Chlorocebus Aethiops Sabaeus Research Articles

Authentication of the green monkey kidney Vero76 cell lines subcultured through many generations

The Vero76 cell lines isolated from the kidney of an African green monkey (Chlorocebus aethiops sabaeus), are commonly used for viral culture, vaccine development, and cytotoxicity assays. However, subculturation of cell lines over multiple generations can lead to a genetic change in these cell lines compared to the original cell. This will lead to deviations in results of research that using the modified cell lines. Therefore, identification and evaluation periodically the genetic stability of cell lines after subculture are necessary. In recently studies, cell authentication by using STR markers (short tandem repeat markers) have been becoming a simple and effective method that used commonly. In this study, we used eight STR markers: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389 for cell lines authentication and evaluation of the genetic stability of subcultured Vero76 cell lines. The STR indicators were amplified, sequenced and compared with the original cell to identify the differences between cell lines in subcultured generations. The evaluation results showed that these cell lines did not have any differ compared with the original cell line, this confirmed that the genetic of the cell lines were stability. The results also suggested that STR markers can be used to authenticate for Vero76 cell lines.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies

BackgroundDengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles.MethodsWe tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans.ResultsAll of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals.ConclusionSylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

Identification of Schistosoma mansoni Infection in a Nonhuman Primate from St. Kitts More than 50 Years after Interruption of Human Transmission.

.Transmission of Schistosoma mansoni was interrupted on St. Kitts, a Caribbean island, in the 1950s. With no reported cases since that time and most Biomphalaria spp. snail populations eliminated based on surveys in the 1970s, S. mansoni has been considered eliminated on St. Kitts. In 2019, S. mansoni eggs were found in an African green monkey (Chlorocebus aethiops sabaeus) that originated from St. Kitts. Nonhuman primate (NHP) infections have been considered incidental to human infections, with infections in NHPs resolving with the elimination of S. mansoni in the human population. An NHP with S. mansoni infection suggests that the NHP may be able to maintain a reservoir sylvatic cycle. Alternatively, S. mansoni transmission was not eliminated or S. mansoni has been reintroduced to St. Kitts. The occurrence of an infected NHP from St. Kitts supports the need for continuous monitoring in areas where S. mansoni is considered eliminated.

Biomarkers of senescence in non-human primate adipose depots relate to aging.

Forty-three female African green monkeys (Chlorocebus aethiops sabaeus) were selected to represent young adult to advanced geriatric ages (7-24years) to exhibit a wide range of obesity status (8-53% body fat) and diverse metabolic syndrome criteria such as diabetes, dyslipidemia, and hypertension. Subcutaneous and visceral adipose tissues were collected and evaluated for the presence of senescence cells in both whole tissue and single-cell isolates from subcutaneous sources, utilizing senescence-associated β-galactosidase (SAβ-gal) staining. Plasma samples were analyzed for selected metabolic and inflammatory biomarkers related to the senescence-associated secretory profile. Our results indicated that tissue staining scores did not differ between subcutaneous and intra-abdominal visceral depots and were highly related within individuals. Tissue staining was significantly associated with chronological age; however, no associations with fatness or metabolic syndrome criteria were observed. Associations with age were unchanged when obesity status was included in regression models. Isolated cell staining did positively relate to age but not tissue staining, suggesting some of the SAβ-gal-positive cells were stromal vascular cells or small adipocytes, but that mature large adipocytes, filtered out in the cell isolation process, are also likely to exhibit positive SAβ-gal staining. Plasminogen activator inhibitor-1 (PAI-1) concentration in circulation was the sole inflammation-related biomarker that positively associated with age and is considered to be a marker of senescent cell burden. Our study is the largest, most comprehensive assessment of adipose SAβ-gal staining in a relevant animal model of human aging, and confirms that this senescence-associated biomarker specifically indicates an age-related process.

Advanced maternal age impacts physiologic adaptations to pregnancy in vervet monkeys.

The trend to delay pregnancy in the USA has resulted in the number of advanced maternal age (AMA) pregnancies to also increase. In humans, AMA is associated with a variety of pregnancy-related pathologies such as preeclampsia (PE). While AMA is known to be a factor which contributes to the development of pregnancy-induced diseases, the molecular and cellular mechanisms giving rise to this phenomenon are still very limited. This is due in part to lack of a preclinical model which has physiologic relevance to human pregnancy while also allowing control of environmental and genetic variability inherent in human studies. To determine potential physiologic relevance of the vervet/African green monkey (Chlorocebus aethiops sabaeus) as a preclinical model to study the effects of AMA on adaptations to pregnancy, thirteen age-diverse pregnant vervet monkeys (3-16 years old) were utilized to measure third trimester blood pressure (BP), complete blood count, iron measurements, and hormone levels. Significant associations were observed between third trimester diastolic BP and maternal age. Furthermore, the presence of leukocytosis with enhanced circulating neutrophils was observed in AMA mothers compared to younger mothers. Moreover, we observed a negative relationship between maternal age and estradiol, progesterone, and cortisol levels. Finally, offspring born to AMA mothers displayed a postnatal growth retardation phenotype. These studies demonstrate physiologic impairment in the adaptation to pregnancy in AMA vervet/African green monkeys. Our data indicate that the vervet/African green monkey may serve as a useful preclinical model and tool for deciphering pathological mediators of maternal disease in AMA pregnancy.

