Characterisation of Mhc class I and class II DRB polymorphism in red-bellied tamarins (Saguinus labiatus).

The infection of red-bellied tamarins (Saguinus labiatus) with GB virus B (GBV-B) is an important surrogate model of hepatitis C virus infection in man. To fully exploit the value of this model, we have characterised MHC class I G and class II DRB alleles in eight tamarins representing a cross-section of a UK breeding colony. The results indicated a high degree of classes I and II DRB allele sharing. Each animal transcribed three to four putative surface-expressed class I alleles and two to four class II DRB alleles. Most animals also transcribed at least one class I allele predicted to result in a C-terminal truncated protein. These results represent the first description of MHC polymorphism in this species and provide a foundation for characterisation of MHC diversity in breeding populations of red-bellied tamarins. The data will facilitate the identification of associations between MHC polymorphism and control of viral infections, and detailed dissection of cellular immune responses against GBV-B.

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.

Selection of monoclonal antibodies for the identification of lymphocyte surface antigens in the New World primate Saguinus oedipus oedipus (cotton top tamarin).

32 monoclonal antibodies reactive with human CD antigens were tested against tamarin peripheral blood lymphocytes, ConA blasts and lymphoblastoid B cell lines derived from tamarin cells. Reagents that cross-react with MHC class I and II, B cells (CD20, -21 and -23), monocytes (CD14) and NK cells (CD16, -56) have been identified. In addition monoclonals that cross-react with T cells (CD2, CD3), the CD4/CD8 subsets of T cells and the IL-2 receptor (CD25) are reported. A monoclonal against the beta chain of LFA-1 (CD18) cross-reacted strongly, but there was only a very poor cross-reaction with a monoclonal against the alpha chain of CD11a. Two monoclonals tested against ICAM-1(CD54) were negative.

A naturally occurring bone-marrow-chimeric primate. I. Integrity of its immune system.

The New World primate species Saguinus oedipus, the cotton-top tamarin, has a high incidence of spontaneously occurring adenocarcinoma of the colon and develops a fatal lymphoproliferative syndrome following infection with various herpes viruses. Some investigators have linked such disease susceptibilities to abnormalities in the immune function of the cotton-top tamarin that may result from the natural bone marrow chimerism that occurs in this species. The present studies were initiated to establish conditions for studying the immune system of these primates and to assess the integrity of this system. First, we document the state of bone marrow chimerism of S oedipus. We then show that standard in vitro assays of lymphocyte function can be done with cells from this animal. Proliferative T cell assays, cytotoxic T lymphocyte (CTL) assays, and measurements of CTL precursor frequency all indicate that the immune system of this animal functions normally. Finally, we demonstrate in vitro the tolerance of lymphocytes from one S oedipus for the cells of its twin. The ability to manipulate tamarin blood cells in vitro will allow future investigation into mechanisms of immune tolerance and major histocompatibility complex restrictions on cell cooperation in this species.

Independent origin of functional MHC class II genes in humans and New World monkeys.

In previous studies, major histocompatibility complex (MHC) class II DP, DQ, and DR families of genes were characterized in different primate species mostly on the basis of their second exon sequences. Resemblances were found between Old World monkey (OWM) and New World monkey (NWM) genes and were interpreted as being the result of transspecies evolution. Subsequent analysis of intron sequences of catarrhine and platyrrhine DRB genes, however, revealed that the amplifiable genes were not, in fact, orthologous. To test other DRB genes and other families of the class II region Southern blot hybridizations were carried out with tamarin genomic DNA using probes specific for the third exons of the tamarin DQA, DQB, DPB, and DRB genes. The hybridizing bands were extracted from the gel and the third exons of the genes were amplified by PCR, cloned, and sequenced. With two exceptions, all NWM class II genes were found to group separately from the human sequences. Only the sequences of one nonfunctional DQB locus appeared to be more closely related to human genes than to other platyrrhine DQB genes. In the DRB family one gene was found that grouped with sheep and strepsirhine DRB sequences and might represent an old gene lineage. To extend the sequences to the second exon, long PCRs were performed on tamarin genomic DNA. This approach was successful for five of the ten third exon sequences. From these data, we conclude that at least the functional MHC class II genes have expanded independently in catarrhines and platyrrhines.

