Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are detected, like bacterial or viral DNA, as a danger signal by the vertebrate immune system. CpG ODN synthesized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been relatively inactive on primate leukocytes in vitro. Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CpG ODN for their in vitro and in vivo immune effects in mice and identified in vitro activation of B and NK cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate proliferation and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence, number, and spacing of individual CpG motifs contribute to the immunostimulatory activity of a CpG phosphorothioate ODN. An ODN with a TpC dinucleotide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separated by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinated once against hepatitis B with this CpG ODN adjuvant developed 15 times higher anti-hepatitis B Ab titers than those receiving vaccine alone. In conclusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.     

Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells.

MRL/1pr mice demonstrate anatomic specificity in their development of vasculitis including the small- and medium-sized muscular arteries of the mesentery. To define the functional role of endothelium in vasculitis, we have cloned endothelial cells derived from inflamed small- and medium-sized arteries. Primary cells were derived by enzymatic dispersement and endothelial cells were selected by utilizing a combination of specific culture conditions. Cloned endothelium were developed utilizing limiting dilution cultures supplemented by endothelial cell growth factor. The cloned endothelial cells express many structural features of mature endothelial cells including Factor VIII-RA, non-muscle-specific actin, and Weibel-Palade bodies. Functionally, the clones express functional receptors for the scavenger pathway for LDL metabolism. The cells do not express Class I MHC antigens; however, IFN-beta and IFN-gamma stimulate Class I MHC expression after 24 h, which induces lysis of virus-infected cloned endothelium by Class I-restricted virus-primed T cells. In direct contrast to site-identical vascular smooth muscle cells (VSMCs), endothelial cells do not spontaneously express Class II MHC antigens, nor do they secrete biologically relevant levels of IL-1 unless triggered by lipopolysaccharide. The availability of site-specific cloned endothelium along with cloned VSMCs from autoimmune mice should resolve major experimental controversies involving the pathophysiology of inflammatory vascular disease.     

Delayed response task performance as a function of age in cynomolgus monkeys (Macaca fascicularis)

We compared delayed response task performance in young, middle-aged, and old cynomolgus monkeys using three memory tests that have been used with non-human primates. Eighteen cynomolgus monkeys—6 young (4–9 years), 6 middle-aged (10–19 years), and 6 old (above 20 years)—were tested. In general, the old monkeys scored significantly worse than did the animals in the two other age groups. Longer delays between stimulus presentation and response increased the performance differences between the old and younger monkeys. The old monkeys in particular showed signs of impaired visuo-spatial memory and deteriorated memory consolidation and executive functioning. These results add to the body of evidence supporting the utility of Macaca fascicularis in studies of cognition and as a potential translational model for age-associated memory impairment/dementia-related disorders.     

Cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis)

We have examined the motility, morphology, and cryopreservation of epididymal spermatozoa collected by needle biopsy from cynomolgus monkeys (Macaca fascicularis). At collection, epididymal sperm (23 x 10(6) +/- 4 x 10(6) sperm/sample; 611 x 10(6) +/- 116 x 10(6) sperm/ ml; n = 18) were alive (79 +/- 2%), motile (67 +/- 2%), and exhibited intact membranes (65 +/- 2%). Sperm maintained at room temperature in handling medium exhibited decreased motility over time, but head-to-head agglutination was limited. Tris egg-yolk extender containing 6% glycerol and dimethylsulfoxide (DMSO) did not significantly affect functional morphology, whereas extender containing propanediol significantly reduced motility, survival, and membrane integrity. Cryostorage reduced all measures of functional morphology independent of cryoprotectant. Post-thaw motility was superior for glycerol and DMSO compared to propanediol. Variation in glycerol concentration (4, 6, and 8%) produced equivocal effects on sperm functional morphology post-thaw. Needle biopsy may be a useful technique for laboratory and field-based collection of spermatozoa from nonhuman primates.     

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis)

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.     

