Immunosuppression in nonhuman primates

Nonhuman primate organ transplant models have been and continue to be an excellent conduit for the development of immunosuppressive therapies for use in human organ transplantation. These experimental therapies often make use of immunosuppressive drugs currently used in clinical transplantation and combine them in new and exciting ways with cutting-edge reagents and strategies. This review summarizes our own research experience and the experience of other leaders in the field using a variety of immunosuppressants in nonhuman primate transplant models. Information on drug dosing and safety is included.     

Strange osteosclerosis at femoral neck.

A 4 (1/2) yr follow-up of bone mineral density of a young woman revealed a dramatic 84% increase at the femoral neck, whereas the increase at the trochanter was only 11%. This finding suggested a very substantial accumulation of bone mineral in the neck region only. In the immediate repeated DXA scan, however, all the signs of this osteosclerosis disappeared. The primary reason for this strange DXA finding was a discrete contact failure in the control electronics of the scanner that confused the analysis of the scan data. The standard quality control was unable to detect this malfunction.     

Small intestinal transplantation in nonhuman primates

Small intestinal transplantation represents a potentially therapeutic procedure for individuals with short gut syndrome. The purpose of this study was to develop a model for small intestinal transplantation in primates that is: technically feasible without microsurgery; consistent in the prevention of allograft rejection; functional in terms of nutrient absorption; and compatible with harvest for multiple organ procurement. First, autotransplantations on four rhesus monkeys were performed in order to study a variety of harvesting techniques and vascular anastomoses. Then, a study was performed with 14 heterotopic allotransplants in 4 baboons and 10 rhesus primates. The successful donor model consisted of division of the pancreas, harvesting the small bowel with a superior mesenteric artery and portal vein pedicle. The allograft vascular pedicle was anastomosed to the recipient's common iliac vessels in end-to-side fashion. The graft was transplanted as an out-of-continuity loop, both ends being exteriorized as stomas providing access for absorption studies and biopsy. Three immunosuppressive regimens were tested: (1) cyclosporine A (CyA) 20 mg/kg/d, solumedrol (SML) 2 mg/kg/d, and graft irradiation (150 rad) (n = 4); (2) CyA 20 mg/kg/d and SML 2 mg/kg/d (n = 3); and (3) CyA 40 mg/kg/d, SML 2 mg/kg/d, and azathioprine 5 mg/kg/d (n = 3). There were 4 deaths due to technical error in the first 24 hours. Weekly graft biopsy, serum CyA levels, complete blood count, and automated 24-channel serum analysis were performed. Grafts surviving greater than 14 days underwent absorption study via luminal perfusion with sucrose, maltose, dextrose, Pregestimil, xylose, and cyclosporine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bone induction in nonhuman primates. An experimental study on the baboon.

Development leading to local bone differentiation in response to intramuscular and subcutaneous implantation of demineralized bone matrix has been predominantly investigated in rodents. To investigate bone differentiation by induction in primates, diaphyseal bone was harvested from ten male baboons and chemosterilized to obtain autolyzed, antigen-extracted allogeneic (AAA) bone containing the bone morphogenetic protein essential for osteoinduction. A total of 96 AAA bone diaphyseal cylinders were implanted intramuscularly in 24 adult male baboons and harvested at three, six, and nine months. Histologic analysis showed that the matrix had undergone considerable resorption, particularly at six and nine months. Seventy-three implants showed variable amounts of newly formed bone at the internal and external surfaces of the chemosterilized matrix. Numerous specimens showed florid bone formation, and newly formed woven bone persisted in association with the matrix for as long as nine months. Coating the AAA bone matrix with an allogeneic fibrin-fibronectin protein concentrate prepared from fresh-frozen baboon plasma did not significantly increase the amount of induced bone. Bone formation was confirmed by intravital double tetracycline labeling of the mineralization fronts. The unequivocal demonstration of bone formation by induction in a large series of adult nonhuman primates provides evidence that long-lived higher vertebrates retain the bone-inductive proteins in the extracellular matrix of bone and the crucial set of responding mesenchymal cells capable of transformation and differentiation into osteoblastic cell lines.     