Postpartum Hypertensive Nonhuman Primates Have Decreased Plasma Osmolality without Proteinuria

Gestational hypertensive disorders are a leading cause of pregnancy‐related mortality. Postpartum hypertension (PPHT) is a related condition characterized by elevated blood pressure following delivery in previously normotensive (NT) females that returns to normotension within 6 weeks. PPHT is poorly characterized due to its development during a maternal care period lacking direct clinical supervision. We have previously reported spontaneous PPHT in African Green Monkeys (AGM; Chlorocebus aethiops sabaeus). We hypothesize that PPHT in AGM is associated with altered renal function and proteinuria that may lead to long term renal dysfunction. Systolic blood pressures (SBP) were obtained by forearm plethysmography under ketamine sedation (15 mg/kg i.m.) followed by venous blood collection. AGM were classified as NT (SBP ≤ 120 mmHg) or PPHT (NT before and during pregnancy but SBP ≥ 140 mmHg postpartum). Animals were placed in metabolic pens and acclimated for at least 1 week before 3 consecutive days of urine collection. Animals were fed standard nonhuman primate chow and water ad lib. Protein concentrations were measured using a bicinchoninic acid assay. Measurements were taken when nonpregnant (NP), during 3rd trimester of pregnancy (3T, ~14–21 weeks gestation), 1 day postpartum (PP1), 14 days postpartum (PP14), and 42 days postpartum (PP42). Body weights did not differ between NT and PPHT. Water intake for NT and PPHT in NP, 3T and PP did not differ. In NT animals, SBP remained similar to NP SBP during pregnancy and postpartum (NP 104 ± 4.7 mmHg; 3T 99 ± 4.4 mmHg; PP1 102 ± 5.2 mmHg; PP14 101 ± 6.8 mmHg; PP42 97 ± 6.0 mmHg, n=27 per time point). PPHT SBP increased in PP1 compared to NP and 3T (NP 114 ± 10.0 mmHg, n=8; 3T 107 ± 10.0 mmHg, n=8; PP1 144 ± 9.5 mmHg*, n=9; PP14 131 ± 9.0 mmHg*, n=10; PP42 117 ± 10.0 mmHg, n=8; * p≤0.05 vs NP; ANOVA, post hoc SNK). NT protein excretion remained constant though PP14 (NP 365.2 ± 48.5 mg/day, n=26; 3T 473.0 ± 99.0 mg/day, n=8; PP1 501.0 ± 171.4 mg/day, n=6; PP14 387.0 ± 140.0 mg/day, n=7) but declined at PP42 compared to NP (PP42 143.3 ± 171.4 mg/day, n=8; p≤0.05). Furthermore, in PPHT animals protein excretion did not change (NP 421.4 ± 75.5, n=8; 3T 375.4 ± 87.2, n=6; PP1 544.2 ± 71.2, n=9; PP14 578.5 ± 75.5, n=8; PP42 560.8 ± 80.7, n=7). Plasma osmolality was unchanged in NT animals (NP 308.5 ± 4.6 mOsm/kg, n=13; 3T 304.7 ± 6.7 mOsm/kg, n=6; PP1 290.5 ± 8.2 mOsm/kg, n=5; PP14 311.0 ± 11.6 mOsm/kg, n=5; PP42 299.0 ± 16.4 mOsm/kg, n=5). Plasma osmolality of PPHT animals decreased in 3T compared to NP (NP 315.0 ± 7.2 mOsm/kg, n=4; 3T 289.3 ± 5.1 mOsm/kg, n=8; PP1 297.6 ± 4.5 mOsm/kg, n=10; PP14 309.9 ± 5.1 mOsm/kg, n=8; PP42 293.0 ± 5.4 mOsm/kg, n=7; p≤0.05) but returned to NP values by PP14 (p≤0.05). Thus, PPHT in AGM is not associated with renal dysfunction but displays a decrease in plasma osmolality at 3T and elevated postpartum SBP. This decreased plasma osmolality may result from elevated ADH during 3T and may contribute to PPHT. We conclude that PPHT has characteristics of gestational hypertension without altered renal function or 3T hypertension. These data support the AGM as a translational model for acute and long‐term studies of cardiovascular disease in women that develop postpartum hypertension.

Female African Green Monkeys Exhibit Protection Against Glucose Loading Indicative of Type 2 Diabetes Mellitus

Over 30 million Americans have diabetes, roughly 90% of which exhibit the type 2 form. Overall, males have a higher prevalence of Type 2 Diabetes Mellitus (T2DM) and glucose intolerance than females. A co‐morbidity of T2DM and the resultant elevated blood glucose is decreased kidney function and elevated plasma creatinine. African Green Monkeys (AGM; Chlorocebus aethiops sabaeus) are known to express glucose intolerance similar to T2DM and renal pathologies making them an optimal nonhuman primate experimental model. Given the AGM genetic homology to humans, we hypothesized that female AGM would have protection against T2DM compared to males. A total of 9 healthy, age matched AGM were utilized in the study (male: 4.96±0.47 kg, n=4; female: 3.10±0.34 kg, n=5). Systolic blood pressure (SBP) measurements were taken by forearm plethysmography. Glucose handling was measured via a standard oral glucose tolerance test protocol. Fasted (12 hours) AGM were anesthetized (ketamine, 15mg/kg, i.m.) and baseline glucose was obtained via finger prick. AGM were gavaged with a 5% glucose load (15ml/kg) in water. Plasma glucose measurements were taken at 15, 30, 60, 90, and 120 minutes after gavage. Venous blood samples were obtained at baseline, 30, 60, and 120 minutes after glucose administration. Females had lower SBP and were normotensive, while males were borderline hypertensive (99±4.8 mmHg; 132±8.6 mmHg, respectively; p≤0.05). Females had higher baseline plasma glucose levels than males (female: 95±6.5 mg/dl; male: 84±9.8 mg/dl), but a lower peak at 30 minutes post glucose load (female: 108±33.2 mg/dl; male: 145±86.4 mg/dl; p≤0.05). Females also had a lower area under the curve (AUC) than males measured over the 120 minutes post‐glucose administration (female: 11,670±722.9 min*mg/dl; male: 14,411±1,143.2 min*mg/dl; p≤0.05). Plasma osmolality was not different between male and female AGMs prior to glucose administration (females: 297 ±9.2 mosm/kg; males: 314±7.6 mosm/kg; p≥0.05). Fasting female AGM had lower plasma creatinine levels than males (female: 1.28±0.23 mg/dl; males: 2.16±0.23 mg/dl; p≤0.05). These data demonstrate that peak plasma glucose at 30 minutes following an oral glucose load in females was decreased compared with age matched males. This reduced female response occurred despite female AGM fasting glucose being greater than that of male AGM. Furthermore, female AUC was reduced compared with male AUC over two hours following the oral glucose load. Females also had lower plasma creatinine levels than males. The higher creatinine levels indicate decreased kidney function in the males. These data coupled with the borderline hypertension demonstrate both cardiovascular and renal dysfunction in these male AGMs. Together, these data suggest increased efficiency of glucose handling by female AGM, retention of normal kidney function, and maintenance of normotension compared to age matched males. Overall, the data suggest that female AGM are doubly protected from T2DM and cardiovascular disease. Future studies will include conscious glucose and insulin measurements of AGM, and insulin/glucose clamp studies to understand the metabolomic differences between male and female AGM.

