Clinicopathologic Characteristics,Prevalence, and Risk Factors of Spontaneous Diabetes in Sooty Mangabeys (Cercocebus atys)

In 2008, clinical observations in our colony of sooty mangabeys (Cercocebus atys) suggested a high frequency of type 2 diabetes. Postmortem studies of diabetic animals revealed dense amyloid deposits in pancreatic islets. To investigate these findings, we screened our colony (97 male mangabeys; 99 female mangabeys) for the disease from 2008 to 2012. The overall prevalence of diabetes was 11% and of prediabetes was 7%, which is nearly double that reported for other primate species (less than 6%). Fructosamine and triglyceride levels were the best indicators of diabetes; total cholesterol and glycated hemoglobin were not associated with disease. Increasing age was a significant risk factor: prevalence increased from 0% in infants, juveniles, and young adults to 11% in adults and 19% in geriatric mangabeys. Sex, medroxyprogesterone acetate exposure, and SIV status were unrelated to disease. Weight was marginally higher in prediabetics, but body condition did not indicate obesity. Of the 49 mangabeys that were necropsied after clinical euthanasia or death from natural causes, 22 were diabetic; all 22 animals demonstrated pancreatic amyloid, and most had more than 75% of islets replaced with amyloid. We conclude that type 2 diabetes is more common in mangabeys than in other primate species. Diabetes in mangabeys has some unusual pathologic characteristics, including the absence of altered cholesterol levels and glycated hemoglobin but a robust association of pancreatic insular amyloidosis with clinical diabetes. Future research will examine the genetic basis of mangabey diabetes and evaluate additional diagnostic tools using imaging and serum markers.Abbreviations: HbA1c, glycated hemoglobin; MPA, medroxyprogesterone acetate; YNPRC, Yerkes National Primate Research CenterSooty mangabeys (Cercocebus atys) are Old World NHP that are native to West Africa. Historically their use in research has been limited to infectious disease studies, leprosy studies, and behavioral research.^14,25 Over the past 20 to 30 y, they have been used in HIV–AIDS research. Mangabeys are natural hosts of SIV_smm, which is recognized as the origin of HIV2 infection in humans.^7,8,30,36,42 SIV typically is nonpathogenic in mangabeys despite high levels of virus replication, which makes this species a unique and invaluable model in AIDS research.^7,30,36,42 Our facility maintains a colony of approximately 200 sooty mangabeys. In 2008 clinical observations of relative hyperglycemia, glucosuria, and weight loss in our colony suggested that type 2 diabetes mellitus occurred at a relatively high frequency in this population. Spontaneous diabetes was found in 10% of the colony, and 5% of animals were prediabetic; this incidence is higher than that typically reported for other NHP species, such as cynomolgus macaques (less than 1% to 2%)²² and chimpanzees (less than 1%).³⁷ The prevalence of spontaneous diabetes in humans is typically 8.3%.^2,6,22,37 In addition, necropsies revealed that many affected animals had dense amyloid deposits in pancreatic islet cells. Insular amyloidosis was seen on histology, with a total replacement of islets by amyloid deposition in advanced diabetes. Advanced diabetes was determined by increased weight loss and severity of relative hyperglycemia. The increased clinical prevalence of diabetes in our mangabey colony prompted additional characterization of the clinicopathologic profile, risk factors, and prevalence of diabetes in our mangabey colony.The form of diabetes in this mangabey colony is characterized as type 2 diabetes mellitus, as they have hyperglycemia, hypertriglyceridemia, and islet amyloidosis. Type 2 diabetes mellitus is the most common of the 3 forms of diabetes, and has been documented in humans and NHP,^22,31,37,55 including rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), Celebes crested macaques (Macaca nigra), bonnet macaques (Macaca radiate), pigtailed macaques (Macaca nemestrina), vervet monkeys (Chlorocebus pygerythrus), squirrel monkeys (Saimiri sciureus), chimpanzees (Pan troglodytes), and woolly monkeys (Lagothrix spp.).^{1,24,31,52,55} Type 2 diabetes is a chronic metabolic disorder in which insulin resistance occurs in liver, muscle, and adipose tissue. As type 2 diabetes progresses, it also can be characterized as a relative insulin deficiency.^{1,6,15,22,29,31,37,55} The initial clinical presentation of diabetes in humans and NHP includes polydipsia, polyuria, polyphagia, weight loss, and lethargy.^{1,6,22,27,31,37,55} Similar presentation was observed in our colony of diabetic mangabeys.Diagnostic criteria of diabetes in NHP species is similar to that for humans and is based on clinical symptoms and routine lab tests, including serum chemistry panel to evaluate persistent fasting hyperglycemia, hypertriglyceridemia, and hypercholesterolemia.^{2,6,11,16-18,21,22,29,31,37,48-50,52,55} Hypertriglyceridemia and hypercholesterolemia frequently are elevated due to diabetes and therefore are used as supportive diagnostic markers. In addition, the disease is characterized by transient hyperinsulinemia followed by insulin deficiency subsequent to glucose challenge. Urinalysis is used to evaluate glucosuria and ketonuria. These tests are not exclusive for diagnosing diabetes and can be inconsistent between species, thus making conclusive diagnosis challenging. For example, hyperglycemia can be a transient finding associated with recent food intake or stress associated with restraint for blood sample collection or anesthetic access, whereas hypertriglyceridemia can be seen in obese animals and those with other metabolic diseases such as pancreatitis and hypothyroidism.^1,22,37,55The typical clinical approach to the diagnosis of diabetes in NHP and other veterinary patients includes evaluation of fructosamine and glycated hemoglobin (HbA1c) levels and glucose tolerance testing. These tests are indices of glycemic control and are used in clinical settings primarily to assess prognosis and response to treatment; they are also useful for the initial diagnosis of diabetes when used in parallel with serum chemistry markers. Fructosamine and HbA1c can both provide information on long-term glycemic control, because fructosamine reflects average blood glucose levels over 2 to 3 wk whereas HbA1c reflects average blood glucose over 2 to 3 mo preceding blood collection. HbA1c is the primary test for diabetes in human medicine,^6,31,35,37 whereas fructosamine is commonly used in veterinary medicine. Glucose tolerance testing provides an indirect measure of insulin sensitivity, but it is not frequently used clinically in NHP because of the requirement for prolonged physical restraint or sedation.^{1,21,22,26,27,34,37,55}Prevention and management of diabetes in NHP and humans can be achieved by identifying potential risk factors, including age, weight, sex, genetics, hormone drug exposure, and viral status.^{1,6,15,22,29,31,37,42,55} Advanced age, obesity, sex, and genetics are associated with diabetes in some species of NHP and humans.^{1,6,15,22,29,31,37,55} In addition, exposure to drugs such as medroxyprogesterone acetate (MPA) is suspected to be linked to diabetes due to the hormonal effects of progesterone impacting glucoregulatory function.^{1,6,10,22,23,31,34,55} MPA exposure is of interest, because it is used regularly in our mangabey colony as both a contraceptive and as therapy for endometriosis. In addition, SIV status is being evaluated as a risk factor, because a portion of our colony is SIV positive. Although HIV is not thought to be associated with diabetes in people, SIV pathogenesis in mangabeys differs; therefore it was of interest to explore the possible association of SIV and diabetes in mangabeys.^7,30,36,42 Pancreatic insular amyloidosis has been documented to be associated with type 2 diabetes in several species. Amyloidosis is a group of disorders that are caused by extracellular deposition of misfolded proteins that can result in impaired function of any organ.^{15,20,23,28,32,43,45,48,49} Because a high incidence of pancreatic insular amyloid was noted at necropsy, we sought to document the relationship with clinical diabetes in mangabeys.Spontaneous type 2 diabetes mellitus has been well documented in several species of NHP. Because the literature contains little information regarding the clinicopathologic features (the ‘profile’), risk factors, and prevalence of spontaneous diabetes mellitus in sooty mangabeys, the primary aims of the current study were 1) to determine whether elevated levels of fasting blood glucose, fructosamine, HbA1c, triglycerides, and total cholesterol levels are reliable diagnostic markers of type 2 diabetes mellitus in this NHP species; 2) to determine whether age, sex, MPA exposure, and SIV status influence the risk of diabetes; 3) to determine whether body weight influences diabetic status; 4) to evaluate the relationship between pancreatic amyloidosis and diabetes mellitus; and 5) to characterize the prevalence of diabetes mellitus in the mangabey population at our institution. To our knowledge, this report is the first to describe the natural occurrence of type 2 diabetes mellitus within a captive colony of sooty mangabeys. We hypothesized that blood glucose, fructosamine, HbA1c, triglyceride, and total cholesterol would be reliable diagnostic markers and that age, sex, and MPA exposure would influence the risk of diabetes in this species.     

