Molecular Cloning,Sequence, and Phylogenetic Analysis of T Helper 1 Cytokines of Pashmina Goats

Cytokines play an important role in regulation of immune responses either in health or disease. In the present study, the cDNAs encoding mature Interleukin (IL)-2, interferon gamma (IFN-γ), and IL-12 p35 and p40 of Pashmina goat were cloned and sequenced. The amino acid sequence was deduced from nucleotide sequence and compared with those available in GeneBank. Mature forms of goat IL-2, IFN-γ, IL-12 p35, and IL-12 p40 composed of 135, 143, 196, and 305 amino acid residues, respectively. Comparison of amino acid sequence of goat IL-2 with sheep, buffalo, cattle, pig, camel, cat, and human sequences showed homology percentages of 100, 97.8, 96.3, 72.4, 72.4, 67.2, and 64.7, respectively. Amino acid sequence of goat IFN-γ showed 98.6, 95.8, 81.1, 81.8, 80.4, and 62.9 percent homology with sheep, bovine, pig, horse, dog, and humans, respectively. Homology ranging from 81.6 to 99% for IL-12 p35 sequences and 85.6 to 100% for IL-12 p40 sequences at amino acid level were observed across these species. Multiple sequence alignment and phylogenetic analysis of goat cytokines revealed close relationship with sheep sequence.     

Partial characterization of the CD45 phosphatase cDNA in the owl monkey (Aotus vociferans)

CD45 is a protein tyrosine phosphatase implicated in T and B cell activation, differentiation, and development. It dephosphorylates specific tyrosine residues on its substrates, principally on the Src-family of protein tyrosine kinases, thus regulating T cell or B cell activation during the immune response. In this study, we present the partial CD45 nucleotide and deduced amino-acid sequences for the owl monkey (Aotus vociferens). There is 97% identity in the nucleotide sequence and 96% in the amino acid sequence with the human counterpart. Aotus CD45 undergoes alternative splicing on the extracelular N-terminal tail, and has several conserved features characteristic of other species. This includes the two Tyr phosphatase domains and some residues and/or motifs involved in docking of signaling molecules, intramolecular interactions, and CD45 activity and activity regulation (YINAS, GXGXXG, WPD, and YWP motifs, and the Cys residues). This suggests that the Aotus CD45 molecule is a functional enzyme and that initial lymphocyte activation in Aotus monkeys and humans is very similar. Together with previous reports from our laboratory, this work supports the contention that immune responses in Aotus are similar to those of humans, and supports the strategy for using this experimental model for studies on activation of T lymphocytes in response to specific antigens.     

Molecular Characterization of Pro-Inflammatory Cytokines Interleukin-1β and Interleukin-8 in Asian Elephant (Elephas maximus)

Interleukin (IL)-1β and IL-8 are pro-inflammatory cytokines produced primarily by monocytes and macrophages in response to a variety of microbial and nonmicrobial agents. As yet, no molecular data have been reported for IL-1β and IL-8 of the Asian elephant. In the present study, we have cloned and sequenced the cDNA encoding IL-1β and IL-8 of the Asian elephant. The open reading frame (ORF) of Asian elephant IL-1β is 789 bp in length, encoded a propeptide of 263 amino acid polypeptide. The predicted protein revealed the presence of IL-1 family signature motif and an ICE cut site. Whereas, IL-8 contained 321 bp of open reading frame. Interestingly, the predicted protein sequence of 106 aa, contains an ELR motif immediately upstream of the CQC residues, common in all vertebrate IL-8 molecules. Identity levels of the nucleic acid and deduced amino acid sequences of Asian elephant IL-1β ranged from 68.48 (Squirrel monkey) to 98.57% (African elephant), and 57.78 (Sheep) to 98.47% (African elephant), respectively, whereas that of IL-8 ranged from 72.9% (Human) to 87.8% (African elephant), and 63.2 (human, gorilla, chimpanzee) to 74.5% (African elephant, buffalo), respectively. The phylogenetic analysis based on deduced amino acid sequenced showed that the Asian elephant IL-1β and IL-8 were most closely related to African elephant. Molecular characterization of these two cytokines, IL-1β and IL-8, in Asian elephant provides fundamental information necessary to progress the study of functional immune responses in this animal and gives the potential to use them to manipulate the immune response as recombinant proteins.     