Increased Pulmonary Function Following Administration of a CBD‐Containing Compound NCMB‐1 in Fibrotic Lungs of African Green Monkeys.

The endocannabinoid system is a neuromodulatory system that is conserved among vertebrate species. Cannabidiol (CBD) is a modulator of cannabinoid (CB) receptor action both in the central nervous system and in the periphery. CB receptors are involved in inflammatory responses in peripheral organs, including lung tissue. CBD administration has been shown to reduce airway inflammation and fibrosis in vitro and in experimentally induced rodent models of asthma through modulation of the immune system. However, little is understood regarding the relationship between pulmonary disease and CBD modulation of general CB receptors in vivo. The African Green Monkey (Chlorocebus aethiops sabaeus; AGM) has bronchiolar tissue and tracheal branching remarkably similar to that in human lungs, therefore making it a translational model for the study of CBD treatment on pulmonary fibrosis‐related lung dysfunction. We hypothesize that administration of NCMB‐1, a hemp extract with CBD, will increase the tidal volume, inspiratory flow rate, and respiration rate of animals with previously induced pulmonary fibrosis. Baseline tidal volume, inspiration flow rate, and respiration rate of 16 male AGM with induced lung fibrosis were measured. Animals were divided into a vehicle and 3 treatment groups: 50 μL of undiluted, 5X dilution, and 25X dilution NCMB‐1 administered orally twice daily via banana sections for two weeks. Compared to control (n=4, 206.6 ± 36.4 mL), 5X NCMB‐1 treated animals increased tidal volume (n=4, 484.0 ± 54.6 mL; p<0.05) from baseline at two weeks of treatment. Inspiratory flow rate also increased in 5X treated animals (n=4, 412.0 ± 49.9 mL/s; p<0.05) compared to control (n=4, 212.3 ± 50.7 mL/s) after two weeks of administration. Respiration rates increased from baseline in 1X treated animals (n=4, 33.0 ± 4.5 breaths/min) and 5X treated animals (n=4, 30.8 ± 3.9 breaths/min) compared to control (n=4, 28.5 ± 4.1 breaths/min; p<0.05). Treatment with 25X dilution of NCMB‐1 did not alter pulmonary function at either one or two weeks of treatment compared to vehicle treated animals. Thus, administration of 5X diluted NCMB‐1 to AGM with induced pulmonary fibrosis increased tidal volume, inspiratory flow rate, and respiration rate from compared to vehicle treated AGM. These results indicate CBD as a potential modulator of CB receptors improving pulmonary function and suggest a potential for therapeutic value of CBD in the treatment of restrictive pulmonary disease. Future directions include identification of active CBD compounds within the extract responsible for changes in airway resistance, expression of specific pulmonary genes involved in reduction of inflammation, and immunohistochemistry of CB receptors in the hypothalamus of AGM with induced pulmonary fibrosis. CBD crossing the blood brain barrier in animals treated with NCMB‐1 may cause up or downregulation of CB1 and/or CB2 receptors that could positively impact lung function.Support or Funding InformationFunding provided by Primates Plus, LLC.

Adolescent Offspring of Preeclamptic Nonhuman Primate Mothers Have Impaired Glucose Tolerance and Proteinuria

