Protocol for collection and separation of bone marrow mononuclear cells in chlorocebus aethiops / Protocolo para colheita e isolamento de células mononucleares de medula óssea em chlorocebus aethiops

Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.

Chlorocebus aethiops é uma espécie de primata não humano frequentemente utilizados em pesquisa biomédica. Algumas pesquisas envolve esta espécie como modelo experimental para várias doenças e possível tratamento com células-tronco. A medula óssea é uma das principais fontes destas células e proporciona fácil acesso. O objetivo deste estudo foi o de padronizar o protocolo de coleta e separação de medula óssea em C. aethiops. Dez animais foram submetidos a punção de medula óssea com acesso à crista ilíaca e separação de células por gradiente de densidade. A medula óssea de C. aethiops tinha uma média de 97% de viabilidade. A partir dos resultados obtidos, podemos concluir que C. aethiops é um excelente modelo para obter e isolar células mononucleares da medula óssea, promovendo vários estudos no campo da terapia celular.

Assessing the pulsatility of luteinizing hormone in female vervet monkeys (Chlorocebus aethiops sabaeus).

Specific alterations in the pulsatility of luteinizing hormone (LH) are linked to obesity-related subfertility in ovulatory women. Vervet monkeys (Chlorocebus aethiops sabaeus) are an Old World nonhuman primate that develops obesity and has a menstrual cycle similar to humans. We evaluated follicular-phase LH pulses in 12 adult normal-weight female vervets. Serum was collected every 10 min for 4 h by using a tether device in conscious, freely moving monkeys on menstrual cycle days 2 through 5. Serum estradiol was collected daily during the follicular phase to identify the luteal-follicular transition. For comparison, we used data from 12 ovulatory normal-weight women who had undergone frequent blood sampling of early-follicular LH. LH pulse frequency was similar, with 2.8 ± 0.7 LH pulses during 4 h in vervets compared with 2.3 ± 0.7 LH pulses during 4 h in women. The LH pulse mass (percentage change in the pulse peak over the preceding nadir) was 123.2% ± 27.4% in vervets and 60.9% ± 14.9% in humans. The first day of low serum estradiol after the follicular-phase peak was denoted as the day of the luteal-follicular transition. Luteectomy was performed on luteal days 7 through 9, and corpora lutea were confirmed by histology. We demonstrate that follicular LH patterns in vervets are similar to those in humans and that the luteal phase is easily identified by monitoring daily serum estradiol. These findings demonstrate that vervet monkeys are a suitable animal model for evaluating LH pulse dynamics longitudinally in studies of diet-induced obesity.

Aged vervet monkeys developing transthyretin amyloidosis with the human disease-causing Ile122 allele: a valid pathological model of the human disease.

Mutant forms of transthyretin (TTR) cause the most common type of autosomal-dominant hereditary systemic amyloidosis. In addition, wild-type TTR causes senile systemic amyloidosis, a sporadic disease seen in the elderly. Although spontaneous development of TTR amyloidosis had not been reported in animals other than humans, we recently determined that two aged vervet monkeys (Chlorocebus pygerythrus) spontaneously developed systemic TTR amyloidosis. In this study here, we first determined that aged vervet monkeys developed TTR amyloidosis and showed cardiac dysfunction but other primates did not. We also found that vervet monkeys had the TTR Ile122 allele, which is well known as a frequent mutation-causing human TTR amyloidosis. Furthermore, we generated recombinant monkey TTRs and determined that the vervet monkey TTR had lower tetrameric stability and formed more amyloid fibrils than did cynomolgus monkey TTR, which had the Val122 allele. We thus propose that the Ile122 allele has an important role in TTR amyloidosis in the aged vervet monkey and that this monkey can serve as a valid pathological model of the human disease. Finally, from the viewpoint of molecular evolution of TTR in primates, we determined that human TTR mutations causing the leptomeningeal phenotype of TTR amyloidosis tended to occur in amino acid residues that showed no diversity throughout primate evolution. Those findings may be valuable for understanding the genotype-phenotype correlation in this inherited human disease.

Phenotype and function of myeloid dendritic cells derived from African green monkey blood monocytes.