Retrospective Analysis of the Incidence of Retained Placenta in 3 Large Colonies of NHP

During 1999 through 2014, retained placenta was the most common cause of clinical admission for reproductive complications in breeding colonies of baboons (approximate colony size, 2000 animals), cynomolgus macaques (approximately 1000), and rhesus macaques (approximately 500) at the Southwest National Primate Research Center. Retained placentas occurred in 2.7% of baboons, 3.3% of cynomolgus macaques, and 1.0% of rhesus macaques. Apparent risk factors for retained placenta included stillbirth or abortion and at least one prior cesarean section. There was a significant association between stillbirth and retained placenta in all species. Cesarean sections were performed routinely for baboons to meet research objectives but occurred only as needed for cynomolgus and rhesus macaques. Having had at least one prior cesarean section was an incidence factor for retained placenta in 37.0% of baboons and 4.7% of cynomolgus macaques; none of the rhesus macaques with retained placentas had undergone cesarean section previously. More than 90% of dams with retained placenta returned to a successful reproductive life or assignment to a nonbreeding research protocol. Advances in reproductive management will benefit from prospective studies that capture additional data from all members of a breeding group prior to reproductive complications.Abbreviations: Southwest National Primate Research Center (SNPRC), retained placenta (RP), cesarean section (C-section)Retained placentas and other reproductive problems are unavoidable complications encountered in association with NHP production colonies and are difficult to diagnose, because most births occur at night, when human observation is minimal, and because the dams routinely consume the placenta.^41,44,45 There is extensive information regarding retained placentas in dairy cattle,^7,25,20 and humans,^{3,13,24,35,36,40} but little is documented about this problem''s health hazard and effect on future reproduction in NHP,^16,17,23,49 with 3 documented cases reported as isolated reports between 1991 and 2009.^16,17,23 The first documented case of a retained placenta in a NHP was that of a female chimpanzee (Pan troglodytes).¹⁷ Clinical signs for that case included depression, poor maternal care, and vaginal hemorrhage 1 wk after delivery. The chimpanzee was treated successfully, and the infant was returned to the dam with no further complications. Another documented case involved a golden lion tamarin (Leontopithecus rosalia),¹⁶ which had experienced prolonged labor resulting in a stillbirth. After continued placental retention, the tamarin was hysterectomized. Histopathologic evaluation revealed that placental infiltration into the myometrium was the cause of retention.¹⁶ The third documented case occurred in a bonobo (Pan paniscus) housed in a sanctuary in the Democratic Republic of Congo.²³ The animal was treated with oxytocin injections; after manual removal of the placenta, the dam and infant were reunited, and maternal recovery was uneventful.In addition, in an unpublished case, one of the current authors (TH) treated a mandrill (Mandrillus sphinx) by using oxytocin injection and manual removal of the placenta, after which the dam and infant were reunited, and maternal recovery was uneventful. Several other unpublished cases documented in the medical records of the breeding colonies at the Southwest National Primate Research Center (SNPRC) involved species baboons (Papio spp.), cynomolgus macaques (Macaca fascicularis), and rhesus macaques (Macaca mulatta). Clinical veterinarians had deemed all of these animals to be healthy prior to their placement into breeding groups and were rechecked semiannually concurrent with tuberculin testing. Body condition varied from lean to obese, but body condition score was not recorded. We retrospectively reviewed these records to determine the risk factors and incidence of retained placenta within the 3 NHP colonies over 15 y.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Effects of Maternal and Infant Characteristics on Birth Weight and Gestation Length in a Colony of Rhesus Macaques (Macaca mulatta)

A retrospective study using maternal and birth statistics from an open, captive rhesus macaque colony was done to determine the effects of parity, exposure to simian retrovirus (SRV), housing, maternal parity, and maternal birth weight on infant birth weight, viability and gestation length. Retrospective colony statistics for a 23-y period indicated that birth weight, but not gestation length, differed between genders. Adjusted mean birth weights were higher in nonviable infants. Mothers positive for SRV had shorter gestations, but SRV exposure did not affect neonatal birth weights or viability. Infants born in cages had longer gestations than did those born in pens, but neither birth weight nor viability differed between these groups. Maternal birth weight did not correlate with infant birth weight but positively correlated with gestation length. Parity was correlated with birth weight and decreased viability. Increased parity of the mother was associated with higher birth weight of the infant. A transgenerational trend toward increasing birth weight was noted. The birth statistics of this colony were consistent with those of other macaque colonies. Unlike findings for humans, maternal birth weight had little predictive value for infant outcomes in rhesus macaques. Nonviable rhesus infants had higher birth weights, unlike their human counterparts, perhaps due to gestational diabetes occurring in a sedentary caged population. Similar to the situation for humans, multiparity had a protective effect on infant viability in rhesus macaques.Abbreviations: ANCOVA, analysis of covariance; PRL, Primate Research Laboratory; SRV, simian retrovirusThe rhesus macaque (Macaca mulatta) is a useful animal model for human female reproduction studies because the comparative physiology between the 2 species is nearly identical.^1.5,49 Some factors that affect birth weight and neonatal viability in both humans and macaques include maternal birth weight, maternal age, maternal parity, and the presence of underlying maternal disease. Even experimentally induced simulated human lifestyle factors can affect neonatal outcome.^{10,16,17,25,44}In humans, maternal birth weight correlates with infant birth weight such that low birth weight mothers themselves have low birth weight infants.^8,19,28,30 A similar association has been shown in the macaque.^38,39 Because low birth weight is associated with increased neonatal mortality in humans and in macaques, this correlation, if present, may have important predictive value.^{11,20,21,32,45,47,53} One objective of this study was to establish whether maternal birth weight correlated with neonatal birth weight and viability in this colony of rhesus macaques.The relationship between parity, age, and birth outcomes in humans is controversial because multiparous and grand multiparous women tend to be of lower socioeconomic status, older, and have many confounding lifestyle factors.^2,24,27,56 In macaques, low parity and young age are associated with reproductive failure.⁵⁰ In pigtailed macaques (Macaca nemestrina), increased parity was associated with decreased neonatal viability but increased birth weight. Despite their lower parity, younger mothers in the colony of pigtailed macaques produced lower birth-weight infants, but more viable infants, compared with those of older mothers.¹⁷ The positive correlation between birth weight and viability merits further investigation in rhesus macaques. One objective of the current study was to determine whether maternal parity and age affected birth weight and neonatal viability in our rhesus macaque colony.The lifestyle factors of alcohol consumption, cigarettes, caffeine, drug use, diabetes and exercise have all been shown to influence birth weight and gestation length in humans and macaques.^{4,7,15,22,26,35,40,42,44,51,55} Captive animals can become obese and develop insulin-resistant diabetes, which prolongs gestation and produces oversized infants that are less healthy.^21,46,51 Because exercise is a preventative lifestyle factor for obesity and diabetes, it would be useful to compare active animals with sedentary ones.³⁰ Previous retrospective colony studies in pigtail macaques show that cage type, location, and social housing have significant effects on birth weight and birth outcome.^18,19 Another objective of the current study was to determine whether housing in cages (sedentary animals) or group pens (active animals) influenced gestation length, birth weight, and viability in our rhesus macaques.Another factor in birth outcome is the disease status of the mother. Viral infections, particularly of adenoviruses and immunosuppressive retroviruses, are associated with low birth weight and infant mortality in humans and nonhuman primates.^{13,21,25,33, 34,52,53} A previous report describes maternal transmission of simian retrovirus in a colony of pigtailed macaques with concurrent immunosuppression, low birth weight, and increased infant mortality in viremic mothers.³³ However, some evidence suggests that lentiviral antibodies in amniotic fluid may protect against in utero infection.²³ Further confounding the effects of retroviruses on reproductive outcome, animals infected horizontally can be viremic but serologically negative, and animals with sufficient, detectable immune responses may have provirus latent in their tissues.³³ Because simian retrovirus (SRV) was endemic in the subject rhesus colony and most data were retrospective thus preventing confirmation of viremia, another objective was to determine whether seropositivity of the dam was associated with neonatal viability, gestation length, and infant birth weight.     

