Reactivity of HLADR antibody manifests expression of surface MHC II molecules on peripheral blood T lymphocytes in new world monkeys.

BACKGROUND: Major histocompatibility complex (MHC) class II molecules expressed on B cells, monocytes and dendritic cells present processed peptides to CD4+ T cells as one of the mechanisms to combat infection and inflammation. AIM: To study MHC II expression in a variety of nonhuman primate species, including New World (NWM) squirrel monkeys (Saimiri boliviensis boliviensis), owl monkeys (Aotus nancymae), common marmosets (Callithrix spp.), and Old World (OWM) rhesus (Macaca mulatta), baboons (Papio anubis). METHODS: Two clones of cross-reactive mouse anti-human HLADR monoclonal antibodies (mAb) binding were analyzed by flow cytometry to evaluate MHC II expression on NHP immune cells, including T lymphocytes in whole blood (WB) and peripheral blood mononuclear cells (PBMC). RESULTS: MHC class II antibody reactivity is seen with CD20+ B cells, CD14+ monocytes and CD3+ T lymphocytes. Specific reactivity with both clones was demonstrated in T lymphocytes: this reactivity was not inhibited by purified CD16 antibody but was completely inhibited when pre-blocked with purified unconjugated MHC II antibody. Freshly prepared PBMC also showed reactivity with T lymphocytes without any stimulation. Interestingly, peripheral blood from rhesus macaques and olive baboons (OWM) showed no such T lymphocyte associated MHCII antibody reactivity. DISCUSSION & CONCLUSION: Our results from antibody (MHC II) reactivity clearly show the potential existence of constitutively expressed (with no stimulation) MHC II molecules on T lymphocytes in new world monkeys. These results suggest that additional study is warranted to evaluate the functional and evolutionary significance of these finding and to better understand MHC II expression on T lymphocytes in new world monkeys.

Effects of relocation on immunological and physiological measures in female squirrel monkeys (Saimiri boliviensis boliviensis).

In the present study, we have quantified the effects of transport, relocation and acclimate/adapt to their new surroundings on female squirrel monkey. These responses are measured in blood samples obtained from squirrel monkeys, at different time points relative to their relocation from their old home to their new home. A group of squirrel monkeys we transported, by truck, for approximately 10 hours. Peripheral blood mononuclear cells (PBMCs) were assayed in order to evaluate the phenotype of lymphocyte subsets by flow, mitogen-specific immune responses of PBMCs in vitro, and levels of cytokines at various time points including immediately before transport, immediately upon arrival, and after approximately 150 days of acclimation. We observed significant changes in T cells and subsets, NK and B cells (CD4+, CD8+, CD4+/CD8+, CD16+, and CD20+). Mitogen specific (e.g. PHA, PWM and LPS) proliferation responses, IFN-γ by ELISPOT assay, and cytokines (IL-2, IL-4 and VEGF) significant changes were observed. Changes seen in the serum chemistry measurements mostly complement those seen in the hematology data. The specific goal was to empirically assess the effects of relocation stress in squirrel monkeys in terms of changes in the numbers and functions of various leukocyte subsets in the blood and the amount of time required for acclimating to their new environment. Such data will help to determine when newly arrived animals become available for use in research studies.

Molecular and immunological tools for the evaluation of the cellular immune response in the neotropical monkey Saimiri sciureus, a non-human primate model for malaria research.

BACKGROUND: The neotropical, non-human primates (NHP) of the genus Saimiri and Aotus are recommended by the World Health Organization as experimental models for the study of human malaria because these animals can be infected with the same Plasmodium that cause malaria in humans. However, one limitation is the lack of immunological tools to assess the immune response in these models. The present study focuses on the development and comparative use of molecular and immunological methods to evaluate the cellular immune response in Saimiri sciureus. METHODS: Blood samples were obtained from nineteen uninfected Saimiri. Peripheral blood mononuclear cells (PBMC) from these animals and splenocytes from one splenectomized animal were cultured for 6, 12, 18, 24, 48, 72 and 96 hrs in the presence of phorbol-12-myristate-13-acetate and ionomycin. The cytokine levels in the supernatant were detected using human and NHP cytometric bead array Th1/Th2 cytokine kits, the Bio-Plex Pro Human Cytokine Th1/Th2 Assay, enzyme-linked immunosorbent assay, enzyme-linked immunospot assays and intracellular cytokine secretion assays. Cytokine gene expression was examined through TaqMan® Gene Expression Real-Time PCR using predesigned human gene-specific primers and probes or primers and probes designed based on published S. sciureus cytokine sequences. RESULTS: The use of five assays based on monoclonal antibodies specific for human cytokines facilitated the detection of IL-2, IL-4 and/or IFN-γ. TaqMan array plates facilitated the detection of 12 of the 28 cytokines assayed. However, only seven cytokines (IL-1A, IL-2, IL-10, IL-12B, IL-17, IFN-ß, and TNF) presented relative expression levels of at least 70% of the gene expression observed in human PBMC. The use of primers and probes specific for S. sciureus cytokines facilitated the detection of transcripts that showed relative expression below the threshold of 70%. The most efficient evaluation of cytokine gene expression, in PBMC and splenocytes, was observed after 6-12 hrs of culture, except for LTA in PBMC, whose expression was best analysed after 24 hrs of culture. CONCLUSIONS: Real-time PCR facilitates the analysis of a large number of cytokines altered during malaria infection, and this technique is considered the best tool for the evaluation of the cellular immune response in S. sciureus.