Genetic characterization of long-tailed macaques (Macaca fascicularis) on Tabuan Island Indonesia

Protein and mitochondrial DNA variations (D-loop region PCR-RFLP) were analyzed for 7 serum and 40 clot samples collected from long-tailed macaques (Macaca fascicularis) living on Tabuan Island, Indonesia. Protein polymorphisms were examined electrophoretically for 5 and 12 kinds of protein in serum and erythrocytes, respectively. Each of the protein loci tested showed a monomorphic pattern. Polymorphisms were detected in the analysis of the D-loop-containing region of mtDNA (PCR-RFLP) using 32 restriction endonucleases. Two haplotypes, differing 1.03% in sequence divergence were observed, and both were previously undetected in other local populations. Based on genetic features and differences in pelage color as outlined inFooden's (1995) morphological analysis, the present results suggest that long-tailed macaques on Tabuan Island are a unique population. From the genetic analyses performed here, Tabuan monkeys are considered to be the same species group as those populations of Sumatra and Java (Fooden, 1995).     

Hematological values and parasite fauna in free-rangingMacaca hecki and theM. hecki/M. tonkeana hybrid group of Sulawesi Island, Indonesia

Hematological values and parasitological fauna of free-rangingMacaca hecki and the hybrid group betweenM. hecki/M. tonkeana of Sulawesi Island, Indonesia, were investigated. The hematological values, especially the red cell number (RBC), were lower than those of other macaque species, indicating that Sulawesi macaques are slightly anemic. Several parasites including Plasmodium sp., trombiculid mites, andTrichuris trichiura were identified. Although infection by Plasmodium was observed with considerable frequency, no clear relationship between its infection and the occurrence of anemia was found. Trombiculid mites and eggs ofAnatrichosoma sp. were detected in foci of the ears of most monkeys. The infection with a trombiculid mite is the first recorded occurrence in free-ranging wild Sulawesi macaques. Gastrointestinal parasites were identified from their eggs in fecal samples, where five species of nematoda and one trematoda species were found.     

Modeling managed monkey populations: sustainable harvest of longtailed macaques on a natural habitat island

Computer simulation of population dynamics can be useful in managing harvested populations of monkeys on islands. Between 1988 and 1991, 420 adult female and 58 adult male simian retrovirus-free Macaca fascicularis were released onto Tinjil Island, Indonesia, to provide the nucleus for a free-ranging breeding colony. Natural habitat breeding facilities are excellent alternatives to wild trapping and compound breeding, maximizing the health and well-being of animals destined for essential biomedical research. To avoid a population crash, the number of offspring that can be harvested annually must be based on life table characteristics such as age-specific natality and mortality. We used a modified Leslie matrix to model changes in female population size over 26 years. First, we assumed that all 420 females were released simultaneously and varied the annual birth rate (50%, 60%, 70%), survival rate, and number of offspring harvested per year. Assuming high survival and birth rates vs. low rates, about four times as many female offspring could be harvested annually from a stable population (87 vs. 20 offspring). Terminal population size after 26 years did not differ much across rates modeled (568–696 females). Second, we modeled the number of females actually released (including the recent addition of 42 new female breeders) and harvested (averaging 49 annually 1991–1994), and projected the population through 2014. This indicated that threshold harvest rates and terminal population sizes increased considerably over the first model, assuming intermediate (78 harvested, 952 females) and high (152 harvested, 1,331 females) rates of survivorship and natality, but were unchanged assuming low rates (20 harvested, 559 females). A review of the literature and field observations on Tinjil suggest that actual birth and survival rates resemble the intermediate values modeled. If so, the present density on the island, projected to be ∼215 males and females per square kilometer, is approaching carrying capacity. The high values are realistic upper limits. If actual survivorship and birth rates are at the high end of those modeled, the island's population may be on the verge of rapid expansion, requiring increased harvest and provisioning. © 1996 Wiley-Liss, Inc.