High-Resolution Quantitative Mapping of Macaque Cervicovaginal Epithelial Thickness: Implications for Mucosal Vaccine Delivery.

Vaginal mucosal surfaces naturally offer some protection against sexually transmitted infections (STIs) including Human Immunodeficiency Virus-1, however topical preventative medications or vaccine designed to boost local immune responses can further enhance this protection. We previously developed a novel mucosal vaccine strategy using viral vectors integrated into mouse dermal epithelium to induce virus-specific humoral and cellular immune responses at the site of exposure. Since vaccine integration occurs at the site of cell replication (basal layer 100-400 micrometers below the surface), temporal epithelial thinning during vaccine application, confirmed with high resolution imaging, is desirable. In this study, strategies for vaginal mucosal thinning were evaluated noninvasively using optical coherence tomography (OCT) to map reproductive tract epithelial thickness (ET) in macaques to optimize basal layer access in preparation for future effective intravaginal mucosal vaccination studies. Twelve adolescent female rhesus macaques (5-7kg) were randomly assigned to interventions to induce vaginal mucosal thinning, including cytobrush mechanical abrasion, the chemical surfactant spermicide nonoxynol-9 (N9), the hormonal contraceptive depomedroxyprogesterone acetate (DMPA), or no intervention. Macaques were evaluated at baseline and after interventions using colposcopy, vaginal biopsies, and OCT imaging, which allowed for real-time in vivo visualization and measurement of ET of the mid-vagina, fornices, and cervix. P value ≤0.05 was considered significant. Colposcopy findings included pink, rugated tissue with variable degrees of white-tipped, thickened epithelium. Baseline ET of the fornices was thinner than the cervix and vagina (p<0.05), and mensing macaques had thinner ET at all sites (p<0.001). ET was decreased 1 month after DMPA (p<0.05) in all sites, immediately after mechanical abrasion (p<0.05) in the fornix and cervix, and after two doses of 4% N9 (1.25ml) applied over 14 hrs in the fornix only (p<0.001). Histological assessment of biopsied samples confirmed OCT findings. In summary, OCT imaging allowed for real time assessment of macaque vaginal ET. While varying degrees of thinning were observed after the interventions, limitations with each were noted. ET decreased naturally during menses, which may provide an ideal opportunity for accessing the targeted vaginal mucosal basal layers to achieve the optimum epithelial thickness for intravaginal mucosal vaccination.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Research methodology for in vivo measurements of resting energy expenditure, daily body temperature, metabolic heat and non-viral tissue-specific gene therapy in baboons.

A large number of studies have shown that the baboon is one of the most commonly used non-human primate (NHP) research model for the study of immunometabolic complex traits such as type 2 diabetes (T2D), insulin resistance (IR), adipose tissue dysfunction (ATD), dyslipidemia, obesity (OB) and cardiovascular disease (CVD). This paper reports on innovative technologies and advanced research strategies for energetics and translational medicine with this NHP model. This includes the following: measuring resting energy expenditure (REE) with the mobile indirect calorimeter Breezing®; monitoring daily body temperature using subcutaneously implanted data loggers; quantifying metabolic heat with veterinary infrared thermography (IRT) imaging, and non-viral non-invasive, tissue-specific ultrasound-targeted microbubble destruction (UTMD) gene-based therapy. These methods are of broad utility; for example, they may facilitate the engineering of ectopic overexpression of brown adipose tissue (BAT) mUCP-1 via UTMD-gene therapy into baboon SKM to achieve weight loss, hypophagia and immunometabolic improvement. These methods will be valuable to basic and translational research, and human clinical trials, in the areas of metabolism, cardiovascular health, and immunometabolic and infectious diseases.

Indices of muscle and liver dysfunction after surviving hemorrhage and prolonged hypotension.