Owl monkey MHC-DRB exon 2 reveals high similarity with several HLA-DRB lineages

One hundred and ten novel MHC-DRB gene exon 2 nucleotide sequences were sequenced in 96 monkeys from three owl monkey species (67 from Aotus nancymaae, 30 from Aotus nigriceps and 13 from Aotus vociferans). Owl monkeys, like humans, have high MHC-DRB allele polymorphism, revealing a striking similarity with several human allele lineages in the peptide binding region and presenting major convergence with DRB lineages from several Catarrhini (humans, apes and Old World monkeys) rather than with others New World monkeys (Platyrrhini). The parallelism between human and Aotus MHC-DRB reveals additional similarities regarding variability pattern, selection pressure and physicochemical constraints in amino acid replacements. These observations concerning previous findings of similarity between the Aotus immune system molecules and their human counterparts affirm this specie’s usefulness as an excellent animal model in biomedical research.Experiments carried out in this work complied with current Colombian Ministry of Health law and regulations governing animal care and handling.An erratum to this article can be found at     

Chromosomal distribution of interstitial telomeric sequences in nine neotropical primates (Platyrrhini): possible implications in evolution and phylogeny

To localize interstitial telomeric sequences (ITSs) and to test whether their pattern of distribution could be linked to chromosomal evolution, we hybridized telomeric sequence probes (peptide nucleic acid, PNA) on metaphases of New World monkeys: Callithrix argentata, Callithrix jacchus, Cebuella pygmaea, Saguinus oedipus, Saimiri sciureus, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae), Lagothrix lagotricha (Atelidae) and Callicebus moloch (Pithecidae), characterized by a rapid radiation and a high rate of chromosomal rearrangements. Our analysis of the probe signal localization allowed us to show in all the species analysed, as normally, the telomeric location at the terminal ends of chromosomes and unexpected signal distributions in some species. Indeed, in three species among the nine studied, Aotus lemurinus griseimembra, Aotus nancymaae (Cebidae) and Lagothrix lagotricha (Atelidae), we showed a high variability in terms of localization and degree of amplification of interstitial telomeric sequences, especially for the ones found at centromeric or pericentromeric positions (het‐ITS). A comparative analysis, between species, of homologous chromosomes to human syntenies, on which we have found positive interspersed PNA signals, allowed us to explain the observed pattern of ITS distribution as results of chromosomal rearrangements in the neotropical primates analysed. This evidence permitted us to discuss the possible implication of ITSs as phylogenetic markers for closely related species. Moreover, reviewing previous literature data of ITSs distribution in Primates and in the light of our results, we suggest an underestimation of ITSs and highlight the importance of the molecular cytogenetics approach in characterizing ITSs, which role is still not clarified.     

Sequence and expression of MHC-DPB1 molecules of the New World monkey Aotus nancymaae,a primate model for Plasmodium falciparum

Aotus nancymaae represents an animal model for the pre-clinical evaluation of blood-stage vaccine candidates against Plasmodium falciparum and Plasmodium vivax. We present here the nucleotide sequences of exon 2 and 3 of MHC-DPB1 genes. In a group of seven unrelated animals captured in the wild, three alleles of MHC-DPB1 exon 2 could be identified. Phylogenetic analysis shows that in contrast to Aona- DRB and - DQB, the Aona-DPB1exon 2 amino acid sequences cluster in a species-specific manner. No evidence could be found for the conservation of allelic lineages pre-dating the divergence of Old and New World monkeys. Additionally, two nucleotide sequences of MHC-DPB1 exon 3 could be identified differing in one synonymous base exchange. Phylogenetic analysis of Aona-DPB1exon 3 amino acid sequence shows that it clusters together with human sequences separately from the New World monkey Saguinus oedipus. Aona-DP heterodimers are expressed on the surface of Aotus cells, as detected by staining with a cross-reactive monoclonal antibody, and can therefore present antigenic peptides to the cellular immune system.     

