Generation of a familial hypercholesterolemia model in non-human primate.

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder that is associated with a high plasma level of low-density lipoprotein (LDL) cholesterol, leading to an increased risk of cardiovascular diseases. To develop basic and translational research on FH, we here generated an FH model in a non-human primate (cynomolgus monkeys) by deleting the LDL receptor (LDLR) gene using the genome editing technique. Six LDLR knockout (KO) monkeys were produced, all of which were confirmed to have mutations in the LDLR gene by sequence analysis. The levels of plasma cholesterol and triglyceride were quite high in the monkeys, and were similar to those in FH patients with homozygous mutations in the LDLR gene. In addition, periocular xanthoma was observed only 1 year after birth. Lipoprotein profile analysis showed that the plasma very low-density lipoprotein and LDL were elevated, while the plasma high density lipoprotein was decreased in LDLR KO monkeys. The LDLR KO monkeys were also strongly resistant to medications for hypercholesterolemia. Taken together, we successfully generated a non-human primate model of hypercholesterolemia in which the phenotype is similar to that of homozygous FH patients.

Duration of Vascular Endothelial Growth Factor Suppression After Intravitreal Injection of Brolucizumab and Aflibercept in Macaque Eyes.

Purpose: To compare the duration of vascular endothelial growth factor (VEGF) suppression in the aqueous humor of macaque eyes after intravitreal injection of brolucizumab and aflibercept. Methods: Clinical dose of intravitreal brolucizumab (IVBr; 6.0 mg/50 µL) or intravitreal aflibercept (IVA; 2 mg/50 µL) was injected into the right eye of each of 8 macaques. Aqueous humor samples (150 µL) from both eyes were obtained just before injection and on days 1, 3, 7, 14, 21, 28, 42, 56, 84, and 112 after IVBr injection or IVA injection. VEGF concentrations were measured using enzyme-linked immunosorbent assays. Results: In the injected eyes, the mean VEGF suppression durations (range) were 4.9 (3-8) weeks for IVBr injection and 6.8 (6-8) weeks for IVA injection (P = 0.04). The VEGF concentrations returned to the preinjection level in the aqueous humor at 12 weeks both after IVBr and IVA injection. In the noninjected fellow eyes, the aqueous VEGF concentrations had decreased least at 1 day after IVBr injection and at 3 days after IVA injection, but were still detectable. The VEGF concentrations in the fellow eyes returned to the preinjection level in the aqueous humor at 1 week after IVBr injection and at 2 weeks after IVA injection. Conclusions: The duration of VEGF suppression in the aqueous humor after IVBr injection may be shorter than that after IVA injection, which may be related with clinical usage.

A new primate model of hypophyseal dysfunction.

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

Pharmacokinetics of Intravitreal Vancomycin and Ceftazidime in Silicone Oil-Filled Macaque Eyes.

Purpose: This study evaluated the pharmacokinetics of intravitreal vancomycin and ceftazidime in the aqueous humor of macaque eyes filled with silicone oil in the vitreous cavity. Methods: Intravitreal vancomycin (1 mg/0.1 mL) and ceftazidime (2 mg/0.1 mL) were injected into four normal macaque eyes, four vitrectomized aphakic macaque eyes, and four previously vitrectomized aphakic macaque eyes filled with silicone oil (silicone oil-filled eyes). Aqueous humor samples (0.1 mL) were obtained just before injection and at 2 and 5 hours and 1, 2, 3, 5, 7, and 10 days after injection. In each group, corneal endothelial cell density (ECD) measurements and electroretinogram (ERG) recordings were obtained before injection and after 1 month. Results: The half-lives of vancomycin in the aqueous humor of normal, vitrectomized, and silicone oil-filled eyes were 29.4, 21.1, and 6.8 hours, respectively, and those of ceftazidime were 20.4, 5.2, and 3.1 hours, respectively. The maximum vancomycin aqueous humor concentrations of normal, vitrectomized, and silicone oil-filled eyes were 151.4, 205.6, and 543.5 µg/mL, respectively, and the maximum ceftazidime aqueous humor concentrations are 64.6, 260.0, and 1176.3 µg/mL, respectively. There was no change in ECD, and ERG was not declined after intravitreal injection in all groups. Conclusions: The half-lives of vancomycin and ceftazidime in the aqueous humor were shorter in silicone oil-filled eyes than in normal and vitrectomized eyes. High antibiotic concentrations in silicone oil-filled eyes seemed to be well tolerated. Translational Relevance: This study aids in estimating how often an antibiotic should be intravitreally injected for endophthalmitis of silicone oil-filled eyes.