Strategies for tolerance induction in nonhuman primates

Recent advances in the field of reconstructive surgery and immunology resulted in increased interest in composite tissue allograft (CTA) transplantation. Up to date, more than 50 CTA transplants have been reported in humans. A significant number of experimental studies on CTA transplants under different protocols of tolerance-inducting strategies have been reported in small-animal models. There is however, a limited number of CTA transplants performed in nonhuman primates. To reach the ultimate clinical success in CTA transplantation, more experimental studies on tolerance induction in nonhuman primates are needed to apply these immunomodulatory protocols to CTA transplants in humans. In this review, strategies for tolerance induction in the nonhuman primate model in solid organ and CTA transplants are presented in 3 major categories: chimerism induction, T-cell depletion, and costimulatory receptor blockade.     

Metastable tolerance in nonhuman primates and humans

It is clear that both humans and nonhuman primates can do without immunosuppression for long periods of time before rejecting their allogeneic organ transplants. Immune cell depletion, particularly lymphocyte depletion, is an effective clinical strategy for achieving a tolerant state. Based on nonhuman primate and human studies, evidence suggests that metastable tolerance develops over time in a donor-specific manner and is mediated at least in part by donor antigen-specific regulatory T cells. Suppression of effector T cells by CD8+ and CD4+ T-regulatory cells is mediated by transforming growth factor (TGF)-beta or interleukin 10, and the presence of CD4+ TGF beta(latent) T-cell infiltrates may be a useful diagnostic marker for metastable tolerance. Loss of these cells has been shown to correlate with loss of tolerance. Future efforts to withdraw immunosuppressive drugs and establish a tolerant state will depend importantly on development of such diagnostic immunologic monitoring tools. Some of the reasons that tolerance remains such a challenging goal in clinical transplantation are discussed herein.     

Experimental prostatitis in nonhuman primates: II. Ascending acute prostatitis

Bacterial prostatitis is a common cause of urinary tract infection in males, but little is known of its pathophysiology. To study this, we developed a nonhuman primate model using a wild-type clinical isolate of Escherichia coli. Primates have a prostatic anatomy that is similar to humans, which makes them ideal as an animal model of this disease. The monkeys had a urethral inoculation of this organism and were then followed with urine, blood, and semen cultures, white blood counts, and renal scans. They were sacrificed at from 10 days to 4 weeks, and their genitourinary tracts histologically examined. The prostatitis paralleled that reported in humans, and we conclude that the infection occurs by the ascending route. The organisms causing the infection in man do so in our primate model, and the histologic change is also the same. Thus, the primate model holds promise for studies to help us understand this disease.     

Experimental prostatitis in nonhuman primates: I. Bacterial adherence in the urethra

Both man and monkey possess urothelial (transitional cell) receptors for P-fimbriae of Escherichia coli; however, the male urethra has pseudostratified columnar cells. We studied adherence using scanning electron microscopy and found that P-fimbriae were the principal mediators of adherence to these cells as well. The monkey, therefore, should be a good model for the study of the ascending route of infection in prostatitis, the route thought to occur in man.     

Eight years experience with Permalens intracorneal lenses in nonhuman primates.

