and 0 of four sera from animals without colitis almost completely inhibited the binding of 7E12H12 to the colonic epithelium. Four of these 10 sera inhibited the binding of 7E12H12 to the autologous colon. These results show the presence of circulating autoantibodies in S oedipus with colitis against an epitope(s) on Mr 40,000 protein shared by human and S oedipus colon.

Evolution of the MHC class I genes of a New World primate from ancestral homologues of human non-classical genes.

The products of the classical human major histocompatibility complex (MHC) class I genes (HLA-A, -B, -C) are highly polymorphic molecules that bind peptides and present them to T lymphocytes. The non-polymorphic, non-classical MHC class I gene products (HLA-E, -F, -G) are not restricting elements for the majority of T lymphocytes. The evolutionary relationship of the non-classical and classical MHC class I genes is unclear. Here we present the cloning and sequencing of the MHC class I genes of a New World primate, the cotton-top tamarin (Saguinus oedipus). The expressed MHC class I genes of this species are more closely related to the human non-classical HLA-G gene than they are to genes of the human classical HLA-A, -B, and -C loci. These observations imply that classical and non-classical genes do not necessarily constitute mutually exclusive groups over evolutionary time.

Genetically distinct cell populations in naturally occurring bone marrow-chimeric primates express similar MHC class I gene products.

The cotton-top tamarin (Saguinus oedipus) is a naturally occurring "A" + "B"----"A" bone marrow-chimeric species. These primates usually are born as dizygotic twins and, due to placental vascular anastomoses, develop sharing each others' bone marrow elements. Strikingly, almost 50% of the PBL of a member of a twin pair are derived from the hematopoietic stem cells of its cotwin. To clarify the mechanisms underlying the maintenance of tolerance in these stable chimeras, MHC gene products have been biochemically characterized in cloned, genetically distinct, male and female lymphocytes from two male/female cotton-top tamarin twin pairs. Extensive MHC class II sharing between the genetically distinct cell populations was not seen in the two twin pairs. This was consistent with the MHC class II polymorphism seen in the species. However, the MHC class I gene products expressed by one member of a twin pair were almost identical to those expressed by its cotwin. A human minisatellite probe demonstrated restriction fragment length polymorphism in DNA from these animals, indicating extensive polymorphism. Thus, MHC class I sharing did not occur due to inbreeding in these animals. Additionally, another bone marrow-chimeric primate species, the common marmoset (Callithrix jacchus), expresses MHC class I molecules with low levels of variation. These studies suggest that the stable chimerism of bone marrow-chimeric primates may be facilitated by MHC class I similarity between the genetically distinct bone marrow derived-cell populations in their circulation.

Immunoregulation in the common marmoset, Calithrix jaccus: functional properties of T and B lymphocytes and their response to human interleukins 2 and 4.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.

Monoclonal antibodies from Epstein-Barr virus-transformed lymphocytes of common marmosets (Callithrix jacchus) immune to malaria.

The B lymphocytes of the common marmoset Callithrix jacchus can be immortalized by infection with Epstein-Barr virus (EBV) in vitro (Desgranges et al., 1976). C. jacchus is susceptible to infection with the blood stages of several species of malaria parasite including the line designated MVF1 (Mitchell et al., 1988) from which it recovers and shows immunity to reinfection. By exploiting these two phenomena, EBV-transformed, marmoset lymphoblastoid cell lines secreting antibodies to malaria parasite antigens have been generated and cloned. We believe this to be the first time that monoclonal antibodies (MAbs) have been raised from common marmosets. Since numerous and diverse human pathogens can infect this small primate in the laboratory, these methods may prove generally applicable for the generation of MAbs whose specificities derive from immune responses to infection.

Expression of immune sensitization to epithelial cell-associated components in the cotton-top tamarin: a model of chronic ulcerative colitis.