BACKGROUND: This study determined the long-term effects of prolonged hypotension (PH) on liver, muscle, and kidney dysfunction. The hypothesis was that longer duration of PH after hemorrhage will result in greater organ dysfunction. METHODS: Baboons were sedated and hemorrhaged (30% blood volume). Systolic blood pressure greater than 80 mm Hg was maintained for 1 hour (1 hr-PH; n = 5), 2 hours (2 hr-PH; n = 5), or 3 hours (3 hr-PH; n = 5). After PH, hemorrhage volume was replaced. Animals were recovered and monitored for 21 days. Control animals were hemorrhaged and immediately resuscitated (0 hr-PH, n = 3). Data are Mean ± Standard Deviation, and analyzed by 2-way repeated measures ANOVA and Holm-Sidak test. RESULTS: Hemorrhage resulted in mild hypotension. Minimal resuscitation was required during the hypotensive phase, and survival rate was 100%. Significant increases (p < 0.001) in alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase, and lactate dehydrogenase occurred on Day 1 after PH, and were significantly greater (p < 0.001) in the 2 hr- and 3 hr-PH groups than the 0 hr-PH group. Maximum alanine aminotransferase levels (U/L) were 140 ± 56 (0 hr-PH), 170 ± 130 (1 hr-PH), 322 ± 241 (2 hr-PH), and 387 ± 167 (3 hr-PH). Maximum aspartate aminotransferase levels (U/L) were 218 ± 44 (0 hr-PH), 354 ± 219 (1 hr-PH), 515 ± 424 (2 hr-PH), and 711 ± 278 (3 hr-PH). Maximum creatine phosphokinase values (U/L) were 7834 ± 3681 (0 hr-PH), 24336 ± 22268 (1 hr-PH), 50494 ± 67653 (2 hr-PH), and 59857 ± 32408 (3 hr-PH). Maximum lactic acid dehydrogenase values (U/L) were 890 ± 396 (0 hr-PH), 2055 ± 1520 (1 hr-PH), 3992 ± 4895 (2 hr-PH), and 4771 ± 1884 (3 hr-PH). Plasma creatinine and blood urea nitrogen were unaffected by PH (p > 0.10). CONCLUSION: These results indicate that PH up to 3 hours in duration results in transient liver and muscle dysfunction that was most severe after 2 hr-PH and 3 hr-PH. Prolonged hypotension produced minimal effects on the kidney. LEVEL OF EVIDENCE: Basic science research, Level of evidence not required for basic science research.

Anti-HIV IgM protects against mucosal SHIV transmission.

OBJECTIVE: Worldwide, most new HIV infections occur through mucosal exposure. Immunoglobulin M (IgM) is the first antibody class generated in response to infectious agents; IgM is present in the systemic circulation and in mucosal fluids as secretory IgM. We sought to investigate for the first time the role of IgM in preventing AIDS virus acquisition in vivo. DESIGN: Recombinant polymeric monoclonal IgM was generated from the neutralizing monoclonal IgG1 antibody 33C6-IgG1, tested in vitro, and given by passive intrarectal immunization to rhesus macaques 30 min before intrarectal challenge with simian-human immunodeficiency virus (SHIV) that carries an HIV-1 envelope gene. RESULTS: In vitro, 33C6-IgM captured virions more efficiently and neutralized the challenge SHIV with a 50% inhibitory molar concentration (IC50) that was 1 log lower than that for 33C6-IgG1. The IgM form also exhibited significantly higher affinity and avidity compared with 33C6-IgG1. After intrarectal administration, 33C6-IgM prevented viremia in four out of six rhesus macaques after high-dose intrarectal SHIV challenge. Five out of six rhesus macaques given 33C6-IgG1 were protected at a five times higher molar concentration compared with the IgM form; all untreated controls became highly viremic. Rhesus macaques passively immunized with 33C6-IgM with breakthrough infection had notably early development of autologous neutralizing antibody responses. CONCLUSION: Our primate model data provide the first proof-of-concept that mucosal IgM can prevent mucosal HIV transmission and have implications for HIV prevention and vaccine development.