Hypertensive pregnancy disorders are the most common pregnancy complication and are a primary cause of maternal and fetal mortality worldwide. Preeclampsia (PE), defined as de novo maternal hypertension and proteinuria in the 2nd or 3rd trimester, is associated with preterm birth and long‐term cardiovascular risk in both mothers and offspring. The African Green Monkey (AGM; Chlorocebus aethiops sabaeus) develops spontaneous preeclampsia and exhibits fetal growth restriction. We hypothesize that PE pregnancies leading to reduced birthweight may increase the risk for impaired renal and metabolic function in offspring. Offspring of normotensive (NT) pregnancies averaged 335.6 ± 9.6g birthweight, while birthweight of offspring born to PE females was 299.4 ± 9.0g, indicating fetal growth restriction (NT n=12; PE n=24; pδ0.05). To assess glucose tolerance, an oral glucose tolerance test (OGTT) was conducted on a subset of offspring at 1–3 years of age. Glucose was fed to a final concentration of 1.75 g/kg bodyweight. Blood glucose was measured by finger stick with standard glucometry prior to the glucose load and at 15, 30, 60, 90, and 120 minutes post glucose load. Fasting blood glucose was similar between NT and PE juveniles (NT n=9, 77.7 ± 8.4 mg/dl; PE n=11, 82.2 ± 7.6 mg/dl; p>0.05). Blood glucose of NT juveniles peaked at 15 minutes (141.7 ± 18.9mg/dl) and subsequently decreased. In contrast, PE juveniles showed a biphasic glucose response, first peaking at 15 minutes (132 ± 13 mg/dl) and again at 60 minutes (149.4 ± 14.1 mg/dl). The area under the curve of the OGTT was used as an index of glucose tolerance. Glucose tolerance of PE juveniles was impaired compared to NT juveniles (NT n=9, 10994 ± 1390 min*mg/dl; PE n=11, 14831 ± 5761 min*mg/dl; pδ0.05). To monitor water intake and urine flow, juveniles were individually housed (NT n=9; PE n=10). After an acclimation period of 1 week, water intake and urine flow were measured for 3 consecutive days. Urinary protein excretion was greater in PE offspring (NT 152.0 ± 61.7 mg/day vs. PE 354.7 ± 55.8 mg/day; pδ0.05). Sodium excretion was similar between groups (NT 16.10 ± 3.2 mmol/day; PE 18.96 ± 2.9 mmol/day; p>0.05). However, potassium excretion was greater in PE offspring (6.19 ± 0.8 mmol/day vs. NT 2.70 ± 0.9 mmol/day; pδ0.05). Plasma creatinine was similar between offspring of NT and PE females (NT 1.74 ± 0.2 mg/dl; PE 1.65 ± 0.1 mg/dl; p>0.05). An individual with the lowest birth weight of the PE cohort (228.5g vs. mean 314.1 ± 14g) had the highest creatinine level (2.38 mg/dl vs. mean 1.65 ± 0.1 mg/dl) and elevated protein excretion (504.6 mg/day vs. mean 378.5 ± 74.7 mg/day). PE in AGMs is associated with fetal growth restriction resulting in low birthweights. Juveniles born to PE females showed impaired glucose tolerance, proteinuria, and kaliuresis. Exposure to PE in utero may predispose offspring to impaired renal and metabolic function in early adolescence. Continued monitoring of current PE juveniles and phenotyping of 2019 offspring will further assess the influence of maternal pregnancy phenotype on adolescent markers of renal and metabolic impairment.Support or Funding InformationThis work was supported by research funds from Primate Plus, LLC.

Abstract P3038: Reduced Renal Responsiveness and Altered Fluid Volume in an African Green Monkey ( Chlorocebus Aethiops Sabaeus) Preeclampsia Model

Preeclampsia is a prevalent, multi-organ complication of late pregnancy characterized by de novo gestational hypertension (GH) and proteinuria. Our group has demonstrated that arginine vasopressin (AVP) secretion, as measured by copeptin, is predictive of the development of human preeclampsia. Altered regulation of AVP early in gestation has been demonstrated in human preeclampsia. Recently, the nonhuman primate, African Green Monkey (AGM) has been characterized as a spontaneous, translational model of chronic hypertension, GH and preeclampsia. AGMs also have similar gestation and placentation to humans. We hypothesized that the AGM model of preeclampsia would demonstrate similar AVP related plasma and fluid volume changes as humans with preeclampsia. AGMs (n=10) were anesthetized with ketamine during late third trimester of pregnancy to obtain physiologic and obstetric measurements. Compared to normotensive (NT, n=4) third trimester pregnant females, GH females (n=6) had higher systolic and diastolic blood pressures (106 ± 5 vs. 162 ± 11 mmHg; 46 ± 4 vs. 86 ± 12, p=0.01) with no differences in heart rates (158 ± 4 vs. 169 ± 18 bpm, p=0.64). Consistent with other models of GH and preeclampsia, the GH AGM had decreased offspring birthweight in comparison to NT offspring (352 ± 11 vs. 275 ± 12 g, p=0.01). Further, plasma osmolality of GH females was higher in late third trimester compared to NT females (286 ± 11 vs. 312 ± 9 mOsm, p=0.05). This higher plasma osmolality was associated with the GH female’s trend to higher AVP secretion as measured by copeptin (2 ± 2 vs. 4 ± 1 ng/g, p=0.280). In contrast to human pregnancies, late third trimester plasma [Na + ] of GH females was elevated compared to NT females (149 ± 3 vs. 157 ± 2 mMol/L, p=0.04). These data demonstrate that that the AGM model of preeclampsia replicates the plasma volume restricted findings consistent with human preeclampsia and possible insensitivity to control by AVP. Ongoing studies in early pregnancy will quantify changes in renal function during preeclampsia, differences in fluid volumes among NT and GH AGMs, and further the understanding of hormonal regulation of kidney function in the pathogenesis of preeclampsia in this translational AGM nonhuman primate.

Abstract P106: Elevated Fasting Glucose and Proteinuria in Offspring Born to Gestational Hypertensive African Green Monkeys