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

Abstract: There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti-human mAb to T-cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM-CSF, 4–1BB, FLT3, FLX2, common β-chain, IL-1 (type I receptor), and IL-8.     

Use of spatial, visual, and olfactory information during foraging in wild nocturnal and diurnal anthropoids: A field experiment comparing Aotus, Callicebus, and Saguinus

Early in their evolution, the ancestors of anthropoid primates radiated from a nocturnal to a diurnal niche. Foraging during the night differs from foraging during the day in terms of the availability of light and color cues, and in the movement of odor molecules through the canopy. In this study, we compared the ability of nocturnal and diurnal New World monkeys to use perceptual cues (i.e., the sight or smell of food) and spatial information (place predictability) in within-patch foraging decisions. An experimental field study was conducted on wild groups of night monkeys (Aotus nigriceps), tamarins (Saguinus imperator imperator and S. fuscicollis weddelli), and titi monkeys (Callicebus cupreus) at the Zoobotanical Park/UFAC, Rio Branco, Brazil. Our research design included the construction of feeding stations located in the home range of the study groups. Each feeding station consisted of eight visually identical feeding platforms located in a circular arrangement. In all test settings, two platforms at each feeding station contained a food reward (banana), and the remaining six platforms contained a sham reward (yellow plastic or inaccessible banana). In the night-monkey experiments, each feeding platform was illuminated by a 40-W red bulb to aid the researcher in observing their behavior. When the location of reward sites was predictable over time, individuals in all four species successfully relocated food rewards based solely on spatial information. Each species was also successful in using visual information to distinguish real from sham food rewards. However, only night monkeys and one group of emperor tamarins used olfactory information alone to locate food rewards. Overall, the species' performances did not clearly differentiate Aotus from diurnal New World primates in these experiments.     

Immunology of interactions between ticks and hosts

Abstract Infestation with ixodid tick stimulates the immune regulatory and effector pathways of the hosts involving antigen presenting cells, T-lymphocytes, B-lymphocytes, basophils, mast cells, eosinophils and a variety of bioactive molecules like cytokines, antibodies and complement. Tick-mediated immunosuppression has been investigated using cells derived from infested animals and by exposing cells from uninfested animals to tick salivary gland molecules. Tick-induced suppression of host immune defences is characterized by reduced ability of lymphocytes from infested animals to proliferate m vitro in the presence of concanavalin A (Con A), diminished primary antibody responses to T-cell dependent antigen, and decreased elaboration of macrophage (IL-1 and TNF-α) and Th 1 -lymphocyte cytokines (IFN-γ), whereas Th2 cytokines production (IL-4, IL-5 and IL-10) isenhanced. It is known that IL-10 inhibits Thl cell development and also reduces the in vitro T-lymphocyte proliferative response to Con A stimulation. Proteins which inhibited T-lymphocyte in vitro responsiveness to Con A were also isolated from tick salivary glands.     

β 2 -microglobulin in neotropical primates (Platyrrhini)

 Nucleotide sequences for the three exons of the β₂-microglobulin (β₂m) gene (B2m) were determined for 135 animals representing 37 species and all 16 genera of neotropical primates (Platyrrhini). Twenty-eight different nucleotide sequences, encoding for 26 different proteins, were obtained. In comparison with those of other primate species, the β₂-microglobulins of the Platyrrhini form a distinct clade. Individual genera of neotropical primates have distinctive B2m sequences, but within a genera species can have either the same or different B2m sequences. B2m polymorphism was found within three of the species sampled: Callicebus personatus, Saguinus midas, and Aotus azarae. Of these only the polymorphism in A. azarae has an effect upon the mature, functional β₂m protein: residue 4 being either alanine or threonine. The A. azarae B2m allele encoding alanine at position 4 is shared with another species of Aotus (A. infulatus). In pairwise comparison the mature β₂m proteins of neotropical primates differ by 1–9 amino acid substitutions which can occur at 18 positions within the sequence. The substitutions are distributed throughout the primary structure but are more commonly found in loops rather than β strands of the tertiary structure. Of 17 residues of β₂m which hydrogen-bond with the class I heavy chain in human MHC class I molecules, 13 are conserved in the neotropical primates. The overall pattern of sequence variation in the B2m genes of the Platyrrhini is consistent with an evolution by successive selectively neutral events. Received: 13 November 1997 / Revised: 12 January 1998     