A putative CEA moiety is shared by the cotton-top tamarin (Saguinus oedipus) and humans.

CEA-like molecules immunologically distinct from those in humans have been described in non-human primates. These primates do not share the human predilection for colitis and subsequent development of colorectal cancer. CEA expression has not been fully evaluated in a lower-order primate, the cotton-top tamarin (Saguinus oedipus), an animal model for colitis and colorectal cancer. We found increased levels of CEA in both colonic washings and tissues of these animals using a commercially available kit, CEA AIA-PACK (Tosoh Medics, Foster City, CA). In contrast, we observed that other CEA kits failed to detect CEA in tamarins. To elucidate the nature of the CEA-like protein detected, we used the two component monoclonal antibodies used in the CEA AIA-PACK kit, and identified the reactive molecules by Western blotting. A band of approximately M(r) 50,000 was found to be common to samples from both humans and the tamarins. Minimal binding was observed with NCA antibody. We conclude that a CEA-like molecule shared by humans and tamarins may play a role in the pathogenesis of colitis and cancer in both species.

Carcinoembryonic antigen family of adhesion molecules in the cotton top tamarin (Saguinus oedipus).

Humans and the cotton top tamarin, a model for colitis and colorectal cancer, share carcinoembryonic antigen (CEA) moieties. We quantified CEA in colonic washings and extracts in both, and CEA bands were confirmed by Western blot. We compared CEA-family expression in tissues and serum in the tamarin with that of the common marmoset, which develops colitis but not cancer. CEA levels are higher in tamarin washings compared with humans, and higher than in marmosets extracts (P<0.005). CEA molecular species appear to be specific, and human CEA-family member epitopes are also found in these primates. The higher CEA levels in the tamarin may reflect the overall higher cancer prevalence.

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.

[Lymphocyte heterogeneity of the T system: characterization of an allotypic antiserum recognizing a sub-species antigenic determinant in non-human primates]. / Heterogeneidad linfocitaria del sistema T: caracterización de un antisuero alotípico que reconoce un determinante antigénico de sub-especie en primates no-humanos.

To determine the membrane properties in T-lymphocyte population from Saguinus primates or lymphoblastoid cell lines of the same origin, an allotypic antiserum was prepared inoculating two Saguinus illigeri with 3.5 x 10(10) viable 70N2 cells obtained in-vitro, from a lymphoma induced by inoculation of Herpesvirus Saimiri in a Saguinus oedipus. The 70N2 cells carried the virus genome in an episomal way; it did not produce infectious virus and it formed spontaneous rosettes (99%) with sheep erythrocytes. The resulting allotypic antiserum inhibited the formation of E rosettes and specifically reacted with lymphoblastic T-cell lines and peripheral lymphocytes of Saguinus oedipus while it did not cross react with these cells or peripheral lymphocytes of S. illigeri or nigrifons, S. jacchus, Saimiri sciureus , Papio Sp. and human primates. These results show that two antigenic populations exist within Saguinus T-lymphocytes; one is common to all members of the species and the other is specific for subspecies related to the spontaneous rosette receptors and it is present in lymphoblastic cell lines-T of the same origin.

[A trial of a cultured inactivated vaccine against hepatitis A on Saguinus mystax tamarins]. / Ispytanie na tamarinakh Saguinus mystax kul'tural'noi inaktivirovannoi vaktsiny protiv gepatita A.

An experimental batch of inactivated hepatitis A vaccine was prepared using hepatitis A virus (HAV), HAS-15 strain, adapted to cell culture and purified by ultracentrifugation. The vaccine was tested in tamarins immunized intramuscularly three times one month apart. Three tamarins received a vaccine preparation containing 10 ng of immunogen each, three--100 ng each, and three animals were used as controls. The efficacy was judged by the anti-HAV antibody response in the vaccinated animals and development of immunity to subsequent virus challenge two months after the last immunization. The criteria of infection were: elevation of serum alanine aminotransferase, fecal excretion of HAV and production of specific antibody of IgM class. The immune response to 10 ng of the immunogen was lower than to 100 ng, however, both doses produced complete resistance to infection. The booster effect was observed in animals receiving 10 ng of the immunogen. The vaccine batch under study in the indicated doses was shown to have a good immunogenic potency and protective activity for tamarins.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus).