First Successful Delivery after Uterus Transplantation in MHC-Defined Cynomolgus Macaques.

Delivery following uterus transplantation (UTx)-an approach for treating uterine factor infertility-has not been reported in nonhuman primate models. Here, six female major histocompatibility complex (MHC)-defined cynomolgus macaques that underwent allogeneic UTx were evaluated. Antithymocyte globulin and rituximab were administered to induce immunosuppression and a triple maintenance regimen was used. Menstruation resumed in all animals with long-term survival, except one, which was euthanized due to infusion associated adverse reaction to antithymocyte globulin. Donor-specific antibodies (DSA) were detected in cases 2, 4, and 5, while humoral rejection occurred in cases 4 and 5. Post-transplant lymphoproliferative disorder (PTLD) developed in cases 2 and 3. Pregnancy was attempted in cases 1, 2, and 3 but was achieved only in case 2, which had haploidentical donor and recipient MHCs. Pregnancy was achieved in case 2 after recovery from graft rejection coincident with DSA and PTLD. A cesarean section was performed at full-term. This is the first report of a successful livebirth following allogeneic UTx in nonhuman primates, although the delivery was achieved via UTx between a pair carrying haploidentical MHCs. Experimental data from nonhuman primates may provide important scientific knowledge needed to resolve unsolved clinical issues in UTx.

Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments.

Long-Term Outcome and Rejection After Allogeneic Uterus Transplantation in Cynomolgus Macaques.

Uterus transplantation (UTx) is an option for women with uterine factor infertility to have a child, but is still in the experimental stage. Therefore, allogeneic animal models of UTx are required for resolution of clinical issues. In this study, long-term outcomes were evaluated in four recipients (cases 1-4) after allogeneic UTx in cynomolgus macaques. Immunosuppression with antithymocyte globulin induction and a triple maintenance regimen was used. Postoperative ultrasonography and biopsy of the transplanted uterus and immunoserological examinations were performed. All four recipients survived for >3 months after surgery, but continuous menstruation did not resume, although temporary menstruation occurred (cases 1 and 2). All animals were euthanized due to irreversible rejection and no uterine blood flow (cases 1, 2 and 4) and post-transplant lymphoproliferative disorder (case 3). Donor-specific antibodies against MHC class I and II were detected in cases 1, 2 and 4, but not in case 3. Peripheral lymphocyte counts tended to elevate for CD3+, CD20+ and NK cells in conjunction with uterine rejection, and all animals had elevated stimulation indexes of mixed lymphocyte reaction after surgery. Establishment of allogeneic UTx in cynomolgus macaque requires further exploration of immunosuppression, but the clinicopathological features of uterine rejection are useful for development of human UTx.

Paromomycin sulfate is an effective treatment for balantidiasis in captive cynomolgus monkeys.

There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.

Evaluation of allowable time and histopathological changes in warm ischemia of the uterus in cynomolgus monkey as a model for uterus transplantation.

INTRODUCTION: The objective of this study was to examine the allowable warm ischemic time and pathological changes due to ischemia and reperfusion injury in the uterus of the cynomolgus monkey as a model for uterus transplantation. MATERIAL AND METHODS: Six female cynomolgus monkeys were used in the study. The uterus was resected from the vaginal canal and connected through the bilateral ovarian and uterine arteries and veins only. One animal was used as a control. In the other five animals, the bilateral uterine and ovarian vessels were clamped for 0.5, 1, 2, 4 and 8 h, respectively. Biopsy of the smooth muscle tissue of corpus uteri was performed after each ischemic time and after subsequent reperfusion for 3 h. Biopsy samples were observed by light and electron microscopy. Menstruation recovery was monitored. RESULTS: There were no particular findings in both light and electron microscopy after ischemia for up to 2 h and after subsequent reperfusion. There were no marked changes after ischemia for 4 h, but dilated nuclear pores and rough endoplasmic reticulum swelling were found after reperfusion. These changes also occurred, along with mitochondrial swelling and cristae loss after ischemia for 8 h, and plasma membrane loss, nuclear fragmentation and chromatin condensation were found after reperfusion. Periodical menstruation restarted in all animals with ischemia up to 4 h, but the animal with ischemia for 8 h had amenorrhea and uterine atrophy. CONCLUSIONS: The uterus of the cynomolgus monkey tolerates warm ischemia for up to 4 h.