Phenotypic and functional characterization of lymphocytes from different age groups of Bolivian squirrel monkeys (Saimiri boliviensis boliviensis).

Due to many physiological and genetic characteristic similarities to humans, squirrel monkeys provide an ideal animal model specifically for studying malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). While squirrel monkeys three years and older are generally considered adult subjects suitable for use in medical research studies, little is known about the functional properties of lymphocytes in relation to the age of these animals, which could significantly impact the quality and quantity of innate and adaptive immune responses. In this study, we investigated differences in the phenotype and function of lymphocytes subsets of young (3-4 years), adult (8-10 years) and aged (16-19 years) squirrel monkeys. In general, animals in all three age groups exhibited comparable numbers of different lymphocyte subsets except for CD20+ B cells that were significantly lower in aged relative to young animals and T cells subsets expressing both CD4 and CD8 (double positive) were significantly higher in aged relative to young animals. With increasing age, phenotypic differences in central and effector memory T cells subsets were observed, that were more pronounced for the CD8+ T cells. Despite equal proportions of CD3+ T cells among the three age groups, responses of peripheral blood mononuclear cells to T cell mitogens PHA and Con A showed lower IFN-γ producing cells in the aged group than that in the young group. Furthermore, aged animals showed significantly higher plasma levels of inflammatory cytokines IL-6, IFN-γ, TNF-α, IL-10 and IL-12. These findings suggest that while the squirrel monkeys in general share phenotypic and functional similarities of lymphocyte subsets with humans in relation to age, specific differences exist in immune function of lymphocytes between young and old animals that could potentially impact experimental outcomes for which the measurement of immunologic endpoints are critical.

Non-ABO blood group systems phenotyping in non-human primates for blood banking laboratory and xenotransplantation.

Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion.

Species-specific inhibition of foamy viruses from South American monkeys by New World Monkey TRIM5{alpha} proteins.

Foamy virus evolution closely parallels that of the host species, indicating virus-host coadaptation. We studied simian foamy viruses (SFVs) from common marmosets, spider monkeys, and squirrel monkeys, New World monkey (NWM) species that share geographic ranges. The TRIM5alpha protein from each of these NWM species inhibited the replication of at least one of the SFVs associated with the other two species but did not affect the replication of its own SFV. Thus, TRIM5alpha has potentially shaped the evolution of SFVs in NWM hosts. Conversely, SFVs may have influenced the evolution of TRIM5 variants in New World primates.

Irregular xenoantibodies against human red blood cells in Papio anubis, P ursinus, P hamadryas, P papio, Saimiri sciureus, and Macaca mulatta: possible effect on xenotransplantation results.

OBJECTIVE: To assess the presence of irregular xenoantibodies against human red blood cells (RBCs) in 6 primate species used in xenotransplantation and other experimental procedures. MATERIALS AND METHODS: Serum samples from 109 baboons of 4 different species (olive, chacma, sacred, and Guinea), 38 rhesus macaques, and 30 squirrel monkeys were tested for irregular xenoantibodies using an agglutination test using human RBCs of known phenotype for Rh, Kell, Kidd, Lewis, Lutheran, P1, and Duffy antigens, commercially available as RBC I, II, and III. RESULTS: We found hemagglutination for RBC I in 49%, 22%, 100%, 57%, 32%, and 33% of olive, chacma, sacred, and Guinea baboons, rhesus macaques, and squirrel monkey, respectively. The frequency for RBC II was 49%, 50%, 100%, 57%, 37%, and 33%, respectively, and for RBC III was 56%, 37%, 100%, 79%, 34%, and 33%, respectively. There were differences in frequency depending on the sex of the rhesus macaques; all 3 RBCs tested were higher in the females: 44% vs 0%, P = .008; 48% vs 1%, P = .02, and 44% vs 9.1%, P = .04 for RBC I, II, and III, respectively. There were differences due to age in only olive baboons, and a higher frequency in younger animals compared with juvenile, subadult, and adult animals for all 3 human RBCs. CONCLUSIONS: Assessment of irregular antibodies in the presence of primate serum should be taken into account during any experimental xenotransplantation protocol.