Preeclampsia (PreE), or gestational hypertension (GH) concurrent with proteinuria, is a leading cause of maternal and fetal mortality worldwide. PreE often leads to fetal growth restriction resulting in low birth weight offspring. Low birth weight infants are at higher risk of developing cardiovascular disease later in life. The African Green Monkey (AGM; Chlorocebus aethiops sabaeus ) develops spontaneous GH with proteinuria and fetal growth restriction. We hypothesize that GH will developmentally program offspring to reduced kidney function and metabolic disease. Maternal systolic arterial pressure (SAP) was measured via forearm plethysmography prior to pregnancy and during the 3 rd trimester of pregnancy. At 1-3 years of age, offspring were single-housed for water intake and urine flow rate collections followed by blood pressure measurement and venous blood sampling. Animals were grouped by pregnancy phenotype. SAP for normotensive (NT, n=23) mothers did not change in pregnancy, but increased during third trimester for GH (Δ=35 ± 7 mmHg, n=34). NT pregnancies resulted in a 4% stillbirth rate (1/23) compared with 29% in GH (10/34). Surviving GH offspring had reduced birth weight (NT 336 ± 11g vs. GH 295 ± 8g, p<0.05). Fasting glucose was higher in progeny born to GH pregnancies (NT 69 ± 8 mg/dl, n=13; GH 92 ± 6 mg/dl, n=20). Urinary protein excretion was higher in offspring born to GH pregnancies (NT 152 ± 27 mg/day; GH 318 ± 46 mg/day). SAP of offspring was similar between maternal phenotype (NT 107 ± 8 mmHg; GH 109 ± 5 mmHg). Juvenile SAP was unrelated to fasting glucose or urinary protein excretion, indicating that these factors are determined by maternal SAP during pregnancy, rather than juvenile phenotype. Thus, GH is associated with fetal growth restriction in the form of high stillbirth rates and lower surviving-offspring birth weights in the AGM. GH offspring have elevated fasting glucose and possible decreased kidney function as evidenced by proteinuria. GH could be programming offspring for early life metabolic risk factors and declining renal function. Future studies include oral glucose tolerance tests and additional markers of renal dysfunction to further assess in utero programming of the pathogenesis of cardiovascular and metabolic disease.

Type-2-Diabetes Alters CSF but Not Plasma Metabolomic and AD Risk Profiles in Vervet Monkeys.

Epidemiological studies suggest that individuals with type 2 diabetes (T2D) have a twofold to fourfold increased risk for developing Alzheimer’s disease (AD), however, the exact mechanisms linking the two diseases are unknown. In both conditions, the majority of pathophysiological changes, including glucose and insulin dysregulation, insulin resistance, and AD-related changes in Aβ and tau, occur decades before the onset of clinical symptoms and diagnosis. In this study, we investigated the relationship between metabolic biomarkers associated with T2D and amyloid pathology including Aβ levels, from cerebrospinal fluid (CSF) and fasting plasma of healthy, pre-diabetic (PreD), and T2D vervet monkeys (Chlorocebus aethiops sabaeus). Consistent with the human disease, T2D monkeys have increased plasma and CSF glucose levels as they transition from normoglycemia to PreD and diabetic states. Although plasma levels of acylcarnitines and amino acids remained largely unchanged, peripheral hyperglycemia correlated with decreased CSF acylcarnitines and CSF amino acids, including branched chain amino acid (BCAA) concentrations, suggesting profound changes in cerebral metabolism coincident with systemic glucose dysregulation. Moreover, CSF Aβ40 and CSF Aβ42 levels decreased in T2D monkeys, a phenomenon observed in the human course of AD which coincides with increased amyloid deposition within the brain. In agreement with previous studies in mice, CSF Aβ40 and CSF Aβ42 were highly correlated with CSF glucose levels, suggesting that glucose levels in the brain are associated with changes in Aβ metabolism. Interestingly, CSF Aβ40 and CSF Aβ42 levels were also highly correlated with plasma but not CSF lactate levels, suggesting that plasma lactate might serve as a potential biomarker of disease progression in AD. Moreover, CSF glucose and plasma lactate levels were correlated with CSF amino acid and acylcarnitine levels, demonstrating alterations in cerebral metabolism occurring with the onset of T2D. Together, these data suggest that peripheral metabolic changes associated with the development of T2D produce alterations in brain metabolism that lead to early changes in the amyloid cascade, similar to those observed in pre-symptomatic AD.

African Green Monkeys Born to Mothers with Hypertensive Pregnancy Disorders have Higher Blood Pressures and Altered Water Balance in Early Adolescence

Hypertensive pregnancy disorders are a leading cause of maternal and fetal mortality worldwide. Preeclampsia (PE), or de novo maternal hypertension, is associated with high incidences of preterm birth. Lower birth weight and exposure to hypertension (HTN) in utero have been shown to increase the risk of HTN and cardiovascular disease later in life. The African Green Monkey (AGM; Chlorocebus aethiops sabaeus) spontaneously develops gestational hypertension (GH) with pathophysiological similarities to PE, including proteinuria and fetal growth restriction. The AGM also spontaneously develops chronic HTN not associated with pregnancy. We tested the hypothesis that GH and chronic HTN in pregnancy will developmentally program offspring to HTN in adolescence. Male and female juvenile AGMs between the ages of 1 and 2 years were single housed. After a 1‐week acclimation, urine excretion and water intake were measured daily for 3 consecutive days. Blood pressure was measured by forearm plethysmography and fasting blood glucose was assessed. Juvenile AGMs were characterized by maternal systolic blood pressure (SBP) during pregnancy: normotensive (NP; SBP < 120 mmHg throughout pregnancy); hypertensive (HP; SBP > 140 mmHg before, during, and after pregnancy); and gestational hypertensive (GHP; SBP < 120 mmHg before and > 140 mmHg in the second half of pregnancy). Juvenile growth rate was based on body weight from birth to 1–2 years of age. Juvenile growth rates were reduced for GHP and HP compared to NP (NP 1303 g/year; GHP 1083 g/year; HP 946 g/year; p < 0.05). Although animals born to GHP and HP have hindered growth from birth through early adolescence, growth within the first 2 weeks postpartum was greater in GHP animals (34 g/week) compared to NP (15 g/week) and HP (14.6 g/week). Juvenile SBP was higher for HP offspring compared to NP (HP 131.3 ± 11.7 mmHg, n = 3; NP 100.2 ± 9.1 mmHg, n = 5; p < 0.05) but not GHP offspring (GHP 103.4 ± 6.4 mmHg, n = 7; p > 0.05). Diastolic blood pressure (DBP) was higher for HP animals compared to both NP and GHP (HP 67.3 ± 5.7 mmHg, n = 3; NP 45.4 ± 4.4 mmHg, n = 5; GHP 50.7 ± 3.7 mmHg, n = 7; p < 0.05). Fasting glucose tended to be higher in GHP AGM offspring compared to NP and HP, (GHP 111.6 ± 12.1 mg/dL, n = 7; NP 75.4 ± 14.3 mg/dL, n = 5; HP 75.7 ± 18.5 mg/dL, n = 3; p = 0.07). Water intake was similar between NP and GHP (NP 457.5 ± 43.9 ml/day, n = 4; GHP 425.0 ± 13.1 ml/day; p > 0.05). Urine flow was lower in GHP animals compared to NP (NP 146.3 ± 52.0 ml/day vs GHP 77.5 ± 28.2 ml/day, p < 0.05). Thus, adolescent AGMs born to GH pregnancies may have dysregulation of water balance, favoring water retention. This suggests in utero developmental programming may contribute to increased blood pressures later in life. In conclusion, HTN in AGM pregnancy adversely impacts offspring into early adolescence. These insults include hindered growth and susceptibility to HTN. In pregnancy‐induced hypertension, offspring display altered water homeostasis in early adolescence. Continued studies of these NP, HP and GHP offspring will include long‐term blood pressure and heart rate measurements, regulation of sodium and potassium balance, and the further phenotyping of over 50 additional offspring from 2018 births.Support or Funding InformationAHA 17PRE33670127This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Structured Breeding Groups for the Transmission of Spontaneous Hypertension in African Green Monkeys