BACKGROUND: For the past 8 years, three independent laboratories have been researching the biocompatibility and performance of Permalens intracorneal lens implants in the corneas of nonhuman primates. Both myopic and hyperopic corrections have been achieved. This article describes the evolution of the intracorneal lens design and manufacturing process. METHODS: During this time period, 63 surgeries were performed on various species of nonhuman primates. Follow-up examination extended between 30 months and 8.2 years. Objective measures of refractive performance, as well as biocompatibility were made using slit lamp, retinoscopy, autorefractor, specular microscope, etc. Additionally, histopathology was performed on many of the specimens, both acute and chronic. RESULTS: Surgically successful implants were achieved in between 60% and 100% of eyes in the various series of lens implants outlined in the article. Levels of contamination in the preparation of hydrogels were felt to be responsible for many of the surgical failures. The removal of silicone and other contaminants seems to have significantly improved the biocompatibility of these materials within the cornea. The major histopathological finding was that there appeared to be some epithelial thinning over the implants, but in general excellent biocompatibility was obtained over the 8-year period outlined in this paper. CONCLUSIONS: Although extensive studies of biocompatibility have been completed, the future of the performance of these materials remains to be proven in the human subject. Additionally, empirical relationships between lens implant power and refractive results will have to be determined in humans, prior to their general clinical usage.     

Surgical complications in kidney transplantation in nonhuman primates

Surgical complications are important causes of graft loss in the nonhuman primate kidney transplantation model. We reviewed the incidence and intervention methods in 182 kidney transplantations performed in our lab recently 2 years in Cynomolgus monkeys. There were six renal artery thromboses (3.3%), eight urine leakages (4.4%), and five ureteral stenoses (2.7%). All renal artery thrombosis cases were found within 3 days after surgery. Urine leakage appeared from the 5th to 12th day after surgery and all cases were caused by ureter rupture. Reexploration was performed in five cases to reanastomose ureter with stent. Four cases reached long‐term survival. The rest one died of graft rejection. Ureteral stenoses were found in long‐term survival cases. Ureter reanastomoses with stent were performed in two cases. The postoperative renal functions of these two monkeys recovered to normal and they survived until study termination. From this large number of study, our experience indicated that kidney transplantation in the nonhuman primate is a safe procedure with low complications. Reexploration is recommended for salvage of the graft with urine leakage and ureteral stenosis. © 2010 Wiley‐Liss, Inc. Microsurgery, 2010.     

Bone remodeling at the endocortical surface of the human femoral neck: a mechanism for regional cortical thinning in cases of hip fracture.

Endocortical remodeling and wall thickness (W.Th.) were measured in femoral neck bone from 12 female fracture cases (81.3 +/- 1.5 years) and 12 sex-matched controls (81.9 +/- 1.9 years). Regionally, osteoid and eroded surface were increased, whereas W.Th. was reduced. These processes likely contribute to cortical bone loss seen in hip fracture. INTRODUCTION: Because periosteal expression of alkaline phosphatase was similar between cases and controls, we hypothesized that the mechanism causing the marked femoral neck cortical thinning associated with hip fracture may be net endocortical bone loss. METHODS: Twelve female cases of femoral neck fracture (mean age = 81.3 +/- 1.5 years) and 12 age- and sex-matched postmortem controls (mean age = 81.9 +/- 1.9 years) were included in the study. Samples of their femoral neck bone were embedded in methyl methacrylate, sectioned at 10 microm, and stained with Solochrome cyanine R and Goldner's trichrome for the detection of osteoid (%OS/BS) and resorption surfaces (%ES/BS) respectively. In addition, wall thickness (W.Th.) and lamellar thickness (Lm.Th.) data were also collected from identifiable endocortical bone packets as a measure of formative potential. RESULTS AND CONCLUSIONS: %OS/BS was significantly elevated in the anterior (control = 3.4 +/- 0.7: fracture = 11.0 +/- 2.3; p = 0.0001), inferior (3.4 +/- 1.0: 9.9 +/- 3.0; p = 0.0009), and posterior quadrants (3.2 +/- 0.8: 9.1 +/- 2.3; p = 0.0021). Only for anterior region was increased %ES/BS demonstrated in the fracture group (2.8 +/- 0.6: 5.3 +/- 0.7; p = 0.055). W.Th. (mm) was reduced only in the inferior region of the fracture cases (control = 33.7 +/- 1.2: fracture = 30.6 +/- 0.9; p = 0.013), whereas Lm.Th. was also reduced inferiorly (control = 2.7 +/- 0.08: fracture = 2.5 +/- 0.08; p = 0.042). These data suggest that an endocortical remodeling imbalance involving reduced bone formation within inferior region coupled with elevated anterior resorption may make an important contribution to the cortical thinning observed in cases of femoral neck fracture.     