Observations on the Uganda I strain of Plasmodium malariae and Plasmodium brasilianum in Aotus and Saimiri monkeys and Anopheles mosquitoes.

Splenectomized Aotus lemurinus griseimembra, A. azarae boliviensis, A. nancymaae, A. vociferans, and Saimiri boliviensis monkeys were infected with the Uganda I/CDC strain of Plasmodium malariae. The maximum parasite counts were lower if the animals had been previously infected with Plasmodium vivax. Mosquito infection was concentrated in the 12 days following the rise in count above 1,000/microl. Mosquito infection and parasite counts were highest with A. l. griseimembra. Anopheles freeborni was more readily infected than An. gambiae, which was more readily infected than An. stephensi. Parasite counts and mosquito infection with P. brasilianum were much higher in S. boliviensis monkeys than with the Uganda I strain of P. malariae in this host, suggesting marked differences between the host-parasite-vector relationships and indicating that P. brasilianum in S. boliviensis monkeys may be a better reflection of the relationship of P. malariae in the human host.

An outbreak of Toxoplasmosis amongst squirrel monkeys in an Israeli monkey colony.

An outbreak of Toxoplasmosis in a colony of squirrel monkeys (Saimiri sciureus) in Israel is described. Serological, pathological, and molecular findings of monkeys, as well as rodents and pigeons from the vicinity are summarized. Seventy-nine percent (19/24) of monkeys were T. gondii seropositive at titer 1:16 whilst 4% (1/24) were also seropositive at titer 1:64 using the Modified Agglutination Test (MAT). Eighty four percent (21/25) of rats were positive at titer 1:16 and 8% (2/25) of rats were positive at titer 1:32. DNA amplification of a 529bp repeated sequence of T. gondii was detected in the liver and lungs of all monkeys tested, 6/7 in myocardial extractions and 5/6 in brain extractions. Sequence analysis of the SAG2 locus disclosed that T. gondii detected was of Type III genotype. The source of disease was thought to be contamination of feed with infective feline oocysts. As a result of this study, the implementation of a program to capture and remove resident feral cats, to discontinue the feeding of stray cats, and to control rodent populations in the park was introduced.

Observations on the sporozoite transmission of Plasmodium vivax to monkeys.

Saimiri boliviensis monkeys were infected via sporozoites with the Salvador I strain of Plasmodium vivax that had been stored frozen for periods ranging from 12 to 5,312 days. Prepatent periods ranged from 16 to 53 days.

Blood typing in Saimiri sciureus monkeys: influence of anti-red blood cell alloantibodies on Plasmodium falciparum parasitaemia in vivo.

The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.

Flow cytometry identification and characterization of mononuclear cell subsets in the neotropical primate Saimiri sciureus (squirrel monkey).

BACKGROUND: The neotropical primate squirrel monkey is used in many areas of biomedical research including neuroendocrinology, immunology and infectious diseases. However, research has been hampered by the lack of immunological tools for this primate. METHODS: A series of 67 commercially available monoclonal antibodies to human CD antigens or cytokines were tested on Saimiri mononuclear cells and the specificity was assessed by double staining using flow cytometry. RESULTS: Monoclonal antibodies defining the main mononuclear cells subsets (monocytes, B, T, including CD4 and CD8 T cells) as well as activation markers have been identified. The conditions to specifically identify the various cell subsets using two color flow cytometry and establish their relative proportions have been set-up. We also have established normal values of the main circulating mononuclear cell subsets for adult Saimiri sciureus monkeys from the breeding unit of Institut Pasteur in French Guiana. The distribution between spleen, blood and lymph nodes has been compared. CONCLUSIONS: These tools allow documenting the phenotype of most Saimiri mononuclear cell subsets and assessing their activation level. This opens new perspectives for vaccinology and immunopathology research in this experimental non-human primate host, in particular for malaria research.

Molecular characterization of major histocompatibility complex class 1 (MHC-I) from squirrel monkeys (Saimiri sciureus).

Little is known about the major histocompatibility complex (MHC) class 1 in squirrel monkeys ( Saimiri sciureus). We cloned, sequenced and characterized two alleles and the cDNA of the coding region of MHC class 1 in these New World monkeys. Phylogenetic analyses showed that these sequences are related to HLA class 1 genes ( HLA-A and HLA-G). The structure and organization of one of the two identified clones was similar to that of a class 1 MHC gene ( HLA-A2). All the exon/intron splice acceptor/donor sites are conserved and their locations correspond to the HLA-A2 gene. The sequences of the newly described cDNAs reveal that they code for the characteristic class 1 MHC proteins, with all the features thought necessary for cell surface expression. Typical sequences for the leader peptide, alpha(1), alpha(2), alpha(3), transmembrane and cytoplasmic domains were found.