Nonhuman primate model in clinical modeling of diseases for stem cell therapy.

Nonhuman primates (NHPs) are alike humans in size, behavior, physiology, biochemistry, and immunology. Given close similarities to humans, the NHP model offers exceptional opportunities to understand the biological mechanisms and translational applications with direct relevance to human conditions. Here, we evaluate the opportunities and limitations of NHPs as animal models for translational regenerative medicine. NHP models of human disease propose exceptional opportunities to advance stem cell-based therapy by addressing pertinent translational concerns related to this research. Nonetheless, the value of these primates must be carefully assessed, taking into account the expense of specialized equipment and requirement of highly specialized staff. Well-designed initial fundamental studies in small animal models are essential before translating research into NHP models and eventually into human trials. In addition, we suggest that applying a directed and collaborative approach, as seen in the evolution of stroke NHP models, will greatly benefit the translation of stem cell therapy in other NHP disease models.

Successful pharmaceutical-grade streptozotocin (STZ)-induced hyperglycemia in a conscious tethered baboon (Papio hamadryas) model.

BACKGROUND: Non-human primate (NHP) diabetic models using chemical ablation of ß-cells with STZ have been achieved by several research groups. Chemotherapeutic STZ could lead to serious adverse events including nephrotoxicity, hepatotoxicity, and mortality. METHODS: We implemented a comprehensive therapeutic strategy that included the tether system, permanent indwelling catheter implants, an aggressive hydration protocol, management for pain with IV nubain and anxiety with IV midazolam, moment-by-moment monitoring of glucose levels post-STZ administration, and continuous intravenous insulin therapy. RESULTS: A triphasic response in blood glucose after STZ administration was fully characterized. A dangerous hypoglycemic phase was also detected in all baboons. Other significant findings were hyperglycemia associated with low levels of plasma leptin, insulin and C-peptide concentrations, hyperglucagonemia, and elevated non-esterified fatty acids (NEFA) concentrations. CONCLUSIONS: We successfully induced frank diabetes by IV administering a single dose of pharmaceutical-grade STZ safely and without adverse events in conscious tethered baboons.

Successful ß cells islet regeneration in streptozotocin-induced diabetic baboons using ultrasound-targeted microbubble gene therapy with cyclinD2/CDK4/GLP1.

Both major forms of diabetes mellitus (DM) involve ß-cell destruction and dysfunction. New treatment strategies have focused on replenishing the deficiency of ß-cell mass common to both major forms of diabetes by islet transplantation or ß-cell regeneration. The pancreas, not the liver, is the ideal organ for islet regeneration, because it is the natural milieu for islets. Since islet mass is known to increase during obesity and pregnancy, the concept of stimulating pancreatic islet regeneration in vivo is both rational and physiologic. This paper proposes a novel approach in which non-viral gene therapy is targeted to pancreatic islets using ultrasound targeted microbubble destruction (UTMD) in a non-human primate model (NHP), the baboon. Treated baboons received a gene cocktail comprised of cyclinD2, CDK, and GLP1, which in rats results in robust and durable islet regeneration with normalization of blood glucose, insulin, and C-peptide levels. We were able to generate important preliminary data indicating that gene therapy by UTMD can achieve in vivo normalization of the intravenous (IV) glucose tolerance test (IVGTT) curves in STZ hyperglycemic-induced conscious tethered baboons. Immunohistochemistry clearly demonstrated evidence of islet regeneration and restoration of ß-cell mass.

Stillbirths in Macaca fascicularis.