The African Green Monkey (AGM; Chlorocebus aethiops sabaeus) is a nonhuman primate native to the Caribbean island nation of Saint Kitts and Nevis with genetic ancestry in West Africa. We have shown that the AGM develops spontaneous hypertension (HTN) with renal glomerular pathologies and left ventricular hypertrophy. The AGM is also a translational animal model of hypertensive pregnancy disorders and the study of APOL1 genetic variation in HTN and end‐stage renal disease. Our St. Kitts colony of AGMs consists of over 230 wild‐caught animals. This study assesses the selective breeding of normotensive and spontaneously hypertensive animals and strategies for success in breeding paradigms for the potential transmission of HTN. Over the course of 3 years, the breeding groups have produced more than 80 purpose‐bred offspring. We tested the hypothesis that larger breeding groups would lead to greater reproductive success in both percent gravid females and number of live births for socially complex AGM groups. Breeding was established with one male and either 15–25 (large group) or 2–9 (small group) females. We assessed pregnancies and live births over one annual breeding season (November – March). Systolic blood pressure (SBP) and heart rates (HR) were measured via doppler stethoscope and forearm plethysmography. Breeding groups had phenotypes of hypertensive (HT; systolic blood pressure > 140 mmHg) or normotensive (NT; systolic blood pressure < 120 mmHg) male and female adults ages 5–15 years. The percentage of pregnant females for small breeding groups (70.5 ± 38.2%) was similar to that of females for large breeding groups (68.7 ± 21.6%, p>0.05). The average number of annual births for small breeding groups (2.8 ± 2.6 births) was fewer than that for large breeding groups (13.2 ± 10.6 births, p<0.05). Furthermore, when a new male was introduced to a breeding group, the percentage of gravid females was unchanged (p > 0.05). Offspring from breeding groups have been phenotyped for blood pressure and heart rate at 1 and 2 years of age (juveniles before sexual maturity). All juveniles were derived either from a NT mother and HT father (NT x HT) or two HT parents (HT x HT). SBP of NT x HT offspring did not change with age (1.2 years = 106.9 ± 3.4 mmHg, n = 19 vs 2.0 years 103.8 ± 6.8 mmHg, n = 4; p > 0.05). SBP of HT x HT offspring also remained unchanged with age (1.2 years 114.6 ± 7.2 mmHg, n = 5 vs 2.2 years 111.0 ± 9.6 mmHg, n = 3; p > 0.05). HR decreased with age in both groups (NT x HT: 1.2 years 182.5 ± 3.7 bpm, 2.0 years 165.5 ± 7.5 bpm; HT x HT: 1.2 years 188.0 ± 8.0 bpm, 2.2 years 159.0 ± 10.5 bpm; p<0.05). Offspring from HT x HT parents tended to have greater blood pressures than that of NT x HT parents. In conclusion, these data suggest that breeding group size does not affect the rate of successful mating and progeny production in AGMs. With limited space, smaller breeding groups are shown to be effective, but larger breeding groups are useful when space is not a factor. Juvenile age between 1 and 2 years does not affect changes in SBP regardless of maternal parent blood pressure phenotype when paternal phenotype is HT. Future studies will further assess the effect of both NT x NT and HT x HT parental phenotype, including genetic assessment on the development of adolescent HTN.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the Klebsiella pneumoniae Proteome

Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A β-lactamase+ invasive strain of Klebsiella pneumoniae that was isolated from a lethal outbreak within laboratory colonies of Chlorocebus aethiops sabaeus monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic. Drug-specific changes to proteomes included proteins for receptor-mediated membrane transport and sugar utilization, central metabolism, and capsule production, whereas mechanisms common to both antibiotics included elevated scavenging of reactive oxygen species and turnover of misfolded proteins. Resistance to combined antibiotics presented integrated adjustments to protein levels as well as unique drug-specific proteomic features. Our results demonstrate that dampening of Klebsiella pneumoniae susceptibility involves global remodeling of the bacterial proteome to counter the effects of antibiotics and stabilize growth.