Osteochondroma of the femoral neck.

Osteochondromas of the femoral neck represent intraarticular lesions and are difficult to access for surgical resection, especially when located posteriorly. A versatile surgical approach with dislocation of the femoral head is described through which reliable resection of the osteochondroma can be done respecting the crucial blood supply to the femoral head, the deep branch of the medial femoral circumflex artery. Surgical femoral head dislocation offers the possibility of excellent visualization, circumferential access to the femoral neck, and complete intraarticular inspection. This approach has been used in four patients with osteochondroma of the femoral neck who presented with pain, restricted range of motion, and a limp. Femoroacetabular impingement of the bulky osteochondroma against the acetabular rim could be verified in all patients. In two patients labral lesions were found at the impingement site. All patients had prompt bleeding intraoperatively from a 2.0-mm drill hole of the femoral head after resection of the osteochondroma. There were no or minimal symptoms after a median followup of 34 months (range, 18-48 months) and no clinical or radiographic signs of avascular necrosis of the head.     

Correlates to traumatic brain injury in nonhuman primates

BACKGROUND: Traumatic brain injury (TBI) is a major health problem, both in terms of the economic cost to society and the survivor's quality of life. The development of devices to protect against TBI requires criteria that relate observed injury to measurements of head kinematics. The objective of this study is to find the best statistical correlates to impact-induced TBI in nonhuman primates using a qualified, self-consistent set of historical kinematic and TBI data from impact tests on nonhuman primates. METHODS: A database was constructed and qualified from historical head impact tests on nonhuman primates. Multivariate logistic regression analysis with backwards stepwise elimination was performed. Variables considered are the peak rotational acceleration (Omegamax), the peak linear acceleration (Amax), and the number of impacts (N). RESULTS: Bivariate combinations of angular acceleration and the number of impacts are the best correlates to all modes of TBI considered, i.e., concussion, subarachnoid hemorrhage, brain contusion, and subdural hematoma. For a nonhuman primate with 100-g brain mass, the criteria that the probability of TBI is less than 10% by injury mode are:Concussion: OmegamaxN(0.84) < 70 krad/s/s SAH: OmegamaxN(0.70) < 160 krad/s/s Contusion: Omegamax N(0.35) < 160 krad/s/s SDH: Omegamax N(0.60) < 280 krad/s/s CONCLUSIONS: Based on this dataset, the best statistically based risk factor for all modes of TBI in nonhuman primates is the bivariate combination of rotational acceleration and number of impacts.     

Mapping quantitative trait loci for cross-sectional geometry at the femoral neck.

A genome-wide linkage scan was performed in a sample of 79 multiplex pedigrees to identify genomic regions linked to femoral neck cross-sectional geometry. Potential quantitative trait loci were detected at several genomic regions, such as 10q26, 20p12-q12, and chromosome X. INTRODUCTION: Bone geometry is an important determinant of bone strength and osteoporotic fractures. Previous studies have shown that femoral neck cross-sectional geometric variables are under genetic controls. To identify genetic loci underlying variation in femoral neck cross-sectional geometry, we conducted a whole genome linkage scan for four femoral neck cross-sectional geometric variables in 79 multiplex white pedigrees. MATERIALS AND METHODS: A total of 1816 subjects from 79 pedigrees were genotyped with 451 microsatellite markers across the human genome. We performed linkage analyses on the entire data, as well as on men and women separately. RESULTS: Significant linkage evidence was identified at 10q26 for buckling ratio (LOD = 3.27) and Xp11 (LOD = 3.45) for cortical thickness. Chromosome region 20p12-q12 showed suggestive linkage with cross-sectional area (LOD = 2.33), cortical thickness (LOD = 2.09), and buckling ratio (LOD = 1.94). Sex-specific linkage analyses further supported the importance of 20p12-q12 for cortical thickness (LOD = 2.74 in females and LOD = 1.88 in males) and buckling ratio (LOD = 5.00 in females and LOD = 3.18 in males). CONCLUSIONS: This study is the first genome-wide linkage scan searching for quantitative trait loci underlying femoral neck cross-sectional geometry in humans. The identification of the genes responsible for bone geometric variation will improve our knowledge of bone strength and aid in development of diagnostic approaches and interventions for osteoporotic fractures.     