Chronic colitis is present in up to 50% of adult cotton-top tamarins, but the etiology is unknown. To explore one putative immunopathogenic pathway for the chronic colitis, we determined whether immune sensitization to macromolecules associated with mucosal epithelium of intestine (designated ECAC) had occurred in this primate species. Specifically, we sought to define (a) whether antigenic determinants associated with ECAC are present on tamarin gut epithelium in vivo; (b) if immunoglobulin, capable of binding ECAC, is detectable in tamarin serum; (c) whether the presence of ECAC-specific immunoglobulin is positively correlated with age of the animal or the severity of the colitis, or both; and (d) the number of glycoprotein fractions composing ECAC (denoted as P1 through P4) that are reactive with tamarin immunoglobulin. Expression of ECAC was found by immunofluorescence using characterized oligo-specific or monoclonal antibody: tamarin intestinal epithelium--but not lamina propria, muscularis mucosae, subserosa, or glycocalyx--demonstrated determinants of the ECAC antigen system. Furthermore, coded sera from 10 tamarins with biopsy-proven inflammation involving colonic intestinal mucosa and in which disease activity was moderate to severe showed ECAC-specific cytotoxicity of 3.6% +/- 1.6%. Test values for 9 of 10 of those animals were above the upper limit of normal for the assay (2.1%), and exceeded the level of lysis found with serum from histologically normal tamarins less than 2 yr old (less than 0.3%). When the data were reanalyzed by age of the animal, the incidence of ECAC-specific cytotoxicity correlated with age greater than 2 yr (r = 0.86, p less than 0.001). Epithelial cell-associated component-specific serum binding was confirmed by a second methodology (enzyme-linked immunosorbent assay), where the A405 for tamarins with lesions of moderate-severe grade (0.35 +/- 0.26) clearly exceeded that for young tamarins who were histologically normal (0.02 +/- 0.039) (p less than 0.05). Most of the reactivity was directed toward the P1 fraction of ECAC. Thus, immune sensitization to a fraction of macromolecules associated with colonic epithelium has been found in the cotton-top tamarin, analogous to findings in humans with chronic inflammatory bowel disease.

Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains.

Most studies using rabbit or mouse antisera failed to detect CRI between human IgM directed to MAG. We show here that 9 of 10 such IgM express a public CRI as defined by a nonhuman primate antiserum. Shared idiotype is likely involved in (or close to) the combining site of those IgM since antiidiotypic serum inhibited the binding of IgM to MAG and reacted with IgM having different variable regions of light and heavy chains. Partial aminoterminal sequence of heavy and light chains showed that anti-MAG IgM use either lambda chains (one IgM) or kappa light chains (six IgM) of different variability subgroups (V kappa IV in three instances, V kappa I in two, and V kappa II in one), whereas heavy chains belong to the VHIII (six IgM) or to the VHII (1 IgM) subgroup. These features distinguish these IgM from other human monoclonal IgM with a defined antibody activity, such as rheumatoid factors or cold agglutinins.

Analysis of T lymphocyte subsets in tamarins with colitis and colon cancer.

The cotton-top tamarin, Saguinus oedipus, serves as an animal model for the study of human colon cancer. This New World monkey has a high incidence of colitis and colon cancer that develops spontaneously. Evidence suggests that these diseases may be the result of a virally induced immunodeficiency. We have shown that T4+/T8+ cell ratios are significantly altered in tamarins with acute colitis and colon cancers. The T4+/T8+ ratios were 1.50 +/- 0.09, 0.70 +/- 0.05, and 0.48 +/- 0.05 for negative controls, acute colitis, and cancer positive tamarins, respectively. Statistical analysis showed a significant difference (p less than or equal to .0005) between negative controls vs. acute colitis and cancer positive groups.

Colon cancer cells in peripheral blood of cancerous tamarins.

Diagnosing colon cancer in its early stages would lower the mortality rate. The cotton-top tamarin, Saguinus oedipus, serves as a model for the study of human colon cancer. This New World monkey has a high incidence of colitis and colon cancer. The mouse anti-human monoclonal antibody BR55.2, with specificity for human colon adenocarcinoma, was biotinylated. Peripheral blood mononuclear cells (PBMC) from animals with colon cancer were fluorescently stained with the biotinylated BR55.2. These results showed the cross-reactivity of mouse anti-human colon cancer monoclonal antibody to the PBMC of cancerous tamarins. Antibodies from either cancerous or chronic colitis tamarins were also biotinylated. Fluorescently labeled cells were detected when PBMC from cancerous tamarins were incubated with biotinylated antibodies from cancerous tamarins. Cytofluorographic analysis also showed a significant 4.5-fold difference in the percentage of fluorescently labeled PBMC between cancerous and chronic colitis tamarins when stained with biotinylated antibodies from cancerous tamarins. DNA flow cytometry analysis showed that PBMC from cancerous tamarins have a higher percentage of aneuploid cells than PBMC from chronic colitis tamarins.

Demonstration in vitro of cell mediated immunity to Epstein-Barr virus in cotton-top tamarins.