BACKGROUND: Stillbirths in non-human primates are a major problem and represent failure of the maternal-fetal-placental unit to maintain normal relationships because of various endogenous, undetermined or environmental factors. METHODS: Records of 236 stillborns and their dams in a Macaca fascicularis colony during a 7-year period were reviewed retrospectively. RESULTS: The 7-year stillbirth incidence was 11.99% (236 stillbirths, 1967 live births). Most (61.02%, n = 144) were of undetermined etiology. Fetal causes included trauma (22.46%, n = 53), fetal pneumonia (0.85%, n = 2) and congenital anomalies (0.42%, n = 1). Maternal causes included dystocia (9.75%, n = 23) and uterine rupture (0.42%, n = 1). Forty-nine placentas were available for histologic evaluation; there was placentitis in five, necrosis in five and placental abruption in two. Most stillbirths occurred close to term. First stillbirths usually occurred in 8- to 12-year-old animals during the first six pregnancies. CONCLUSIONS: Most stillbirths were of undetermined etiology. Fetal trauma was the most common cause.

Nephroblastomatosis and nephroblastoma in nonhuman primates.

Wilms' tumors, or nephroblastomas, are renal embryonal malignancies with a high incidence in humans. Nephroblastomas are uncommon in nonhuman primates. This report describes three cases of spontaneous proliferative renal tumors in young monkeys: two cases of unilateral kidney nephroblastomas in baboons and a nephroblastomatosis in a cynomolgus macaque. Histologically, both baboon tumors were typical of Wilms' tumors found in humans, with proliferative epithelial cells forming tubules and aborted glomeruli, nephrogenic rests and proliferative fibrovascular tissue. The left kidney of the macaque was markedly enlarged and histologically similar to the baboon tumors, although normal kidney architecture was completely effaced by primitive tubules and occasional glomeruli surrounded by edematous stromal tissue. Cytogenetic analysis did not detect any macaque or baboon equivalents to human Wilms' tumor chromosomal abnormalities. By human pathology classification, the diffuse nature of the macaque tumor is more consistent with nephroblastomatosis than nephroblastoma. This differentiation is the first to be reported in a species other than human. The nephroblastomas described here are the first nephroblastomas to be reported in baboons. Our observations indicate that nonhuman primate nephroblastomatosis and nephroblastomas develop in a similar way to Wilms' tumors in humans, although no genetic marker has been associated with nephroblastomas of nonhuman primates thus far.

Ontogeny of hematological cell and biochemical profiles in maternal and fetal baboons (Papio species).

The normal ranges of hematological cell profiles and biochemistry are documented in adult non-pregnant, pregnant, juvenile, and neonatal baboons. Despite the extensive use of the baboon as a model for the study of various aspects of pregnancy, there is no data from paired mothers and their fetuses at different stages of gestation. Hematologic and biochemical profile data were obtained from eight non-pregnant female baboons, 37 mothers and 38 fetal baboons at 30 +/- 2, 90 +/- 2, 125 +/- 2, and 175 +/- 2 days of gestation (mean +/- range; dGA; term, 180 dGA). Changes observed in fetal and maternal blood during normal baboon pregnancy were similar to those reported in human pregnancy. The level of alkaline phosphatase was two times higher in fetal blood circulation than that reported in human pregnancy.

Development of a system for individual feeding of baboons maintained in an outdoor group social environment.

We developed a system that allows individual feeding of adult baboons, 8-15 years of age, maintained in an outdoor group social environment. The purpose of the system is to allow careful monitoring and control of individual diet. Baboons were housed in two group cages, 16 females and a single male in one and 12 females and a single male in the other. Baboons exited the group cage once daily and passed along a chute and over a scale into individual cages where they received their individual diets. Food intake was monitored during their 2-hour stay in the individual cages. Baboons rapidly learned to use this system. Food intake and weight were stable within 20 days. Food consumed decreased during the period of sexual receptivity. The maintenance of the group social environment allowed observations on the group's dominance structure and the relationship of dominance to food consumption. Speed of food access in the group cage was related to dominance. Dominance was not related to food consumed in individual cages. The system permits study of many variables related to behavior and food intake while still retaining critical social interactions.