Abundance, diversity, and patterns of distribution of primates on the Tapiche River in Amazonian Peru

This work presents data on the relative diversity, abundance, and distribution patterns of primates in a 20 km2 area of the Tapiche River in the Peruvian Amazon. Population data were collected while the study area was both inundated and dry (March to September 1997) using conventional line-transect census techniques. Survey results reflected the presence of 11 primate species, but population parameters on only eight of the species will be presented, including saddleback tamarins (Saguinus fuscicollis), Bolivian squirrel monkeys (Saimiri boliviensis), brown capuchins (Cebus apella), white-fronted capuchins (Cebus albifrons), monk sakis (Pithecia monachus), red titi monkeys (Callicebus cupreus), red uakaris (Cacajao calvus), and red howler monkeys (Alouatta seniculus). Woolly monkeys (Lagothrix lagotricha), night monkeys (Aotus nancymaae), and pygmy marmosets (Callithrix pygmaea) were also seen in the area. The data for the smaller-bodied primates is similar to that reported almost 18 years earlier, but the data for the larger-bodied primates reflect a loss in the number of animals present in the area. Pressure from hunters and the timber industry may account for declining numbers of large-bodied primates, while it appears that natural features peculiar to the conservation area contribute to the patchy pattern of distribution.     

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.     

Reactivity of human T-lymphocyte-specific antibodies with peripheral blood mononuclear cells and spleen of Aotus azarae ssp. boliviensis (owl monkey)

Aotus monkeys offer one of the few models that can be used for the evaluation of the immunogenicity and efficacy of new vaccine candidates against the human malarias, Plasmodium falciparum and Plasmodium vivax. However, the tools available for evaluation of the immune responses in these New World primates are still limited. In the present study, a previously selected set of monoclonal antibodies that were raised against human T cell determinants and were reactive with at least one other primate species was investigated for its reactivity with Aotus lymphocytes using FACS analysis, indirect immunofluorescence (IFA) and immunohistochemistry. From a panel of 19 mAb, six were found to react consistently with Aotus lymphocytes using FACS analysis. Further evaluation of the mAb using IFA confirmed these findings. Analysis of the selected mAb on spleen sections of Aotus monkeys identified one anti-CD4 and one anti-CD8 mAb that can be used for immunohistochemical studies. The set of mAb identified in this study can be used for the detection of various T lymphocyte markers in peripheral blood and in tissues of Aotus monkeys. Together with data published by others, mAb are now identified for detection of six different markers of Aotus T lymphocytes. These mAb are very valuable for the characterisation of immune responses after vaccination and infection in the Aotus malaria models.     

Development of immunoassays for human interleukin 3 and interleukin 4, some of which discriminate between different recombinant DNA-derived molecules

Two panels of hybridomas were produced that secreted monoclonal antibodies (MAbs) against recombinant DNA-derived human interleukin 3 and interleukin 4 (rhIL-3 and rhIL-4). From each panel, sensitive immunoradiometric assays (IRMAs) were developed which were capable of detecting the recombinant molecule used as the immunogen but were unable to recognize natural or other recombinant forms of the same cytokine. Subsequent studies using the MAbs from each panel showed that a number of the MAbs appeared only to recognize that particular recombinant molecule used as immunogen, with little or no binding to other recombinant forms of the molecule. By using MAbs that were found to be unrestricted in their recognition for different recombinant forms of the cytokines, it was possible to develop an IRMA for IL-4 that was capable of detecting natural IL-4 as well as all the recombinant forms equally. An IRMA was also developed for IL-3 but was not of equivalent sensitivity in detecting the different recombinant forms of IL-3 used in the study. The recombinant DNA-derived cytokine molecules used to raise the two panels of MAbs contained amino acid substitutions relative to the natural sequences, and these findings indicate that caution should be exercised when using immunoassays to estimate natural sequence molecules if antibodies raised to modified rDNA-derived molecules are used.