In the course of developing an effective Epstein-Barr (EB) virus vaccine, the immune responses in cotton-top tamarins to a tumourigenic dose of EB virus were studied. Cell mediated responses were measured using a tissue culture 'growth inhibition' assay where peripheral blood lymphocytes were tested for their ability to inhibit the outgrowth of autologous EB virus transformed lymphoblastoid cells. This system has previously been recognized as a very sensitive assay for detecting cell-mediated responses to EB virus in man. Using this assay no cell-mediated immunity was detected up to the time of death in two tamarins following injection with a tumourigenic dose of EB virus. However, two other animals which had recovered from tumours induced by a first dose of EB virus 18 months previously when subsequently re-stimulated with a second tumourigenic dose did exhibit cell-mediated responses. These latter animals remained healthy following the re-challenge and did not show evidence of EB virus-induced disease.

Marmoset red blood cell receptor for membrane-associated complement components is not related to human CR1: partial characterization of the C3-binding proteins responsible for the spontaneous rosette formation between marmoset red blood cells and human leukocytes.

Cells from all the human B-lymphoblastoid cell lines tested and most human monocytes form rosettes with marmoset red blood cells (MaRBC). Because previous reports suggested the involvement of complement components in this phenomenon, the mechanism of rosette formation and the eventual similarities between the MaRBC receptor and the CR1 receptor present on human erythrocytes have been analyzed herein. The binding of MaRBC to human leukocytes strongly differs from the immune adherence phenomenon: rabbit anti-human CR1 did not react with MaRBC and the MaRBC receptor-binding activity is Ca2+-dependent. Rosette formation required intact energy metabolism and cytoskeleton integrity of leukocytes. Our attempts to purify the receptor from MaRBC membranes revealed the absence of CR1. Nevertheless, C3-binding proteins were isolated by selective desorption by Sepharose iC3 column chromatography. A three-band pattern was observed under reduced conditions with 74,000, 70,000, and 53,000 molecular weights. It was not possible to further separate these components. This protein complex inhibited the rosette phenomenon between MaRBC and both Raji and U-937 cells, exhibited a very poor cofactor activity, and had no decay-accelerating activity toward the classical C3 convertase. This material did not cross-react with antibodies directed to human proteins. These results showed that erythrocytes from new world monkeys do not express a receptor analogous to the human CR1, but expressed C3-binding protein with low cofactor activity that could recognize membrane-associated complement components.

Hepatitis A virus replication in tamarins and host immune response in relation to pathogenesis of liver cell damage.

Hepatitis A virus (HAV) shedding in the faeces, appearance of HAV-Ag (antigen) in the liver, and development of humoral immunity to HAV have been studied in experimentally infected tamarins. The appearance of liver damage measured by transaminase elevation and histology, in relation to the above variables, suggests that the virus is not cytopathic and the immune system contributes to the production of liver cell damage. Preliminary data suggest that HAV replication may occur in the mucosa of the small intestine and in the liver.

Lymphocyte subsets in the young and aging marmoset (Callithrix jacchus).

T lymphocytes were studied in healthy young and old marmosets (Callithrix jacchus). Data indicate that monoclonal antisera to human T lymphocyte antigens can be used to identify these cells in marmosets. Total T and suppressor T cells were lower in the older group; there were no differences in helper T or in B cells. Helper/suppressor ratios (T4/T8) were increased in the older group. Thus, immune system alternations with aging and/or immune deficiency syndromes may be studied in this animal model using available reagents.

A naturally occurring bone marrow-chimeric primate. II. Environment dictates restriction on cytolytic T lymphocyte-target cell interactions.

Restriction on cytolytic T lymphocyte (CTL)-target cell-interactions are studied in the primate S. oedipus, a naturally occurring A + B----A bone marrow-chimeric species. We show that the T cell, B cell, and myelomonocytic progenitor cell populations are chimeric in this species. We selected animals for study that are populated by fully major histocompatibility complex (MHC)-disparate hematopoietic cell populations, using a functional assay system. We then developed an in vitro system for analyzing at the clonal level the genetic restrictions on the trinitrophenyl-specific CTL-target cell interactions of this species. In this system, we have shown that tolerance to foreign MHC determinants was not, of itself, sufficient to facilitate the generation of CTL specific for target cells expressing those foreign MHC determinants. Rather, a marked preference for the expansion of CTL clones with a restriction for target cells bearing the host animals' MHC determinants was seen. Hematopoietically derived cells did not affect the repertoire of these T lymphocytes. These studies represent the first demonstration of the phenomenon of an environment dictating interactional restrictions on CTL in a naturally occurring bone marrow-chimeric animal. This is also the first demonstration of the profound influence of the environment on the repertoire of the T lymphocyte in a primate species.