Self-Injurious Behavior Secondary to Cytomegalovirus-Induced Neuropathy in an SIV-Infected Rhesus Macaque (Macaca mulatta)

A 3.5-y-old, female rhesus macaque (Macaca mulatta) inoculated with SIVmac239 presented 8 mo later for inappetence and facial bruising. Physical examination revealed a superficial skin abrasion below the left eye, bruising below the left brow, and epistaxis of the left nostril. There were no significant findings on CBC, serum chemistry, urinalysis, or radiographs. Differential diagnoses included infectious etiologies, self-injurious behavior, immune-mediated dermatitis, and neoplasia. Lack of response to antibiotic and analgesic therapy and observations of the macaque made it apparent that the skin lesions were self-inflicted. The excoriations rapidly progressed to extend over the nose, and the left palpebrae became edematous. Euthanasia was elected because the macaque appeared to be experiencing continued discomfort despite analgesic therapy. Histopathologic examination revealed systemic cytomegalovirus (CMV) infection involving the facial nerves, periocular nerves, meninges, and perimesenteric lymph nodes. CMV is a common infection in macaques, with adult seroprevalence close to 100% in most colonies. Infection in immunocompetent animals is usually asymptomatic but can cause significant clinical disease in immunodeficient hosts. CMV is associated with a painful peripheral neuropathy in human AIDS patients, and analgesic treatment is often unsatisfactory. Peripheral neuropathy secondary to CMV should be considered as an underlying cause of self-injurious behavior in SIV-infected macaques. Macaques affected by other diseases and disorders may also be at risk for development of painful peripheral neuropathies.Abbreviations: CMV, cytomegalovirus; HCMV, human CMV; RhCMV, rhesus CMV; SIB, self-injurious behaviorRhesus macaques (Macaca mulatta) are one of the most commonly used NHP species in biomedical research.^9,25 They generally adapt well to captivity, but some develop abnormal behaviors such as stereotypies and self-injurious behavior (SIB).³² Examples of SIB include excessive hair-plucking, head banging, and self-biting that causes wounds in some cases.³² Self-inflicted wounding has been reported in 11% to 14% of individually housed rhesus macaques and can be difficult to manage.^26,32,39 The most commonly discussed risk factors for the development of SIB are related to housing and management, and little is known about potential physiologic causes, such as neuropathic pain.^18,26,32 Compulsive SIB directed toward a specific body part due to neuropathic pain or pruritus occurs in humans.^12,28Rhesus cytomegalovirus (RhCMV) is enzootic in rhesus macaques with close to 100% seroprevalence by 1 y of age in both wild and captive populations.^22,42 As with human cytomegalovirus (HCMV), RhCMV infection is generally subclinical in immunocompetent animals but can cause serious disease in immunodeficient macaques.⁸ RhCMV can be highly pathogenic in SIV-infected animals, and HCMV is the most common viral opportunistic infection in humans with AIDS.^8,37 HCMV infection has been associated with painful peripheral neuropathies in AIDS patients.^17,36,37 We here report a case of SIB associated with RhCMV-induced peripheral neuropathy in an immunocompromised macaque.     

Serologic Evaluation of Clinical and Subclinical Secondary Hepatic Amyloidosis in Rhesus Macaques (Macaca mulatta)

Secondary hepatic amyloidosis in nonhuman primates carries a grave prognosis once animals become clinically ill. The purpose of this study was to establish serologic parameters that potentially could be used to identify rhesus macaques undergoing subclinical development of secondary hepatic amyloidosis. A retrospective analysis was completed by using serum biochemical profiles from 26 histologically diagnosed amyloidotic macaques evaluated at 2 stages of disease, clinical and subclinical (3 to 32 mo prior to clinical signs of disease). Standard serum biochemistry values for cases were compared with institutional age- and gender-specific references ranges by construction of 95% confidence intervals for the difference between means. In addition, 19 histologically diagnosed amyloidotic macaques and 19 age-matched controls were assayed for changes in various parameters by using routinely banked, frozen (–80 °C) sera available from clinical and subclinical time points. Clinically amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyltranspeptidase, and macrophage colony-stimulating factor and significantly decreased quantities of albumin and total cholesterol. Subclinical amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, and serum amyloid A and decreased concentrations of albumin and total cholesterol. The serologic parameters studied indicate a temporal relationship of these factors not previously described, show a clear pattern of disease progression, and could be useful in subclinical disease detection.Abbreviations: mCSF, macrophage colony stimulating factor; SAA, serum amyloid AAmyloid is an eosinophilic substance made of insoluble fibrillar protein.³² When deposited extracellularly, amyloid causes displacement of tissue form and disruption of organ function.³² Persistent accretion of amyloid can result in organ failure and ultimately animal death.²² Clinical signs of disease depend on the tissues affected and the degree of involvement.³² Amyloidosis has been well documented in humans, other mammals, birds, and reptiles.³⁸ In humans, amyloidosis plays a key role in many diseases, including Alzheimer disease, type II diabetes, rheumatoid arthritis, and Down syndrome.^15,20,35,38Amyloidosis generally is classified into 3 categories: primary, secondary, and hereditary. Primary amyloidosis consists of the immunoglobulin- and myeloma-associated types. Secondary (reactive) amyloidosis is associated with chronic inflammation.²⁴ Common causes of secondary amyloidosis in humans include rheumatoid arthritis, idiopathic colitis, infectious diseases, such as tuberculosis and leprosy, and malignant tumors, such as mesothelioma and Hodgkins disease.²⁸ Hereditary amyloid syndromes are rare and include Mediterranean fever, Muckle–Wells syndrome, and familial amyloid cardiomyopathy.^32,38Secondary amyloidosis is the most common form of amyloidosis in animals.³⁸ Amyloidosis occurs in many species of nonhuman primates including the common marmoset (Callithrix jacchus),²³ squirrel monkey (Saimiri sciureus),³⁴ rhesus macaque (Macaca mulatta),^9,10 pigtailed macaque (Macaca nemestrina),^18,27 crab-eating macaque (Macaca fascicularis),²⁷ barbary ape (Macaca sylvanus),⁶ baboon (Papio spp.),¹⁷ mandrill (Papio sphinx), and chimpanzee (Pan troglodytes).^16,39 Although a definitive cause of secondary amyloidosis has not been identified in nonhuman primates, this condition has been associated with chronic inflammation due to rheumatoid arthritis,⁶ viral infection,¹⁸ parasitism,¹ respiratory disease,^27,30 trauma,³⁰ and bacterial enterocolitis.^27,30,31 Shigella spp. have received particular attention as a common etiology linking enterocolitis with amyloidosis.^4,7,38Previous research on amyloidosis in nonhuman primates has yielded clinical and serologic profiles in end-stage amyloidotic animals, but little is known about the serologic status in the subclinical stages of disease. Amyloid can accumulate for as long as 3 y before severe organ disruption occurs¹⁴ and clinical signs of amyloidosis become evident.¹⁶ With appropriate analysis, detection of amyloidosis could occur much earlier than typically now achieved, thus allowing for targeted preventative therapy to potentially halt the progression of this insidious disease.     

Endometrial Decidualization and Deciduosis in Aged Rhesus Macaques (Macaca mulatta)

Superficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates. Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells. Previous reports involving nonpregnant rhesus monkeys have described localized and widespread endometrial decidualization in response to administration of progesterone and synthetic progestogens. Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells are located outside of the endometrium, most often in the ovaries, uterus and cervix but also in various other organs. In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua can be found in the ovary in nearly all term pregnancies. Here we describe pronounced endometrial decidualization in 2 rhesus macaques. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. In one animal, florid extrauterine and peritoneal serosal decidualization was admixed multifocally with carcinomatosis from a primary colonic adenocarcinoma. Cells constituting endometrial and serosal decidualization reactions were immunopositive for the stromal markers CD10, collagen IV, smooth muscle actin, and vimentin and immunonegative for cytokeratin. In contrast, carcinomatous foci were cytokeratin-positive. To our knowledge, this report describes the first cases of serosal peritoneal decidualization in rhesus macaques. The concurrent presentation of serosal peritoneal decidualization with carcinomatosis is unique.Abbreviations: GnRH, gonadotropin-releasing hormone; PAS, periodic acid–Schiff; SMA, smooth-muscle actinSuperficial decidualization of the endometrial stroma is an essential feature of the implantation stage of pregnancy in rhesus macaques and other primates.^13,27,29,37 This process typically begins, and is most prominent, adjacent to the spiral arteries, eventually expanding to affect the endometrium uniformly.³⁵ The endometrial stroma surrounds and supports the endometrial glands and is composed mainly of endometrial stromal cells and blood vessels.³⁵ Decidualization involves proliferation of the endometrial stromal cells, with differentiation into morphologically distinct decidual cells.^7,27,38 Endometrial stromal cells transform into large, polyhedral, cytoplasm-rich cells with large amounts of stored glycogen and are often binucleated or polyploid in character.^{6,13,27,30,35} Ultrastructurally, decidualized cells have numerous ribosomes, prominent rough endoplasmic reticulum and Golgi complexes, and cytoplasmic accumulation of glycogen and lipid droplets.^13,35 Consistent with their stromal origin, decidualized cells express mesenchymal immunohistochemical markers, such as vimentin, desmin, and muscle-specific actin.^{6,7,14,16,20,22}Initiation of decidualization by attachment of the blastocyst to the uterine epithelium depends on previous sensitization by progesterone secretion, after a brief priming by estrogen.^12,13,27 Estrogen and progesterone regulate a series of complex interactions at the interface between the developing embryo and the cells in the stromal compartment, leading to the formation of a differentiated maternal tissue (decidua) that supports embryo growth and maintains early pregnancy.²⁷ Postovulatory levels of circulating progesterone increase and help maintain the differentiation of decidual cells.^{7,13,33,37,38}Ectopic decidua or ‘deciduosis’ describes the condition in which groups of decidual cells reside outside of the endometrium, most often in the ovaries, uterus, and cervix; the fallopian tubes, peritoneum, omentum, diaphragm, liver, skin, spleen, appendix, abdominal–pelvic lymph nodes, renal pelvis, and lungs of women have also been reported as affected.^{6,14,18,20,22,28,29,38} In humans, most cases of deciduosis are associated with normal pregnancy, and ectopic decidua have been reported in the ovary in 90.5% to 100% of term pregnancies.^{6-8,14,20,22,28-30,38} Occasional cases in nonpregnant or postmenopausal women have been attributed to progesterone-secreting active corpora lutea, progesterone secretion by the adrenal cortex, trophoblastic disease, exogenous progestational agents, and pelvic irradiation.^{6-8,14,18,20,22,28,38} Deciduosis is usually an incidental finding that regresses postpartum within 4 to 6 wk; rarely, florid reactions have been reported to cause peritonitis, adhesions, hydronephrosis and hematuria, acute bowel obstruction or perforation (or both), abdominal pain mimicking appendicitis, massive and occasionally fatal hemoperitoneum, vaginal bleeding, and pneumothorax.^{6,7,14,18,20,22,28,29,31}Previous reports involving nonpregnant rhesus macaques have described localized and widespread endometrial decidualization in response to the administration of progesterone, synthetic progestogens, or progesterone-releasing bioactive intrauterine devices and intravaginal rings and have referred to these changes as ‘pseudodecidualization’ to indicate the absence of pregnancy in these animals.^12,33,35,37 In macaques given low (but superphysiologic) levels of progestogens, decidual changes have been noted in localized regions (around spiral arteries and underneath superficial epithelium), whereas high doses of progesterone or synthetic progestagens can cause a more pronounced and extensive reaction.³⁵In cynomolgus macaques, extrauterine decidual cell plaques are rare histologic findings in the subcoelomic mesenchyme of the ovarian cortex.^8,30 Despite the frequency of the condition in women, deciduosis is postulated to be a rarely documented lesion in primates because it is most often observed in conjunction with pregnancy, and pregnant cynomolgus macaques are seldom used in toxicity studies.⁸ Here we describe the pronounced endometrial decidualization of 2 rhesus macaques, one of which also had florid extrauterine and peritoneal decidualization that was admixed multifocally with carcinomatosis. Both macaques had been treated long-term with medroxyprogesterone acetate for presumed endometriosis, which was confirmed in one of the macaques at postmortem examination. To our knowledge, this report describes the first cases of peritoneal decidualization in rhesus macaques as well as the concurrent occurrence of carcinomatosis, endometriosis and peritoneal decidualization in a macaque. The extensive intermixing of the cell populations presented a diagnostic challenge at pathologic examination, and accurate diagnosis was achieved only through the use of multiple immunohistochemical markers.     

Alterations in Cytokines and Effects of Dexamethasone Immunosuppression during Subclinical Infections of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques

Invasive Klebsiella pneumoniae with the hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that also has been attributed to fatal multisystemic disease in African green monkeys at our institution. Combining a cluster of subclinically infected macaques identified in March and April 2008 and the animals documented during a subsequent survey of more than 300 colony nonhuman primates yielded a total of 9 rhesus macaques and 6 cynomolgus macaques that were subclinically infected. In an attempt to propagate the responsible HMV K. pneumoniae strain, a subset of these animals was immunosuppressed with dexamethasone. None of the treated animals developed clinical disease consistent with the multisystemic disease that affected colony African green monkeys. However, cytokine analysis revealed significant alterations of secreted cytokines in macaques subclinically infected with HMV K. pneumoniae when compared with noninfected macaques, thereby calling into question the suitability of animals subclinically infected with HMV K. pneumoniae for use in immunologic or infectious disease research.Abbreviations: HMV, hypermucoviscosity phenotype; rmpA, regulator of the mucoid phenotype gene; magA, mucoviscosity-associated geneKlebsiella pneumoniae is a gram-negative member of the Enterobacteriaceae family that comprises part of the normal fecal and oral flora of many nonhuman primates¹⁹ but also has been implicated in cases of peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates.^17,20,37 Over the past 20 y, strains of invasive K. pneumoniae with a unique hypermucoviscosity phenotype (HMV K. pneumoniae) have been reported to cause community-acquired primary liver abscesses, meningitis, and endophthalmitis in humans in Taiwan and other Asian countries,^{10, 26–31,33,44,48,51} mostly in people with diabetes mellitus.^7,8,44 In addition, HMV K. pneumoniae has caused clinical disease in the United States and other nonAsian countries.^18,30,33 The HMV phenotype is determined based on a positive string test, which is performed by touching a colony with a bacterial loop and gently lifting. If a mucoid ‘string’ of at least 5 mm forms, the string test is considered positive.^3,14,45,51Capsular serotypes K1 and K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^9,15,49,50 The products of the mucoviscosity-associated gene (magA), which encodes a structural outer membrane protein of the K1 serotype, and the regulator of the mucoid phenotype gene (rmpA) have also been proposed as virulence factors.^{16,34,42,52,53}HMV K. pneumoniae has been reported to cause multisystemic abscesses in African green monkeys (Chlorocebus aethiops).⁴⁵ In late 2005 and early 2006, 7 African green monkeys in the research colony at our institution, the US Army Medical Research Institute for Infectious Diseases, were found to have abscesses in multiple locations; all 7 animals either succumbed or were euthanized because of poor prognosis due to surgically nonresectable abdominal abscesses.⁴⁵ The etiology of the final case was determined to be HMV K. pneumoniae with the K2 serotype and rmpA, and all 6 other cases had similar clinical, microbiologic, and pathologic characteristics. Prior to the current study, we believe these 7 cases were the only documented natural infections attributed specifically to HMV K. pneumoniae in nonhuman primates.⁴⁵As a result of those findings, our institution instituted a policy to report K. pneumoniae positive cultures in nonhuman primates during quarantine periods and on routine semiannual examination. Over several months in spring and summer 2008, a group of 19 macaques tested positive on oropharyngeal or rectal culture for HMV K. pneumoniae; 15 of those 19 animals were isolated in a single room for 2 to 4 mo to better characterize the infection.³ None of the animals showed clinical signs of disease during the isolation period, and abdominal palpation failed to suggest the presence of abdominal abscesses like those seen in African green monkeys. Testing of isolates suggested that the macaques harbored subclinical infections and that multiple genotypes of HMV K. pneumoniae were present.³In July 2008, a cynomolgus macaque from the colony that was experimentally challenged with monkeypox virus survived beyond the normal time-to-death window (12 to 16 d after infection). However, on day 22 after infection (6 to 10 d beyond this window), this macaque died unexpectedly. Histopathologic analysis of tissues from this NHP revealed a concurrent gram-negative bacterial infection, based on Gram stains and immunohistochemistry. Although cultures were not available, PCR analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues revealed the presence of K. pneumoniae through the amplification of rmpA,³⁹ which is consistent with the HMV phenotype. This animal was considered to have survived infection with monkeypox based on time to death after infection. Monkeypox is reported to target the mononuclear phagocyte system and associated dendritic cells,⁵⁴ and we theorized that the monkeypox infection in this macaque led to suppression of the immune system, which then allowed development of a fatal HMV K. pneumoniae septicemia.The present project sought to explore the pathophysiology of HMV K. pneumoniae in macaques. We hypothesized that immunosuppression of subclinically infected macaques would produce lesions similar to those observed in the coinfected macaque. In addition, we hypothesized that subclinically infected macaques would have a different immune profile from that of noninfected primates. We measured and analyzed cytokine levels as an indication of altered immune status because such a state potentially could confound research into immunologic responses and infectious disease.     

Adenocarcinoma of the Ileocolic Junction and Multifocal Hepatic Sarcomas in an Aged Rhesus Macaque (Macaca mulatta)

An aged male rhesus macaque in our colony had decreased appetite and a loss of interest in behavioral testing. CBC analysis revealed a regenerative, microcytic, hypochromic anemia with thrombocytosis, consistent with iron deficiency. A fecal occult blood test was positive. Ultrasound imaging revealed numerous, vascularized focal liver lesions that suggested metastases. The macaque''s appetite continued to decrease, and he became more lethargic. At this point, the investigator elected to euthanize the macaque. At necropsy, the ileocolic junction was white and abnormally thickened, and the liver was pale tan with approximately 18 discrete white masses randomly scattered throughout the hepatic parenchyma. Histologically, the mass at the ileocolic junction was identified as an intestinal adenocarcinoma, whereas the liver masses were confirmed to be undifferentiated hepatic sarcomas. This case report describes a rhesus macaque that had 2 unrelated primary neoplasms. A review of the literature indicates that this rhesus macaque is the first reported to have an adenocarcinoma of the ileocolic junction and multiple hepatic sarcomas simultaneously.Rhesus macaques (Macaca mulatta) are genetically similar to humans, have a similar aging phenotype at approximately 3 times the rate of those in humans, and develop spontaneous cancers similar to those in humans.³⁶ In humans, gastrointestinal carcinomas are relatively common, but most of these lesions arise in the colon and rectum with only a small percentage in the small intestine and ileum.^4,12,15,18 Although the ileocolic junction is considered a common site for intestinal adenocarcinomas in aged rhesus macaques, this tumor has also been found in the duodenum, jejunum, distal ileum, cecum, and colon.^{6,13,21-23,25,39} Intestinal adenocarcinomas also occur in aged cynomologus macaques (Macaca fasicularis),³⁹ cotton-top marmosets (Saguinus oedipus),^6,10 common marmosets (Callithrix jacchus),^6,27 and a squirrel monkey (Saimiri sciureus).²⁴ Cotton-top marmosets often develop adenocarcinomas of the colon, including the cecum–colon, and rectum.^6,10 Common marmosets have been reported to develop adenocarcinomas of the small intestine.^6,27 Adenocarcinoma of the cecum in a squirrel monkey has been reported.²⁴Spontaneous hepatic tumors unrelated to carcinogenic factors, such as aflatoxin B1,³³ occur only rarely in nonhuman primates. In the United States, primary malignant hepatic tumors in humans are rare, and fewer than 1% are reported to be hepatic sarcomas.^1,16,40 Review of the nonhuman primate literature revealed reports of hepatic cholangiocarcinoma in a 25-y-old male capuchin monkey (Cebus albifrons),⁷ hepatocellular carcinoma in a 24-y-old male squirrel monkey (Saimiri boliviensis)⁵ and in a female squirrel monkey (Saimiri sciureus) older than 13 y,²⁸ and hepatocellular carcinoma and cholangiocarcinoma in an African green monkey (Cercopithecus aethiops).³⁴ Spontaneous hepatocellular carcinomas were reported to occur in 2 adolescent male cynomologus macaques younger than 5 y.³¹ Hepatic hemangiosarcoma was diagnosed in 3-y-old female rhesus macaque,²⁶ and hepatic cholangiocarcinoma was found in a rhesus macaque that also had an intestinal adenocarcinoma.³⁹The aged male rhesus macaque (Macaca mulatta) in the current case study was found to have adenocarcinoma of the ileocolic junction and multiple, random, discrete neoplasms in the liver, which were identified as undifferentiated sarcomas. No metastases from the intestinal adenocarcinoma were detected, but neoplastic cells similar to those of the undifferentiated hepatic cells were identified in an intestinal artery. The frequency of multiple tumor types in aged nonhuman primates is relevant to the use of older animals in research.     

Epidemiology of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in a Research Colony of Nonhuman Primates

Invasive Klebsiella pneumoniae with hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that, over the past 20 y, has resulted in a distinct clinical syndrome characterized by pyogenic liver abscesses sometimes complicated by bacteremia, meningitis, and endophthalmitis. Infections occur predominantly in Taiwan and other Asian countries, but HMV K. pneumoniae is considered an emerging infectious disease in the United States and other Western countries. In 2005, fatal multisystemic disease was attributed to HMV K. pneumoniae in African green monkeys (AGM) at our institution. After identification of a cluster of subclinically infected macaques in March and April 2008, screening of all colony nonhuman primates by oropharyngeal and rectal culture revealed 19 subclinically infected rhesus and cynomolgus macaques. PCR testing for 2 genes associated with HMV K. pneumoniae, rmpA and magA, suggested genetic variability in the samples. Random amplified polymorphic DNA analysis on a subset of clinical isolates confirmed a high degree of genetic diversity between the samples. Environmental testing did not reveal evidence of aerosol or droplet transmission of the organism in housing areas. Further research is needed to characterize HMV K. pneumoniae, particularly with regard to genetic differences among bacterial strains and their relationship to human disease and to the apparent susceptibility of AGM to this organism.Abbreviations: AGM, African green monkey; HMV K. pneumoniae, invasive Klebsiella pneumoniae with hypermucoviscosity phenotype; NHP, nonhuman primate; RAPD, random amplification of polymorphic DNAKlebsiella pneumoniae is an enteric, gram-negative, lactose-fermenting bacillus with a prominent capsule. This bacterium has been associated with peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates,^10,13,29 although it also is reported to constitute normal fecal and oral flora in many nonhuman primates (NHP).¹² Pathogenic strains associated with the upper respiratory tract typically are heavily encapsulated.¹² Over the past several decades, human medical literature indicates the emergence of an invasive K. pneumoniae disease in Taiwan and other Asian countries, in which community-acquired pyogenic liver abscesses have been attributed to strains of invasive K. pneumoniae with a unique hypermucoviscous phenotype (HMV K. pneumoniae).^{6,17-19,21,26,34} The hypermucoviscous phenotype has also been associated with other serious complications, including bacteremia, meningitis, and endophthalmitis. This strain of Klebsiella has become an emerging cause of pyogenic liver abscesses in some nonAsian countries, including the United States.^16,20,36,39 The majority of clinical cases of HMV K. pneumoniae are in the Asian population, particularly in patients with diabetes mellitus.^3,4,33 Determination of the HMV phenotype typically is based on a positive string test.^8,35,39Several virulence factors have been associated with HMV K. pneumoniae. Klebsiella spp. generally develop prominent polysaccharide capsules which increase virulence by protecting the bacteria from phagocytosis and preventing destruction by bactericidal serum factors. Capsular serotypes K1 or K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^5,8,37,38 In addition, the mucoviscosity-associated gene magA, which encodes a structural outer membrane protein of the K1 serotype, and rmpA (regulator of the mucoid phenotype gene; located on a plasmid) have been proposed as virulence factors.^{9,27,31,40,41} Recently, it was suggested that 2 clones, CC23 ^K1 and CC82^K1, are strongly associated with primary liver abscess and respiratory infection, respectively.²Over a period of several months in 2005 to 2006, 7 African green monkeys (AGM; Chlorocebus aethiops) in the US Army Medical Research Institute of Infectious Diseases research colony developed abscesses in multiple locations and either died or were euthanized when the abscesses were determined to be nonresectable.³⁵ HMV K. pneumoniae of the K2 serotype and carrying rmpA was determined to be the cause of the infection in 1 case, and the 6 other cases had similar clinical and pathologic features. This report³⁵ is the only documentation, to our knowledge, of natural infection with HMV K. pneumoniae in NHP. As a result of these cases, the US Army Medical Research Institute of Infectious Diseases instituted policies to exclude HMV K. pneumoniae from the colony. The organism was included as a specific pathogen-free requirement for vendors, and K. pneumoniae culture results were reported during quarantine periods and on routine semiannual examination for all colony NHP.     

Prevalence of Viremia and Oral Shedding of Rhesus Rhadinovirus and Retroperitoneal Fibromatosis Herpesvirus in Large Age-Structured Breeding Groups of Rhesus Macaques (Macaca mulatta)

We performed a cross-sectional study to estimate the prevalence of 2 gamma-2-herpesviruses, rhesus rhadinovirus (RRV) and retroperitoneal fibromatosis herpesvirus (RFHV), in breeding colonies of rhesus macaques. Of 90 animals selected for sampling, 73 (81%) were positive for RRV, which was detected only in blood in 22 (24%), only in saliva in 15 (16%), and in both blood and saliva in 36 (40%). Detection of RRV DNA in blood and saliva was significantly higher in animals younger than 2 y. In comparison, RFHV was detected in 40 (44%) of the 90 animals: only in blood in 5 (6%), only in saliva in 26 (29%), and in both blood and saliva in 9 (10%). Dual infection was detected in 38 (42%) animals; RFHV was only detected in coinfections. The mean RRV genome copy number in blood was significantly higher than that for RFHV. Age was a significant predictor of RRV copy number in blood and RFHV copy number in saliva. Of the 90 animals, 88 (98%) were positive for rhadinoviral antibodies on an immunofluorescent assay. Both RRV and RFHV are highly endemic in socially housed breeding colonies of rhesus macaques, and their patterns of infection are similar to that for the betaherpesvirus rhesus cytomegalovirus.Abbreviations: CNPRC, California National Primate Research Center; GE, genome equivalents (copy number); KSHV, Kaposi sarcoma-associated herpesvirus; RFHV, retroperitoneal fibromatosis herpesvirus; RRV, rhesus rhadinovirus; OSM, oncostatin M geneThe Rhadinovirus genus of gamma-2-herpesviruses is divided into 2 subgroups, RV1 and RV2, based on genomic sequence comparisons.^36,44 Rhadinovirus infections are generally subclinical in immunocompetent natural hosts, and overt disease is thought to arise only when hosts are immunocompromised.²⁸ In addition, the ability to establish both lytic and latent infections, a hallmark of the Herpesviridae family, occurs during rhadinovirus infections.^1,43 The RV1 subgroup includes Kaposi sarcoma-associated herpesvirus (KSHV; also referred to as human herpesvirus 8)^12,32 the causative agent of Kaposi sarcoma, an angioproliferative lesion composed of a mixed population of endothelial, inflammatory and spindle cells.^19,24 Furthermore, KSHV has been linked etiologically to 2 different B-cell lymphomas: primary effusion lymphoma and multicentric Castleman disease.¹⁷ Retroperitoneal fibromatosis herpesvirus (RFHV) is also a member of the RV1 subgroup and is thought to be the macaque homolog of KSHV.^{4,8,14,36,37,40} DNA sequences specific for RFHV have been detected in retroperitoneal fibromatosis in macaques coinfected with the potentially immunosuppressive simian betaretrovirus type 2.⁷ Histologic similarities between retroperitoneal fibromatosis and KS lesions seen in humans coinfected with KSHV and HIV have been previously described.^7,9,21,37 During outbreaks of simian betaretrovirus type 2 disease at 2 national primate research centers in the 1980s, the incidence of retroperitoneal fibromatosis was reported to be 5% to 7% for animals younger than 2 y and 1% across all age groups.^7,37,45 Since the end of these outbreaks in the late 1980s, retroperitoneal fibromatosis has occurred only rarely in primate colonies. The majority of published RFHV studies have focused on animals with recognized retroperitoneal fibromatosis lesions.^9-11 However, RFHV has proven extremely difficult to isolate and, to date, has not been propagated successfully in vitro, and only a small portion of the RFHV genome has been sequenced.^36,37,40,44 In this study we determined the prevalence of RFHV infection in nondiseased animals and address aspects of the natural history of this virus infection in captive macaque populations.Rhesus rhadinovirus (RRV) is a member of the RV2 subgroup, which naturally infects rhesus macaques.^15,38,44 RRV was isolated independently at 2 national primate research centers in the late 1990s from rhesus macaques.^15,42 Both RRV isolates were shown to have noteworthy sequence similarity to KSHV and RFHV.^2,8,15,42 Unlike RFHV, RRV can be propagated readily in vitro, thus facilitating studies of the lytic replication cycle.^5,6,16 Experimental coinfection of rhesus macaques with SIV and RRV resulted in a lymphoproliferative disease resembling multicentric Castleman disease, but variations in disease outcome between the 2 RRV isolates were noted.^30,49 More recently, RRV has been shown to be associated with nonHodgkin lymphoma and retroperitoneal fibromatosis in SIV-infected rhesus macaques.³⁴ Therefore, RRV infection in macaques is a highly useful animal model for the study of KSHV infection in humans, including studies of viral pathogenesis, factors affecting prevalence of infection, viral shedding, and transmission.^2,25,31,42 In addition, RRV is a persistent virus targeted for elimination in some specific pathogen free (SPF) macaque breeding populations. A better understanding of the natural history of RRV and RFHV infections will lead to improved characterization of host–virus interactions, contribute to the refinement of these nonhuman primate models, and allow more efficient management of SPF colonies.Here we report estimates of the prevalence of viremia and oral shedding of RRV and RFHV in large age-structured breeding colonies of rhesus macaques. Both viruses were highly endemic in the breeding populations we tested, and coinfection with both viruses was common.     

An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV.Abbreviations: BV, B virus (Macacine herpesvirus 1); EU, ELISA units; g, glycoprotein; HSV, herpes simplex virus; tELISA, titration ELISA; UN, uninfected; WBA, western blot analysisB virus (BV; Macacine herpesvirus 1) is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. The virus occurs naturally in macaques (Macaca spp.) and causes a lethal zoonotic infection in 80% of untreated humans. Because biomedical professionals working with macaques, their cells, or tissues are at risk for becoming infected with BV, it is important to know the status of macaques involved in potential BV exposures. Although cases of BV infection after encounters between tourists and macaques have not been reported, any event that involves direct or fomite-associated contact with macaques has inherent risks. Identification of zoonotic BV infection through the detection of antibodies enables timely antiviral intervention, which is critical to reduce or prevent morbidity and mortality. Similarly rapid detection is important to maintain the biointegrity of SPF captive macaque colonies. The identification of BV in clinical specimens is achieved by using cell culture, PCR, or antibody detection methods. Because BV is shed only rarely from peripheral sites, the identification of BV infection in monkeys and humans currently is based on antibody detection (serology).^14,23,28In our laboratory, current serological diagnosis for B virus infections has been based on 2 principal tests: a titration-based (that is, traditional) ELISA (tELISA) as a screening test and western blot analysis (WBA) as a confirmatory test. Each test uses quality-controlled BV antigens that are prepared from lysates of infected cells.^20,22,23 Because BV is the only simplex virus in the Alphaherpesvirinae subfamily that is known to infect macaques,^14,28 antibodies interacting with BV antigens are used to indicate BV infection and not an infection due to a crossreacting virus. In practice, tELISA has identified numerous BV antibody-positive sera, the majority of which are low-titer sera from SPF colonies, which fail to be confirmed by WBA, and therefore, are classified as false positives.²³ We, therefore, searched for other approaches that could be used for confirmation of tELISA results. One reasonable option was the use of BV recombinant proteins as antigens. Numerous investigators have used recombinant-based assays for routine diagnosis of infections with viruses, including cytomegalovirus,³⁶ Epstein–Barr,⁶ herpes simplex (HSV1 and HSV2)^{2,3,17,31,32,34} Crimean–Congo hemorrhagic fever,¹⁰ HIV,³⁶ dengue,^5,11,27 hepatitis C,²⁴ hepatitis B,⁸ West Nile,²⁶ influenza,¹⁶ Ebola, and Marburg³³ viruses.Screening for the presence of serum IgG molecules against an array of defined and purified recombinant antigens has distinct advantages over assays that use the entire complement of viral antigens that are present in virus-infected cells. This is particularly true for pathogens that require BSL4 laboratories.^28,33 The pattern of reactivity obtained against each individual recombinant protein may have diagnostic value, by enabling identification of the stage of infection and the prediction of the prognosis of the disease.^3,4,18 However, using a single or only a few recombinant proteins as ELISA antigens can lead to a false-negative result if the antibody repertoire produced after BV infection reacts with other antigenic determinants that are not represented by the particular recombinant antigens used in the test.^{3,18,28,31,34}Several laboratories have examined the efficacy of using a single BV recombinant antigen (that is, glycoprotein D [gD]) for diagnosing BV infections in macaques^25,37 and humans,¹⁵ and we previously reported the diagnostic potential of an ELISA that incorporated several recombinant BV antigens.²⁸ We chose 4 recombinant BV glycoproteins as candidate antigens: peptides corresponding to the full-length extracellular domain of gB, gC, and gD and the membrane-associated segment of gG (gGm). Among these antigens, gGm was the most BV-specific, because it failed to crossreact with antibodies induced by HSV1 and HSV2. To validate the use of the recombinant BV antigens for the purpose of BV antibody detection, a panel of antibody-negative (n = 40) and antibody-positive (n = 75) macaque sera that were confirmed to be positive by tELISA and WBA were tested against the panel of the 4 B virus recombinant antigens, all of which showed fairly high sensitivity for detecting antibodies to BV.²⁸Here, we examine the performance of the recombinant-based ELISA (rELISA) for BV detection by using numerous (>1000) macaque sera, which have a broad range of antibody titers as determined by tELISA. Because manual ELISA to identify antibodies against an array of antigens are too laborious to be cost-effective, we adapted a previously described high-throughput automated single-antigen ELISA performed in 384-well plates to detect antibodies in macaque sera to multiple BV antigens.²³ This assay format has been adapted to include antigens from other alphaherpesviruses²³ and can be easily modified further for other viruses. We then compared the performance of the rELISA with that of whole-virus tELISA and WBA. The main goal of this study was to determine whether the 384-well rELISA is an effective alternative to WBA as a confirmatory assay for tELISA.     

Osteosarcoma in Baboons (Papio spp)

Bone neoplasms in baboons (Papio spp) are rare, with only one confirmed case of osteosarcoma previously described in the literature. Over a 12-y period, 6 baboons at a national primate research center presented with naturally occurring osteosarcoma; 3 lesions affected the appendicular skeleton, and the remaining 3 were in the head (skull and mandible). The 6 cases presented were identified in members of a large outdoor-housed breeding colony. The subjects were not genetically related or exposed to the same research conditions. Diagnoses were made based on the presentation and radiographic findings, with histologic confirmation.Neoplasia remains a highly prevalent condition across the majority of species. A recent survey of a large baboon colony identified 395 neoplasms among 4297 animals.⁶ The most common neoplasms documented in NHP include lymphosarcoma, adenocarcinoma, and squamous cell carcinoma.⁸ Tumors involving the musculoskeletal system are much rarer than are the other previously mentioned types. The musculoskeletal neoplasms reported most prominently in the literature are osteoma, osteosarcoma, odontoma, and various types of myxoma, with Macaca spp being the NHP affected most often.⁵ However, this apparent prevalence may be artifactual due to the use of far more animals of Macaca spp compared with other NHP species.Osteosarcoma is an infrequently documented neoplasm among NHP, and it is particularly rare in baboons (Papio spp.). Combined-type osteosarcoma in a rhesus macaque,⁴ extraosseous osteosarcoma in a rhesus macaque,¹⁷ and osteoblastic osteosarcoma in a gray mouse lemur have been reported.¹⁸ Among 4 reported cases of bone tumors in baboons, only one was confirmed as osteosarcoma and involved the mandibular ramus of a male baboon.^8,12,24 Another neoplasm, identified on the distal aspect of the right ulna of a baboon, initially was described in a review article as a giant cell tumor²² but was later referred to as a fibrosarcoma²³ and then as an osteosarcoma,¹⁹ thus making the confirmed diagnosis unclear. The remaining 2 known cases of bone tumors in baboons were osteomas present in the tibia and femur.^8,12 Three other osteosarcomas were included in reviews of pathology from the Southwest National Primate Research Center (San Antonio, TX), but detailed clinical and pathologic information was not described.^6,7,9     

Diagnosis of Amyloidosis and Differentiation from Chronic,Idiopathic Enterocolitis in Rhesus (Macaca mulatta) and Pig-Tailed (M. nemestrina) Macaques

Amyloidosis is a progressive and ultimately fatal disease in which amyloid, an insoluble fibrillar protein, is deposited inappropriately in multiple organs, eventually leading to organ dysfunction. Although this condition commonly affects macaques, there is currently no reliable method of early diagnosis. Changes in clinical pathology parameters have been associated with amyloidosis but occur in late stages of disease, are nonspecific, and resemble those seen in chronic, idiopathic enterocolitis. A review of animal records revealed that amyloidosis was almost always diagnosed postmortem, with prevalences of 15% and 25% in our rhesus and pig-tailed macaque colonies, respectively. As a noninvasive, high-throughput diagnostic approach to improve antemortem diagnosis of amyloidosis in macaques, we evaluated serum amyloid A (SAA), an acute-phase protein and the precursor to amyloid. Using necropsy records and ELISA analysis of banked serum, we found that SAA is significantly elevated in both rhesus and pig-tailed macaques with amyloid compared with those with chronic enterocolitis and healthy controls. At necropsy, 92% of rhesus and 83% of pig-tailed had amyloid deposition in either the intestines or liver. Minimally invasive biopsy techniques including endoscopy of the small intestine, mucosal biopsy of the colon, and ultrasound-guided trucut biopsy of the liver were used to differentiate macaques in our colonies with similar clinical presentations as either having amyloidosis or chronic, idiopathic enterocolitis. Our data suggest that SAA can serve as an effective noninvasive screening tool for amyloidosis and that minimally invasive biopsies can be used to confirm this diagnosis.Abbreviations: SAA, serum amyloid AAmyloidosis is a pathologic condition that occurs spontaneously in humans, mammals, birds, and reptiles.⁴⁷ Secondary systemic amyloidosis, also referred to as reactive amyloidosis, is the most common form described in domestic animals.⁴⁶ It is a progressive disease in which an insoluble fibrillar protein consisting of β pleated sheets, amyloid, is deposited inappropriately in multiple organs, eventually leading to dysfunction.^40,46 Secondary amyloidosis is most often the result of chronic infections or inflammatory disease. In humans, it occurs with a wide variety of conditions including inflammatory bowel disease,³ osteoarthritis including rheumatoid and juvenile forms,^20,25 chronic infections such as tuberculosis, and hereditary disease such as familial Mediterranean fever.⁴³ Similarly, in nonhuman primates, the disease has been described with several conditions of chronic infection or inflammation including bacterial enterocolitis,^4,19,30,37 chronic indwelling catheters,⁹ parasitism,^2,4 respiratory disease,^30,37 trauma,³⁷ and rheumatoid arthritis.⁶Despite reported prevalences as high as 30% in rhesus (Macaca mulatta)⁴ and 47% in pig-tailed macaques (Macaca nemestrina),¹⁹ amyloidosis remains a challenge to diagnose. The current diagnostic ‘gold standard’ in macaques is histopathology of the affected organ;¹⁹ however, amyloid can be deposited in tissues for as long as 3 y before the development of clinical signs.¹⁶ Histologic diagnoses of amyloidosis typically are confirmed with Congo red staining, in which amyloid proteins appear apple-green and birefringent under polarized light. In addition, electron microscopy can detect the fibrillar amyloid proteins in tissues, and other histologic stains including methyl violet, sulphonated Alcian blue, and thioflavin S and T can be used but are less specific than is Congo red.³³ Although changes in clinical pathology parameters such as decreases in serum albumin and total protein have been associated with amyloidosis,^19,29 they are often nonspecific and resemble those seen in the frequently comorbid conditions chronic anorexia and chronic, idiopathic enterocolitis. Furthermore, imaging techniques such as abdominal X-ray and ultrasonography have been shown to be nondiagnostic in macaques with amyloidosis.¹⁹ Consequently, at our institution and in other macaque colonies, diagnosis of amyloidosis is often made at necropsy.The current standard of diagnosis in humans is biopsy with histopathology of affected organs, but unlike in nonhuman primates, minimally invasive tissue sampling has been extensively explored.¹⁷ Aspiration or biopsy of the subcutaneous abdominal fat pad has currently replaced many biopsy techniques as the preliminary diagnostic, with reported sensitivities ranging from 66% to 92%.^{5,24,28,39,44} Rectal biopsy was previously the preferred minimally invasive approach and is now often used adjunctively when subcutaneous abdominal fat is negative for amyloid but the clinical suspicion for amyloidosis remains high.^5,17 Additional tissue biopsy sites with limited morbidity such as skin, gingiva, and stomach have been reported with lesser sensitivities.^5,34,39,44 In contrast, limited information is published on the usefulness of minimally invasive biopsy techniques for diagnosing amyloidosis in macaques. One report found endoscopic biopsy of the stomach and colon to be of limited utility in diagnosing amyloidosis in a colony of pig-tailed macaques.¹⁹ Similarly, a single publication reported colonoscopy to be noninformative and labor-intensive in a colony of rhesus macaques.¹⁵ Retrospective studies of macaque colonies have shown a predilection for amyloid deposition in the intestines and liver,^4,30,38 suggesting that endoscopic or percutaneous biopsy of these tissues may reliably provide definitive antemortem diagnosis for amyloidosis.In addition to biopsy, identification of the relevant amyloid precursor protein within the blood is an integral part of the diagnosis of amyloidosis in human patients¹⁷ and holds promise as a screening tool in macaque colonies because of its high throughput potential in comparison to biopsy. Serum amyloid A (SAA), an acute-phase protein, can be found circulating in the blood and is the precursor for amyloid formation and deposition in secondary systemic amyloidosis. Specifically, when elevated SAA persists in the bloodstream, it ultimately progresses to amyloid deposition in tissues.^13,45 Profound elevations in SAA occur in the bloodstream as a result of acute inflammation, but these elevations are transient as SAA then is rapidly degraded and removed from the peripheral circulation.^7,45 Although the exact role of chronic inflammation and SAA in the pathogenesis of secondary, systemic amyloidosis is not well understood, SAA is pathologically persistently elevated in human patients with chronic inflammatory disease that develop secondary systemic amyloidosis. In contrast, serum SAA remains at normal lower levels in human patients without amyloidosis but ongoing chronic inflammatory disease.^13,14,26 Furthermore, quantification of SAA is more effective than are organ function tests as a prognostic measure of amyloid disease and is routinely used to monitor disease progression and response to treatment in humans.¹⁴ In rhesus and pig-tailed macaques, SAA is elevated in subjects with amyloidosis as compared with those that are clinically normal.^8,19 The ability to distinguish between healthy animals and those with subclinical amyloidosis would be clinically useful. Human studies indicate that establishing a diagnosis of secondary amyloidosis in its early stages followed by prompt treatment of the inciting chronic inflammatory process can arrest the progression of amyloidosis and can even result in disease remission in some cases.^{21,23,31,32,36} Of equal interest would be the ability to distinguish amyloidosis from chronic, idiopathic enterocolitis, a common disease among macaque colonies^12,35 that has considerable clinical overlap with the late stages of amyloidosis but different therapeutic options and prognosis than does systemic amyloidosis. Although there is no definitive treatment for amyloidosis in humans or macaques, recent human case reports suggest that antiinflammatory therapy with newer targeted monocolonal antibody medications, such as IL6 receptor antagonists, can successfully reverse the disease. This outcome has been demonstrated in several cases by both the reduction of circulating SAA to normal levels and by the histologic disappearance of amyloid proteins in biopsies of affected tissues.^{21,23,31,32,36} Accurate antemortem diagnosis of amyloidosis in macaques potentially would support further investigations into the novel application of these drugs for the treatment of amyloidosis in both human and macaque patients.We hypothesize that SAA, in addition to being a useful screening method for identifying animals with amyloidosis, can be used to distinguish between macaques with this disease and those with chronic, idiopathic enterocolitis. We further hypothesize that, in agreement with retrospective studies from macaques at other institutions, the intestines and liver will be commonly affected in amyloidotic macaques in our own colonies and that minimally invasive biopsy of these tissues can provide definitive, antemortem diagnosis of amyloidosis.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.