Alendronate treatment results in similar levels of trabecular bone remodeling in the femoral neck and vertebra

Summary  We aimed to determine whether trabecular bone in sites that have different surface-based remodeling rates, the femoral neck and vertebra, are differently affected by alendronate treatment. Alendronate treatment resulted in similar levels of turnover in both sites, suggesting that a lower limit of bone turnover suppression with alendronate may exist. Introduction  Bone turnover suppression in sites that already have a low surface-based remodeling rate may lead to oversuppression that could have negative effects on the biomechanical properties of bone. The goal was to determine how alendronate suppresses bone turnover at sites with different surface-based remodeling rates. Methods  Dynamic histomorphometric parameters were assessed in trabecular bone of the femoral neck and lumbar vertebrae obtained from skeletally mature beagles treated with saline (1 ml/kg/day) or alendronate (ALN 0.2 or 1.0 mg/kg/day). The ALN0.2 and ALN1.0 doses approximate, on a milligram per kilogram basis, the clinical doses used for the treatment of postmenopausal osteoporosis and Paget’s disease, respectively. Results  Alendronate treatment resulted in similar absolute levels of bone turnover in the femoral neck and vertebrae, although the femoral neck had 33% lower pre-treatment surface-based remodeling rate than the vertebra (p < 0.05). Additionally, the high dose of alendronate (ALN 1.0) suppressed bone turnover to similar absolute levels as the low dose of alendronate (ALN 0.2) in both sites. Conclusions  Alendronate treatment may result in a lower limit of trabecular bone turnover suppression, suggesting that sites of low pre-treatment remodeling rate are not more susceptible to oversuppression than those of high pre-treatment remodeling rate.     

Preliminary results of fetal cardiac bypass in nonhuman primates

OBJECTIVE: Fetal cardiac surgery has potential benefits for treatment of some congenital heart defects. However, placental dysfunction as a result of fetal bypass, fetal stress, and fetal exposure to external milieu needs to be overcome to optimize the outcomes of fetal cardiac bypass. In this study we evaluated the technical feasibility of cardiac bypass in the nonhuman primate fetus and the efficacy of different anesthetic approaches. METHODS: Twelve baboon fetuses, average gestation 146 +/- 8 days and weight 696 +/- 184 g, were used. Three fetuses were excluded from the study because of nuchal cord presentations. The animals were separated into two anesthesia groups: isoflurane (n = 6) and fentanyl and midazolam (n = 3). A miniature roller pump circuit without oxygenator was used for fetal bypass for 30 minutes. No blood transfusion was performed. Fetal blood gas samples were collected before bypass, during bypass, and at 15 and 60 minutes after bypass. RESULTS: All fetuses in the isoflurane group were successfully placed on the cardiac bypass circuit. However, 2 animals in the fentanyl and midazolam group were not placed on the bypass circuit because of sustained elevation in maternal uterine tone. All maternal baboons survived. Of the 6 fetuses in the isoflurane group, 5 survived for 60 minutes; however, placental function continued to deteriorate after bypass (Pa o 2 33 +/- 3 mm Hg before bypass, 23 +/- 6 mm Hg 15 minutes after, and 18 +/- 9 mm Hg 60 minutes after). CONCLUSION: The technical feasibility of cardiac bypass in nonhuman primate fetuses weighing less than 1000 g was confirmed. Isoflurane anesthesia appears to be superior to fentanyl and midazolam anesthesia for fetal cardiac surgery because of adequate uterine relaxation.