Sequencing and analysis of genomic DNA and cDNA encoding TNF-alpha in the squirrel monkey (Saimiri sciureus).

If a number of cytokines and growth factors that have been characterized from human cells were investigated in non-human primates, results from such approaches would allow the development of assays to detect and quantitate cytokines in experimental models. Tumor necrosis factor-alpha (TNF-alpha) is an important pluripotent cytokine which plays a crucial role in host defense. As yet, no complete molecular data have been reported for the squirrel monkey TNF-alpha. Polymerase chain reaction (PCR) primers were used to trace introns, by comparing product sizes obtained using cDNA and genomic DNA as templates. The genomic DNA is composed of four exons and three introns with 1793 nucleotides. The corresponding cDNA is 702 nucleotides and phylogenetic analysis showed that the Saimiri sciureus was most closely related to that of the genus Aotus, a new-world primate, compared to old-world primates (genus Macaca and Papio). The deduced TNF-alpha protein consists of 233 amino acids with 82% identity to human, 95% to new-world monkeys and 79% to old-world monkeys. The cloned TNF-alpha cDNA will be useful to quantitate TNF-alpha at the mRNA level.

Molecular cloning, characterization, and quantification of squirrel monkey ( Saimiri sciureus) Th1 and Th2 cytokines.

Primates have long been used as models to study the basic mechanisms of human disease. The squirrel monkey, Saimiri sciureus,is a New World monkey that can be satisfactorily bred in captivity and infected with various human pathogens. The basic immunological parameters of squirrel monkeys, including immunoglobulin subclasses and markers for lymphocyte subsets, have already been characterized. However, immunological reagents and assays specific for the detection and quantification of squirrel monkey cytokines are required to elucidate the immunological and physiopathological changes that occur during experimental infection of these animals with pathogens. We therefore cloned, sequenced, and characterized the cDNAs encoding various squirrel monkey Th1 and Th2 cytokines, including interleukin (IL)-1 beta, IL-2, IL-5, IL-6, IL-10, tumor necrosis factor alpha, and gamma interferon. We found 91.4%-98.1% homology between the nucleotide sequence of the squirrel monkey cytokine genes and published sequences of equivalent human and non-human primate genes. The aligned sequences of cytokines for Saimiriand several New and Old World primates and human are shown, and a phylogenetic analysis of published sequences of selected cytokines in other species and those of non-human primates is given. In addition, we adapted a previously described quantitative, reverse transcriptase, competitive polymerase chain reaction technique to measure the mRNA expression of those cytokines in squirrel monkeys. The primer and competitor plasmids that allowed quantification are described. We showed that the assay is sensitive and reproducible.

Presence of anti-ovalbumin IgE antibody in the sera of laboratory-reared squirrel monkeys (Saimiri sciureus) fed quail eggs.

In this study, we examined serum anti-ovalbumin (OVA) IgE and IgG antibodies in laboratory-reared squirrel monkeys (Saimiri sciureus), that were fed a boiled quail egg everyday. We found that 36 of 95 monkeys (38%) possessed specific IgE and 44% (42/95) had specific IgG against OVA. These antibody titers seemed to increase with age. There was, however, no apparent correlation between the anti-OVA IgE and IgG antibody titers.

Cytokine secretion in squirrel monkeys.

The squirrel monkey, a non-human New World primate, has several endocrine peculiarities, including a 10-fold higher plasma cortisol concentration than Old World primates, such as man. Glucocorticoids are known to have immunomodulatory properties. We therefore measured cytokine levels in supernatants of in vitro cultures of mononuclear cells from the peripheral blood of squirrel monkeys and humans. We stimulated monocytes and lymphocytes with lipopolysaccharide (LPS) and phytohemagglutinin (PHA) in the presence or absence of hydrocortisone. Squirrel monkey monocytes secreted a more than 100-fold lower level of interleukin-1 beta (IL-1 beta) but a four-fold higher level of transforming growth factor beta (TGF-beta) than human monocytes, whereas the secretion of other cytokines, such as tumor necrosis factor alpha (TNF-alpha), TNF-beta and interleukin 2 (IL-2), did not differ between squirrel monkeys and humans. However, in squirrel monkey lymphocytes, the PHA-stimulated secretion of TNF-alpha was much greater than that of TNF-beta. Our results support the view that in squirrel monkeys there are subtle adaptations in some immune functions, particularly linked to the hypothalamic-pituitary-adrenal (HPA) system rather than a global suppression of the immune system.