Should monkeys wash their hands and feet: A pilot-study on sources of zoonotic parasite exposure

Human exposure to zoonotic parasites via contaminated soil is a much studied area. Less research has been performed on exposure via contact with animals and surfaces such as picnic tables with which infected animals might be in contact. On St. Kitts, wild African green monkeys (AGM; Chlorocebus aethiops sabaeus), which are known to have zoonotic parasites, roam freely in areas with outdoor dining facilities and are used in the tourist industry. In this study, the hands and feet of eight AGM and picnic tables where AGM were known to walk on were examined for parasitic organisms. Six of the AGM had parasitic organisms on their hands and/or feet. Trichuris spp. eggs, hookworm larvae and eggs and pinworm eggs were recovered from the tables. The results of this pilot study highlight other potential means of transfer of zoonotic parasites and suggest that surfaces without obvious fecal material can be contaminated with zoonotic parasites.

Abstract 018: High Stillbirth Rate and Altered Maternal Renal Function and Water Balance are Associated With Spontaneous Gestational Hypertension in Nonhuman Primates

Gestational hypertension (GH) is new-onset hypertension specific to pregnancy (PG) that may precede preeclampsia (PE). While the specific etiology of PE is unclear, defective fluid and electrolyte balances have been implicated. The African Green Monkey (AGM; Chlorocebus aethiops sabaeus ) is a novel model of spontaneous GH with pathophysiological consequences of PE. Systolic blood pressure (SBP; forearm plethysmography) was measured in AGMs pre-PG, during each trimester, and postpartum (PP). Gestational age was estimated by fetal lengths at 7 weeks/trimester (21 week gestation). Animals were characterized as normotensive (NT; SBP < 120 mmHg in PG and PP) or gestational hypertensive (GH; SBP ≥ 140 mmHg during PG). SBP was unchanged in NT PG (pre-PG 110 ± 5 mmHg; 1 st trimester 101± 6 mmHg; 3 rd 107 ± 7 mmHg; n = 27). In GH AGMs, SBP increased in the 3 rd trimester from 115 ± 6 mmHg (1 st ) and 117 ± 25 mmHg (2 nd ) to 152 ± 7 mmHg (3 rd ; p<0.05; n = 16). Plasma osmolality did not change during PG for NT AGMs but declined in GH (NT pre-PG 305 ± 5 mOsm/kg; PG 299 ± 5 mOsm/kg; n = 6; GH pre-PG 313 ± 6; PG 293 ± 4 mOsm/kg; p<0.05 ANOVA; n = 15). Maternal body weight increased in GH by the 1 st trimester, while NT body weight did not change until the 3 rd trimester (NT pre-PG 3.8 ± 0.2 kg; 1 st trimester 3.8 ± 0.3 kg; 3 rd 4.4 ± 0.2 kg; GH pre-PG 3.7 ± 0.1 kg; 1 st trimester 4.4 ± 0.2 kg; 3 rd 4.7 ± 0.1 kg; p<0.05). Protein excretion was unchanged in NT AGMs but increased with GH compared to pre-PG and NT PG (NT pre-PG 365 ± 45 mg/day; 3 rd trimester 466 ± 84 mg/day; GH pre-PG 346 ± 138 mg/day; 3 rd trimester 702 ± 69 mg/day; p<0.05). Third trimester estimated GFR (eGFR) did not change in NT AGMs but declined in GH (NT 5.0 ± 0.7 ml/min vs GH 2.5 ± 0.5 ml/min; p<0.05). Infants from GH pregnancies were smaller (NT 327.4 ± 13.7 g, n = 5, vs GH 290.0 ± 8.5 g n = 15; p<0.05) and GH increased fetal stillbirth rate to 40% (6/15). Thus, GH in AGMs has early PG water retention, decreased plasma osmolality and excess weight gain preceding GH. GH AGMs have smaller infants and increased stillbirth rates, showing fetal growth restriction. Elevated SBP during GH may lead to renal dysfunction, proteinuria and reduced eGFR. Thus, the AGM is a novel, translational model of hypertensive pregnancy disorders for the study of acute and long-term effects on fetal development.

Identity Construction and Symbolic Association in Farmer-Vervet Monkey (Chlorocebus aethiops sabaeus) Interconnections in St. Kitts

Primates occupy a liminal space between humans and animals. On the Caribbean island of St. Kitts, translocated vervet monkeys (Chlorocebus aethiops sabaeus) are respected creatures, yet they cause problems. Vervets regularly consume crops on farms and are considered “pests”; still, Kittitians express empathy and understanding for them based largely on the monkeys' display of human-like behaviours. Using data from interviews with 64 Kittitian farmers, we deconstruct the symbolism of the vervet monkey in St. Kitts and analyse how farmers give the monkeys identities that are meaningful only within human social expectations. Our findings reveal that Kittitian farmers consider monkeys to be clever and emotive, displaying complex intentions such as revenge and remorse. Yet, crop-foraging behaviour is a regular and negative experience for the majority of farmers in this study, and the monkeys' presence itself is a constant reminder of the multitude of challenges farmers face in a newly adopted tourism economy that no longer prioritises agriculture. Our results reveal that while vervet crop consumption is a significant problem in St. Kitts, it is the monkeys' boundary-crossing status that drives the growing mentality that “the monkey problem” is completely out of control.