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

Experimental norovirus infections in non-human primates.

Noroviruses, with Norwalk virus as the prototype strain, are the most common cause of viral gastroenteritis in people of all ages. Limited information on the immunology of Norovirus infections has been obtained by studies both in the natural setting and in experimentally infected volunteers. Interpretation of these studies is difficult due to the lack of information on the history of Norovirus exposure and the cross-reactivity of antibodies. An animal model for Norovirus infections would be important to study the immune response, e.g., for vaccine assessment. In the present study the susceptibility of common marmosets, cotton top tamarins, cynomolgus, and rhesus macaques to Norovirus infection was tested. Following oral inoculation, low level replication may have occurred in common marmosets and cotton top tamarins but not in cynomolgus macaques, based on short-term viral shedding; neither clinical symptoms nor antibody responses were observed in these species. In contrast, rhesus macaques were found susceptible to Norwalk virus infection as one animal shed virus for a longer period of time and developed Norwalk virus specific IgM and IgG responses. Further research on Norovirus susceptibility in rhesus macaques may yield an animal model to study the immune response and pathogenesis after Norovirus infection.

Regulated and liver-specific tamarin alpha interferon gene delivery by a helper-dependent adenoviral vector.

Gene therapy approaches based on liver-restricted and regulated alpha interferon (IFN-alpha) expression, recently shown to be effective in different murine hepatitis models, appear promising alternatives to inhibit hepatitis C virus (HCV) replication in patients and minimize side effects. Tamarins (Saguinus species) infected by GB virus B (GBV-B) are considered a valid surrogate model for hepatitis C to study the biology of HCV infection and the development of new antiviral drugs. To test the efficacy of local delivery and expression of IFN-alpha in this model, we have developed HD-TET-tIFN, a helper-dependent adenovirus vector expressing tamarin IFN-alpha (tIFN) under the control of the tetracycline-inducible transactivator rtTA2s-S2. Expression of tIFN was successfully induced both in vitro and in vivo in rodents by doxycycline administration with consequent activation of IFN-responsive genes. More importantly, tIFN efficiently inhibited GBV-B replicon in a Huh-7 hepatoma cell line at low HD-TET-tIFN doses. A certain degree of transcriptional control of tIFN was achieved in tamarins injected with HD-TET-tIFN, but under the conditions used in this study, infection and replication of GBV-B were only delayed and not totally abrogated upon virus challenge. Hepatic delivery and regulated expression of IFN-alpha appear to be a possible approach for the cure of hepatitis, but this approach requires more studies to increase its efficacy. To our knowledge, this is the first report showing a regulated gene expression in a nonhuman primate hepatitis model.

Major-histocompatibility-complex DRB genes of a New-World monkey, the cottontop tamarin (Saguinus oedipus).

The DRB region of the human and great-ape major histocompatibility complex displays not only gene but also haplotype polymorphism. The number of genes in the human DRB region can vary from one to four, and even greater variability exists among the DRB haplotypes of chimpanzees, gorillas, and orangutans. Accumulating evidence indicates that, like gene polymorphism, part of the haplotype polymorphism predates speciation. In an effort to determine when the gene haplotype polymorphisms emerged in the primate lineage, we sequenced three cDNA clones of the New-World monkey, the cottontop tamarin (Saguinus oedipus). We could identify two DRB loci in this species, one (Saoe-DRB1) occupied by apparently functional alleles (*0101 and *0102) which differ by only two nucleotide substitutions and the other (Saoe-DRB2) occupied by an apparent pseudogene. The Saoe-DRB2 gene contains an extra sequence derived from the 3' portion of exon 2 and placed 5' to this exon. This sequence contains a stop codon which makes the translation of the bulk of the Saoe-DRB2 gene unlikely. Preliminary Southern blot hybridization analysis with probes derived from these two genes suggests that both the DRB gene polymorphism and the haplotype polymorphism in the cottontop tamarin may be low. In most individuals the DRB region of this species probably consists of three genes. Comparisons of the Saoe-DRB sequences with those of other primates suggest that probably all of the DRB genes found until now in the Catarrhini were derived from a common ancestor after the separation of the Catarrhini and Platyrrhini lineages. The extant DRB gene and haplotype polymorphism may therefore have been founded in the mid-Oligocene some 33 Mya.