Myeloid dendritic cells probably play an important role in the immune response against HIV and SIV, and in the enhancement of CD4+ T cell infection. Here, we have investigated phenotypic and functional features of myeloid monocyte-derived DC (MDDC) from African green monkeys (AGMs). AGMs are natural hosts of SIV and exhibit no signs of abnormal T cell activation despite high SIV plasma viremia. We identified mAbs that cross-react specifically with homologous molecules expressed on AGM DC. We adapted a protocol to derive AGM MDDC by culture in the presence of GM-CSF and IL-4. The differentiated cells possessed a typical dendritic morphology and the majority were CD11c+ DC-SIGN+. AGM MDDC displayed a high expression of typical maturation markers, such as CD83, CD86 and DC-LAMP, and moderate immunostimulatory capacity, suggesting that the cells were in a semi-mature state. Stimulation resulted in further maturation, as shown by up-regulation of CD80 and decrease of endocytosis ability. However, neither increase of HLA-DR or CD40 expression nor enhanced immunostimulatory capacity was observed. The latter was associated with a low pro-inflammatory cytokine production during mixed lymphocyte reactions and a cytokine balance in favour of IL-10 in contrast to human MDDC. This is the first characterization of AGM MDDC. The tools described here are a crucial step for future studies in vivo or in vitro on the function of myeloid DC using the AGM animal model.

Serological detection of adenoviruses in non-human primates maintained in a colony in Kenya.

BACKGROUND: Adenoviruses are known to cause several human diseases including acute febrile respiratory syndromes, epidemic conjunctivitis and gastroenteritis. These diseases associated with adenovirus infection affect adults and are usually more severe in infants and children. Forty-seven human adenoviruses serotypes have so far been identified adenovirus. The diversity of these viruses has delayed progress on vaccine development due to difficulties in identifying appropriate vaccine targets. To date, limited studies have been done to determine the prevalence of adenovirus infection in non-human primates with the goal of developing a non-human primate model that can be used to study the mechanisms of infection. OBJECTIVE: To determine the prevalence of enteric adenovirus infection in Kenyan non-human primates. DESIGN: A prospective study to investigate the prevalence of enteric andenovirus infection in captive non-human primates maintained in a colony. SETTING: Faecal samples were collected from monkeys trapped from different geographical areas of Kenya and also from the ones maintained in a colony at the Institute of Primate Research (IPR), Kenya. SUBJECTS: Ninety four faecal samples were collected from three species of non-human primates consisting of various ages and sex. Samples were collected from monkeys trapped from different geographical areas of Kenya and also from the ones maintained in a colony at the Institute of Primate Research (IPR), Kenya. All the faecal samples were screened for presence of adenoviruses using a commercial antigen-capture enzyme immunoassay (EIA) kit, this is an enzyme-linked immunosorbent assay (ELISA) kit designed for diagnosis of human enteric adenoviruses in stool samples. RESULTS: The highest prevalence of adenoviruses, detected by EIA kit, was in olive baboons (Papio anubis, 52.9%), followed by vervet monkeys (Cercopithecus aethiops, 48.9%) and the yellow baboons (Papio cynocephalus, 18.8%). Sub-grouping within each species (based on age and sex) indicated no significant differences (p > 0.05) in adenovirus infection signifying equal susceptibility. The prevalence of adenoviruses in vervet monkeys that were also Simian Immunodeficiency virus (SIV) seropositive was determined and shown to be 63.2%. CONCLUSION: The results of this study indicate that adenovirus infection is prevalent among non-human primates in Kenya. These findings suggest that cross species transmission in Kenyan non-human primates may be a common occurrence and there is a possibility of zoonotic transmission of adenoviruses. Furthermore, our results highlight the potential of using these non-human primates as models for testing safety and efficacy of candidate adenovirus vaccines prior to clinical trials in humans.

The effect of a single gavage dose of fumonisin B(2) on the sphinganine and sphingosine concentrations in vervet monkeys.

The disruption in sphinganine (Sa) and sphingosine (So) concentrations in plasma and urine of vervet monkeys (Cercopithecus aethiops) was measured following a single gavage dose of either 1 (low dose) or 10 mg (high dose) fumonisin B(2) (FB(2))/kg body weight. Blood and urine were collected over a 51-day period. In the low-dose monkeys, none of the parameters measured increased significantly above the control values. In the high-dose monkeys the plasma Sa/So ratios were significantly increased (P< 0.05) above the corresponding control ratios after 3 days and continued to be significantly raised for another 27 days, whereafter the ratios declined to control values after 51 days. The plasma aspartate transaminase (AST) activities increased significantly above their control values from day 5 to day 23 and the gamma-glutamyl transferase (GGT) activities from day 7 until the end of the study period. The plasma cholesterol concentration and alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities increased transiently, but not significantly, and returned to control values within the study period. The urinary Sa/So ratio, plasma creatinine and urea values in both groups of monkeys did not increase above the control values. This study demonstrated that a single large dose of FB(2) can cause transient disruption of sphingoid metabolism in vervet monkeys.

Lipids, lipoproteins, and endocrine profiles during pregnancy in the African green monkey (Cercopithecus aethiops).

In an attempt to establish relationships between the endocrine and lipid metabolism during pregnancy, the changes in total plasma cholesterol (TPC) and lipoprotein cholesterol that occur during pregnancy in the African green monkey were investigated longitudinally in ten females in relation to the changes in progesterone, estradiol, and fasting insulin concentrations. Respective means for TPC, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) plus very low-density lipoprotein (VLDL) cholesterol were 343 +/- 35, 108 +/- 9, and 235 +/- 36 mg/dL prior to the estimated date of conception in ten females fed a high-fat, high-cholesterol diet. The concentration of these lipids fell to 225 +/- 31, 54 +/- 4, and 168 +/- 29 mg/dL for TPC (P less than 0.001), HDL cholesterol (P less than 0.001), and LDL + VLDL cholesterol (P less than 0.001), respectively, by midpregnancy (84 days). Progesterone concentrations increased during the first 60 days of pregnancy and were negatively correlated with HDL cholesterol concentrations (r = -0.57, P less than 0.02). After reaching their highest mean value, progesterone concentrations then plateaued at lower concentrations until parturition. The decrease in progesterone concentrations was associated with an initial rise in estradiol concentrations, which reached their highest concentrations in late pregnancy and were inversely correlated with HDL-cholesterol concentrations (r = -.32, P less than 0.01). Although glucose concentrations remained steady during gestation, insulin concentrations were elevated compared to postpartum concentrations (P less than 0.05) suggesting that insulin resistance occurred during the pregnancy in this nonhuman primate.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum aminopeptidases in pregnant vervet monkeys (Cercopithecus aethiops).

Two distinctive aminopeptidase isozymes have been identified in the serum of the vervet monkey. The L-methionine-sensitive cytosol aminopeptidase (AP; E.C. 3.4.11.1) is present in all samples, while the cystyl aminopeptidase (CAP; E.C. 3.4.11.3) isozyme, which is resistant to inhibition by 0.1 M L-methionine, is found only in the serum of pregnant females. The pregnancy-specific CAP isozyme appears in the serum between the fifth and the eleventh weeks of a nearly 22-week pregnancy. The overall aminopeptidase activity and the activity of the L-methionine-insensitive isozymes alone are both significantly greater in the serum of pregnant females than in nonpregnant females. Consequently, the presence of the cystyl aminopeptidase isozyme in the serum is a useful diagnostic indicator of pregnancy in the vervet monkey.

[Isolation of adeno-associated virus type 4 from a culture of green monkey kidney cells]. / Vydelenie adenoassotsiirovannogo virusa tipa 4 iz kul'tury kletok pochek zelenykh martyshek.

In the process of biological control of uninfected green monkey kidney (GMK) cell cultures a thermostable hemagglutinating agent designated No. 5056 was isolated alongside with adenovirus-SV. By its antigenic properties the 5056 strain was identified as adeno-associated virus type 4 (AAV-4). In control of 574 specimens of GMK culture batches, 40 AAV-4 strains were isolated in the presence of a helper adenovirus. Some biological properties of the isolates and their resistance to certain physical and chemical treatments were studied. Two isolates of the satellite virus were examined in the electron microscope. A correlation between the rate of AAV-4 isolation from GMK cultures and the presence of complement-fixing antibody to AAV-4 in monkey sera was observed.

Hemagglutination inhibition studies for the evaluation of blood group antigens in ethanol soluble substances (ESS) obtained from human, baboon and vervet monkey red blood cells.

Soluble blood group substances, isolated from the red blood cells of humans, baboons, and vervet monkeys by ethanol extraction, possessed serologically active specificities for the following antigens: A, B, H, Lea, LebL, P, P19 Pk and I. Human red blood cells lacking any of these specificities by the direct hemagglutination test also lacked the related antigens in their soluble extract. The only exception was in "Bombay" Oh cells, from which soluble H substance could be readily isolated. Soluble substances obtained from baboon and vervet monkey red blood cells, which lack the human variety of A, B, and H antigens on their red blood cells, inhibited both human and lectin anti-H reagents. The detection of "hidden" H activity in Oh cells will pose some important questions regarding membrane characteristics and the role of immune surveilance.