Development and Characterization of a Model for Inducing Fetal Hemoglobin Production in Cynomolgus Macaques (Macaca fasicularis).

Hydroxyurea induces production of fetal hemoglobin (HbF), a tetramer of α and γ globin proteins and corresponding heme molecules, normally found in less than 1% of adult RBC. Increases in circulating HbF are correlated with clinical improvement of patients with hemoglobinopathies, and hydroxyurea, as a daily medication, is the standard treatment for sickle cell anemia. Although olive baboons (Papio anubis) are considered a key model species for HbF induction, cynomolgus macaques (Macaca fasicularis) are another species that conserves the ability to produce HbF into maturity. In this study, moderate anemia was experimentally induced in cynomolgus macaques by phlebotomy, to stimulate accelerated erythropoiesis and HbF production. In contrast to previous studies, vascular access ports were implanted for phlebotomy of conscious monkeys, followed by fluid replacement. As total Hgb levels dropped, reticulocyte counts and the percentage of HbF-expressing cells increased. Once total Hgb levels declined to less than 8 g/dL, 2 courses of oral hydroxyurea (once daily for 5 d) were completed, with a 9-d interval between courses. After hydroxyurea dosing, the percentage of HbF-expressing cells and total HbF were increased significantly. In addition, a significant but transient decrease in reticulocyte count and a transient increase in MCV occurred, replicating the characteristic response of patients receiving hydroxyurea. Daily clinical observations revealed no serious health issues or decreases in food consumption or activity levels. Methods were established for assessing the patency of vascular access ports. This study details a new protocol for the safe and routine induction of moderate anemia in cynomolgus macaques and validates its use in the investigation of novel pharmacologic entities to induce the production of HbF.

Coccidioidomycosis in an Indoor-housed Rhesus Macaque (Macaca mulatta).

Coccidioides spp. are saprophytic, dimorphic fungi that are endemic to arid climates, are capable of infecting many species, and result in diverse clinical presentations. An indoor-housed laboratory rhesus macaque presented with weight loss and decreased activity and appetite. During the diagnostic evaluation, a bronchiolar-alveolar pattern in the cranial lung lobes, consistent with bronchopneumonia, was noted on radiographs. Given the poor prognosis, the macaque was euthanized. Confirming the radiographic assessment, gross necropsy findings included multifocal to coalescing areas of consolidation in the right and left cranial lung lobes. Microscopically, the consolidated regions were consistent with a pyogranulomatous bronchopneumonia and contained round, nonbudding, fungal yeast structures considered to be morphologically consistent with Coccidioides immitis. Culture and colony morphology results were confirmed through additional diagnostic testing. Sequencing of the D1-D2 domain of the 28S large ribosomal subunit positively matched with a known sequence specific to C. immitis. Serology for Coccidioides spp. by both latex agglutination (IgM) and immunodiffusion (IgG) was positive. In this rhesus macaque, the concordant results from histology, culture, DNA sequencing, and serology were collectively used to confirm the diagnosis of coccidioidomycosis. This animal likely acquired a latent pulmonary infection with Coccidioides months prior to arrival, when housed outdoors in a Coccidioides-endemic area. The nonspecific clinical presentation in this macaque, coupled with the recent history of indoor housing and lag between clinical presentation and outdoor housing, can make similar diagnostic cases challenging and highlights the need for awareness regarding animal source when making an accurate diagnosis in an institutional laboratory setting.

Assessment of luteal function in the vervet monkey as a means to develop a model for obesity-related reproductive phenotype.

The objective of the current study was to characterize luteal function in vervet monkeys. Urine from 12 adult female vervets housed at an academic research center was collected for 10 weeks from single-caged monkeys in order to assess evidence of luteal activity (ELA) as determined by urinary excretion of pregnanediol glucuronide (Pdg) and estrone conjugates (E1c). Dual energy X-ray absorptiometry (DXA) was performed on the monkeys to assess body composition, bone density, and fat mass. Menstrual cyclicity was determined using records of vaginal bleeding. ELA was observed in 9 monkeys and was characterized by a late follicular rise in E1c followed by a progressive increase in Pdg excretion. Mean menstrual cycle length was 26.7 ± 3.8 days and the average day of luteal transition was 14 ± 1.8. Three monkeys without ELA had a clearly defined E1c rise (mean 12-fold from nadir) followed by an E1c drop that was not accompanied by Pdg rise and coincided with vaginal bleeding. Among the 9 ELA monkeys, excretion of E1c tended to negatively associate with fat mass, although this finding did not reach statistical significance (r = -0.61, p = 0.08). Similar to women, vervet monkeys experience an increase in E1c late in the follicular phase of the menstrual cycle which is followed by a subsequent luteal Pdg peak. Assessment of urinary reproductive hormones allows for identification of cardinal menstrual cycle events; thus, the similarity of vervet cycles to human menstrual cycles makes them a useful model for obesity-related human reproductive impairment.

Expression and regulation of Kit ligand in the ovary of the hen.

The Kit system, composed of Kit ligand (KL) and its tyrosine kinase receptor, cKit, has been well characterized in mammals. Studies have shown that it is involved in signaling between the oocyte and somatic cells during the process of follicle maturation. We characterized KL mRNA expression during follicle maturation in the domestic hen, examined regulation of KL and a possible function of the Kit system. KL mRNA expression was assessed using quantitative PCR (n=4 replicates) in follicles of various sizes (1, 3, 5, 6-12 mm, F1). Expression of KL mRNA decreased significantly (p<0.01) with follicle development and was highest in <1 mm follicles, which contained the theca as well as granulosa layers, with high levels also found in the granulosa layer of 3 mm follicles and ovarian stroma. To study regulation of KL mRNA, granulosa cells from 6-8 mm follicles (n=4 replicates) were plated in M199 plus 0.1% BSA in the presence of various treatments including: oocyte conditioned medium (OCM), Vitamin D(3), FSH, estradiol, progesterone and testosterone. OCM caused a dose-related increase (p<0.05) in expression of KL mRNA; Vitamin D(3) increased and FSH decreased expression of KL mRNA. cKit was detected (at the expected size) in the theca layer of 3-5 mm follicles and in a lysate of whole <1mm follicles. Culture of granulosa cells in the presence of OCM resulted in a decrease of P4 secretion, an effect blocked by pre-incubation of OCM with cKit antibody. Although OCM caused a dose-related increase in E2 secretion from theca, this was not blocked by cKit antibody.