APOL1‐like gene insertion associated with increased blood pressure in the African Green Monkey

End stage renal disease affects 660,000 adults in the United States and disproportionally affects people of African descent. Studies have identified two alleles in the apolipoprotein L1 (APOL1) gene that are expressed in high frequency in African‐ American populations and may contribute to a 4–5 fold increased incidence of end stage renal disease (ESRD) in this demographic. The African Green Monkey (AGM, Chlorocebus aethiops sabaeus) is an Old World monkey species that develops spontaneous hypertension and is indigenous from the regions of Africa from which the two APOL1 alleles are known to exist in humans. We have recently reported that the AGM exhibits renal glomerular and vascular pathologies in conjunction with significant spontaneous hypertension. Furthermore, we report elsewhere that this spontaneous hypertension in the AGM is associated with significant proteinuria even when the animals are prehypertensive; suggesting that altered renal glomerular and/or tubular function in regulating protein excretion may precede the development of spontaneous hypertension in the AGM. This study hypothesized that gene sequence changes in the APOL1‐like gene region may be expressed in the AGM and are associated with increased blood pressure and altered renal function, similar to the human population. DNA was extracted from whole blood or frozen tissue in 44 adult males and the APOL1‐like gene region was sequenced. Bioinformatic analysis identified a 6 nucleotide insertion at base 329 that was positively associated with increased systolic blood pressures (p<0.05). Within the cohort, 2 animals were found to be homozygous for the sequenced insertion (mean ± SE SBP: 206.6±0.1 mmHg), 10 animals were heterozygous (SBP: 136.6±14.6 mmHg), and 32 animals did not have the insertion (SBP: 135.7± 7 mmHg). This data suggests that the insertion in the APOL1‐like gene may affect blood pressure regulation (and/or kidney function) in the AGM, providing a novel and exciting nonhuman primate model to study the progression of ESRD in a non‐human primate species that expresses variations within the APOL1 gene, which is known to correlate with early onset ESRD in African Americans. Ongoing studies are focused on increasing the AGM cohort size, adding normotensive and hypertensive females to the sequenced cohort, and collection of renal functional data on all sequenced animals to determine the effect of the APOL1‐like insertion on kidney function and blood pressure regulation in the AGM.Support or Funding InformationFunding provided by Biomedical Science Research Group, LLC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Infant Birthweight and Postpartum Hypertension in African Green Monkeys

Postpartum hypertension (PPHT) is characterized by significantly elevated arterial pressure developing within 48 hours following delivery. PPHT affects 2% of all pregnancies and the etiology remains unclear. We have previously reported that the African Green Monkey (AGM; Chlorocebus aethiops sabaeus) develops gestational hypertension with pathophysiological features of preeclampsia. In this study, we report characteristics of PPHT in the AGM that occurs in the absence of hypertension before or after the onset of pregnancy. Systolic blood pressures (SBP) were collected using forearm plethysmography under ketamine sedation (15 mg/kg i.m.). Animals were characterized normotensive (NT; SBP < 120 mmHg) and postpartum hypertensive (PPHT; SBP ≥ 140 mmHg developing within 48 hours following delivery). All PPHT were NT pre‐ and during pregnancy. Twenty‐four hour urine was collected in the third trimester and two weeks postpartum for three consecutive days. SBP for nonpregnant, NT was 98.7 ± 3.7 mmHg (n=18), third trimester SBP for NT was 107.0 ± 4.4 mmHg (n=5), and postpartum day 14 (PP14) SBP for NT was 94.0 ± 12.3 mmHg (n=4; p>0.05). Nonpregnant SBP for PPHT was 115.8 ± 17.6 mmHg (n=8), third trimester SBP was 112.1 ± 13.3 mmHg (n=6), and PPHT, PP14 SBP was 137.2 ± 14.3 mmHg (n=6; p <0.05 PP14 vs. third trimester). Nonpregnant plasma osmolality for NT was 306.3 ± 3.6 mOsm/kg (n=13) and third trimester NT plasma osmolality was 298.7 ± 4.6 mOsm/kg (n=7; p>0.05). Third trimester plasma osmolality for PPHT was 288.8 ± 4.6 mOsm/kg (n=6) and 312.5 ± 5.7 mOsm/kg at PP14 (n=6; p<0.05 vs. third trimester). Nonpregnant protein excretion for NT was 472.5 ± 58.0 mg/day (n=13) and 501.8 ± 104.6 mg/day in third trimester (n=5; p>0.05). In PPHT animals, nonpregnant protein excretion was 684.5 ± 152.1 mg/day (n=1), 472.7 ± 62.1 mg/day in third trimester (n=6), and 171.7 ± 68.0 mg/day at PP14 (n=5; p<0.05 PP14 vs. third trimester). Nonpregnant sodium excretion for NT was 2.4 ± 0.4 mmol/day (n=13) and third trimester for NT was 1.8 ± 0.6 mmol/day (n=5; p>0.05). Nonpregnant sodium excretion for PPHT was 0.3 ± 1.4 mmol/day (n=1), third trimester for PPHT was 2.2 ± 0.5 mmol/day (n=6), and PP14 sodium excretion for PPHT was 3.4 ± 0.6 mmol/day (n=5; p >0.05). Babies born to mothers who ultimately developed PPHT were of a higher birthweight than those born to NT mothers (NT 338.2 ± 25.5 g, n=7; PPHT 404.5 ± 14.1 g, n=6; p<0.05). This data demonstrates that postpartum hypertension in the AGM is associated with decreased protein excretion, and increased SBP after delivery when compared to the third trimester of pregnancy. The mechanisms of this PPHT are largely unknown. Ongoing studies will evaluate changes in kidney function, plasma vasopressin concentrations, and plasma renin activity. The AGM has many physiological and behavioral similarities to humans, making the species an optimal model for the study of pregnancy disorders such as PPHT.Support or Funding InformationBiomedical Science Research Group, American Heart Association Predoctoral Fellowship 17PRE33670127This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.