MHC class I-processed pseudogenes in New World primates provide evidence for rapid turnover of MHC class I genes.

The MHC class I genes of the New World primate, the cotton-top tamarin (Saguinus oedipus), are an exception to the high polymorphism and variability displayed by this multigene family. We report the isolation of the first two processed pseudogenes from the MHC region in primates. These two MHC class I-processed pseudogenes (MHC-PS1 and -PS2) were found in several species of New World primates, suggesting a possible explanation for the cotton-top tamarin's limited MHC class I diversity. The pattern of synonymous and nonsynonymous substitutions in PS1 suggests that the gene that gave rise to this processed pseudogene was once subject to selection for variability in the peptide binding region and might, therefore, have been functional. Additionally, PSI is not closely related to the expressed cotton-top tamarin's MHC class I genes, but does show some similarity to So-N1, a tamarin pseudogene from which no transcript has been found. Thus, PS1 may represent a remnant of a once active MHC class I gene that is no longer functional in the cotton-top tamarin. The MHC class I loci in primates, therefore, appear to be evolving by a continual process of duplication and inactivation. This process seems to be exaggerated in New World primates and may in part be responsible for the cotton-top tamarin's limited MHC class I diversity.

Susceptibility of tamarins (Saguinus) to measles virus.

Tamarins (Saguinus mystax and Saguinus labiatus) were experimentally infected with two strains of measles virus. One of the strains (JM) spread readily among the animals with a high fatality rate. The second strain (Edmonston) appeared to be less pathogenic and less transmissible than strain JM. Aerosol was considered the most probable mode of infection.

Gastrointestinal tract antigenic profile of cotton-top tamarin, Saguinus oedipus, is similar to that of humans with inflammatory bowel disease.

As an animal model for human inflammatory bowel disease and colorectal cancer, the cotton-top tamarin remains controversial. Demonstration of antigenic similarity to the human would enhance its validity. Using colonic extracts and washings, we compared binding of seven monoclonal antibodies reactive with bowel and cancer antigens in both tamarins and humans with inflammatory bowel disease. Additionally, telomerase activity was tested for. Expression of a mucin antigen specific to human cancer was increased in tamarin colonic washings as well as aminoproteoglycans and EGFR in tamarin extracts, as compared to those of humans with inflammatory bowel disease (P < 0.005). An adenoma-associated antigen and k-ras p21 protein were negative in the tamarin. A trend to greater telomerase activity exists in tamarins. The antigenic similarity validates this model for human inflammatory bowel disease and colorectal cancer. A trend to increased telomerase activity in tamarins is consistent with the greater predisposition to cancer in these animals.

Detection of human and marmoset immunoglobulin heavy chain by a polyclonal antiserum to a marmoset immunoglobulin-related T cell product.

We show that a polyclonal rabbit antiserum raised against a purified monoclonal T cell component from a lower primate (cotton-topped marmoset) reacts by immunoblot transfer (Western Blot analysis) with serum immunoglobulin of man and marmoset. The antigenic component had an approximate mass of 68 kilodaltons and was isolated by immune-affinity chromatography from culture fluid in which the marmoset T cell leukemia 70-N2 had been grown. The reaction with human serum immunoglobulin is with a subset of the IgG molecules and is localized to the gamma heavy chain. The reaction with marmoset serum immunoglobulin is predominantly directed against the heavy chain, but slight reactivity is also noted against the light chains. These results substantiate reports of serological cross-reactions between immunoglobulin-like T cell receptors and classical immunoglobulins and illustrate the similarity between immunoglobulins of man and those of a distantly related New World primate.

The T-cell receptor beta chain-encoding gene repertoire of a New World primate species, the cotton-top tamarin.

The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species' unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8(+) T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR beta chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (pN) and synonymous (pS) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in pN in the framework regions compared with pN in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR beta chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts.