Animal Models of Q Fever (Coxiella burnetii)

Q fever, caused by the pathogen Coxiella burnetii, is an acute disease that can progress to become a serious chronic illness. The organism leads an obligate, intracellular lifecycle, during which it multiplies in the phagolytic compartments of the phagocytic cells of the immune system of its hosts. This characteristic makes study of the organism particularly difficult and is perhaps one of the reasons why, more than 70 y after its discovery, much remains unknown about the organism and its pathogenesis. A variety of animal species have been used to study both the acute and chronic forms of the disease. Although none of the models perfectly mimics the disease process in humans, each opens a window onto an important aspect of the pathology of the disease. We have learned that immunosuppression, overexpression of IL10, or physical damage to the heart muscle in mice and guinea pigs can induce disease that is similar to the chronic disease seen in humans, suggesting that this aspect of disease may eventually be fully understood. Models using species from mice to nonhuman primates have been used to evaluate and characterize vaccines to protect against the disease and may ultimately yield safer, less expensive vaccines.Coxiella burnetii is the causative agent of human Q fever. Infection can take several forms and has been described as clinically polymorphic.⁶ In humans, presentation ranges from asymptomatic, through acute disease, to chronic illness. In the majority of cases, acute disease presents as a self-limiting febrile illness, with half of cases also having severe headaches.⁸⁸ In severe cases of acute disease, atypical pneumonia is often found.⁸⁸ A small proportion (2% to 4%) of subjects with symptomatic acute Q fever are admitted to hospital.^70,88 Chronic disease may develop in approximately 5% of those infected;¹⁶ the vast majority of these cases will present as a bacterial culture-negative endocarditis^16,22 often in those with predisposing heart-damage¹⁹ or immunosuppression.¹⁶ Without effective treatment, Q fever endocarditis is generally fatal, but early diagnosis coupled with novel treatment strategies has brought the death rate down to less than 5%.⁶⁹ The 2009 outbreak in the Netherlands involved 2357 human cases, of which more than 400 required hospitalization.⁹⁰ The animal cost in the Netherlands was far higher, with more than 50,000 pregnant goats culled in an attempt to control the epidemic.⁸²Two other clinical manifestations of Q fever are worthy of mention owing to their less-than-satisfactory outcomes with current treatment strategies. These are Q fever during pregnancy and Q fever fatigue syndrome. C. burnetii infection during pregnancy results in premature delivery in almost half of those affected and spontaneous abortion in more than a quarter.¹⁴ There have been few studies in this area, but there are indications that among those infected during the first trimester and treated suboptimally, the abortion rate is 100%.⁶⁸ This effect is compounded by the fact that the frontline bactericidal drugs for treatment (doxycycline and hydroxychloroquine) are contraindicated for use during pregnancy.⁶⁸ A bacteriostatic regimen (cotrimoxazole) has therefore been proposed for use⁶⁸ until delivery. Without satisfactory treatment during and after pregnancy, there is also a high probability for infection to lead to chronic Q fever: an incidence of 70% was reported in a group of pregnant women in France.⁶⁸Post-Q fever fatigue syndrome was first reported in 1996,⁵² but an association between Q fever and chronic fatigue had been observed as early as 1982.⁵² Between 10% and 15% of those who have had acute Q fever develop a chronic fatigue syndrome that can last between 5 and 10 y—and even longer in some cases.⁵³ Some of these patients have been found to have long-term persistence of C. burnetii cell components and LPS associated with traces of genomic DNA,⁵³ suggesting that Q fever fatigue syndrome may be immunologically mediated rather than caused by the organism directly.Q fever is a zoonosis that has been described worldwide,⁵⁶ and human outbreaks are often associated with contact with the birth products of farm animals.⁵⁶ However, outbreaks associated with the birth products of domestic cats have also been reported.⁵⁴ Human infection primarily occurs via the inhalation of infectious aerosols.⁵⁶ Over the past 10 y, outbreaks have been reported in the Netherlands,⁷¹ Slovenia,²⁶ the United Kingdom,^91,97,99 Israel,² Iraq,¹⁸ the United States,¹¹ Germany,²⁴ Bulgaria,⁶³ Croatia,⁵⁸ Spain,²³ Italy,⁸³ and France.⁸⁸A very small number of C. burnetii organisms can cause infection by inhalation. Infection has been predicted to be possible after exposure to only a single organism.³³ This low dosage, coupled with the organism''s ability to cause debilitating disease and high levels of resistance to various means of inactivation^67,77,78 have resulted in it being listed as a category B biologic warfare and bioterrorism agent by the Centers for Disease Control.⁴⁹Prevention of Q fever in man can be achieved by vaccination; the only vaccine available for general use is Q-Vax, which was licensed in Australia in 1989.⁵¹ This vaccine consists of formalin-inactivated C. burnetii whole cells, produced in chick embryos. Its use has been associated with severe local reactions in those with preexisting immunity. As a precaution, prevaccination screening (history, skin test, and serology) must therefore be performed prior to administration.³⁵ Despite this safeguard, severe local reactions to vaccination are reported.⁴⁴ The vaccine is also hazardous to produce, with the organism requiring culture in chick-embryos at biosafety level 3 prior to inactivation.⁵¹ There is, therefore, a need for a vaccine that is safer to produce and safer to use and that does not require prevaccination screening.The organism displays antigenic phase variation often paralleled with the rough-smooth variation seen in Enterobacteriaceae. In C. burnetii, phase variation has been demonstrated to be due to differences in LPS. Phase I has been shown to contain a unique disaccharide galactosaminuronyl glucosamine and 9 unidentified components in addition to the components of phase II LPS.¹ Organisms with the phase I phenotype are the infectious and virulent form found in the environment. Organisms with the phase II phenotype are observed only during repeated subculture in laboratory chick embryo or cell culture systems;²⁷ they have a chemically simpler LPS¹ and several deletions in the genome.^32,92 Phagocytosis of phase I, but not phase II, organisms by macrophages involves an interaction between the bacterial LPS and Toll-like receptor 4. This mechanism also stimulates F-actin reorganization of the host cells and stimulates the release of type 1 cytokines including IFNγ and TNF.³⁰ This interaction appears important in the initial priming of the immune response and could provide an explanation for the limited protection of vaccines based on potential virulence genes (omp1, HspB, Pmm, Fbp, Orf 410, Crc, CbMip, MucZ, P28) singly and in combinations but containing no LPS.^47,89,102In addition to its antigenic phase variation, C. burnetii occurs in 2 morphologic forms, a large-cell variant and a small-cell variant. These forms differ antigenically due to differences in the proteins expressed on their surface. It has been suggested that the resistance of C. burnetii to host defense mechanisms may be enhanced by antigenic differences between the different developmental forms.^57,94 The small-cell morphologic form is highly resistant to destruction by chemical and environmental factors and is likely the transmissible form of the pathogen.^15,67 After infection, which generally occurs by inhalation of the small-cell form, the organisms are taken up by host alveolar macrophages.⁸¹ Morphogenesis from the small-cell to large-cell form then occurs, the large-cell variant being the replicative form of the organism.¹⁵ These organisms then replicate within parasitophorous vacuoles.⁵⁰ As the organisms enter the stationary phase of their growth within the cell, they condense back into the small-cell form.¹⁵ During replication within the host cell, the organism subverts cellular processes though active mechanisms to avoid and modify the host immune response.⁵⁰ C. burnetii possesses a type IV secretion system, and the proteins that cause this subversion are likely delivered to the host cell by this machinery.^50,93Because C. burnetii is an obligate intracellular organism, it has only been possible to study the organism within living animal hosts. Host-cell–free growth of the organism has been reported recently,⁶² but the technique has yet to be exploited fully. Cell-culture–based in vitro systems remain limited in the study of C. burnetii, given that the organism soon reverts to the avirulent (at least in immunocompetent hosts) phase II form (characterized by the loss of the phase I LPS phenotype) in these systems.¹⁰ A key problem in comparing models of C. burnetii infection is related to the organism''s intracellular nature, which complicates attempts to count the organisms used for infection. The literature reflects this difficulty in the fact that there are many different methods used (including plaque assay in primary cell cultures, median infectious doses in chick eggs or mice, and median lethal dose in SCID mice) and no way to directly compare them.     

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.     

Anatomic,Hematologic, and Biochemical Features of C57BL/6NCrl Mice Maintained on Chronic Oral Corticosterone

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic–pituitary–adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 μg/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-μg/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.Obesity and associated metabolic dysfunctions are an increasing public health concern in modern Western society. In humans, obesity and metabolic syndrome heighten the risk of developing debilitating and costly illness including diabetes, cardiovascular disease, stroke, and some forms of cancer.^2,20 Mounting evidence indicates that stress and associated hormones such as cortisol (corticosterone in rodents) contribute to the development of metabolic syndrome. Furthermore, regional glucocorticoid metabolism in adipocytes is proposed to be involved in the pathogenesis of metabolic syndrome.^{6,16,17,27,56} Cushing syndrome, iatrogenic hypercortisolemia, and metabolic syndrome share clinical and physiologic similarities, including central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^{1,2,31,35,41,46} How glucocorticoids contribute to the development of these problems remains unclear.Numerous clinical and experimental studies have linked stress, diet, and lifestyle choices to changes in risk factors associated with the development of metabolic disorders.^{1,3,7,10,21,33,36,42,55} How corticosterone influences this risk remains unclear. Although corticosterone has beneficial short-term effects, long-term corticosterone exposure can result in damage to the physiologic systems it protects acutely.²⁷ Disruption of this physiologic signal occurs in numerous disparate disorders, ranging from depression to Cushing syndrome.^16,22,36,54 Therefore, understanding the effects of chronic high corticosterone on metabolism and physiology is of key importance.To clarify how chronic treatment with corticosterone alters the physiology of an organism, we treated adrenally intact adult male mice with corticosterone in drinking water for 4 wk. Furthermore, we examined the return of physiology 2 wk after withdrawal of chronic corticosterone administration. We used this approach as a rapid (3- to 4-wk), noninvasive method of altering plasma corticosterone levels that enabled us to retain some integrity in the diurnal rhythm present in normal animals.We previously characterized the gross metabolic consequences of exogenous noninvasive corticosterone delivery in the drinking water.^20,28 In those studies, we found that high doses of corticosterone (100 μg/mL) resulted in rapid and dramatic hyperphagia; weight gain; increased adiposity; elevated plasma corticosterone, leptin, insulin, and triglyceride levels; and decreased homecage locomotion.²⁰ Moreover, several studies have shown that a lower dose of corticosterone (25 μg/mL) resulted in an intermediate phenotype in some of these measures but had no effect on others.^{12,14,20,23,28,38,42,47} As such, the high corticosterone dose results in a phenotype that satisfies most of the criteria for metabolic syndrome as defined by the National Heart, Lung, and Blood Institute and the American Heart Association.¹⁵ However, little information is available on the resulting histologic, hematologic, and serum chemical profiles associated with this treatment. We sought to more fully characterize this model to support selection of the model that most accurately reflects the human disease conditions under study. In-depth characterization of the model also provides more precise measurements of response to therapies intended to ameliorate the effects of the treatment.The current study provides a detailed examination of the physiologic effect of 3 dosages of corticosterone—low (25 μg/mL), intermediate (50 μg/mL), and high (100 μg/mL) doses—in drinking water. The goal was to extend the previous findings that established this regimen as a model of metabolic syndrome by exploring the detailed physiologic changes associated with this model and to assess whether and how treated mice recover after withdrawal of the corticosterone treatment. We propose that the physiologic changes observed in the mice treated with high-dose corticosterone approximate changes observed in human patients with metabolic syndrome and that these mice potentially serve as a model for hypercortisolemia and associated obesity. In addition, we hypothesized that 2 wk of recovery from corticosterone treatment would not completely resolve cellular and clinical pathologies characterized during treatment, given the numerous changes in physiology.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Caspase-3 feedback loop enhances Bid-induced AIF/endoG and Bak activation in Bax and p53-independent manner

Chemoresistance in cancer has previously been attributed to gene mutations or deficiencies. Bax or p53 deficiency can lead to resistance to cancer drugs. We aimed to find an agent to overcome chemoresistance induced by Bax or p53 deficiency. Here, we used immunoblot, flow-cytometry analysis, gene interference, etc. to show that genistein, a major component of isoflavone that is known to have anti-tumor activities in a variety of models, induces Bax/p53-independent cell death in HCT116 Bax knockout (KO), HCT116 p53 KO, DU145 Bax KO, or DU145 p53 KO cells that express wild-type (WT) Bak. Bak knockdown (KD) only partially attenuated genistein-induced apoptosis. Further results indicated that the release of AIF and endoG also contributes to genistein-induced cell death, which is independent of Bak activation. Conversely, AIF and endoG knockdown had little effect on Bak activation. Knockdown of either AIF or endoG alone could not efficiently inhibit apoptosis in cells treated with genistein, whereas an AIF, endoG, and Bak triple knockdown almost completely attenuated apoptosis. Next, we found that the Akt-Bid pathway mediates Bak-induced caspase-dependent and AIF- and endoG-induced caspase-independent cell death. Moreover, downstream caspase-3 could enhance the release of AIF and endoG as well as Bak activation via a positive feedback loop. Taken together, our data elaborate the detailed mechanisms of genistein in Bax/p53-independent apoptosis and indicate that caspase-3-enhanced Bid activation initiates the cell death pathway. Our results also suggest that genistein may be an effective agent for overcoming chemoresistance in cancers with dysfunctional Bax and p53.Mammalian cell death proceeds through a highly regulated program called apoptosis that is highly dependent on the mitochondria.¹ Mitochondrial outer membrane (MOM) multiple apoptotic stresses permeabilize the MOM, resulting in the release of apoptogenic factors including cytochrome c, Smac, AIF, and endoG.^{2, 3, 4} Released cytochrome c activates Apaf-1, which assists in caspase activation. Then, activated caspases cleave cellular proteins and contribute to the morphological and biochemical changes associated with apoptosis. Bcl-2 family proteins control a crucial apoptosis checkpoint in the mitochondria.^{2, 5, 6, 7} Multidomain proapoptotic Bax and Bak are essential effectors responsible for the permeabilization of the MOM, whereas anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 preserve mitochondrial integrity and prevent cytochrome c efflux triggered by apoptotic stimuli. The third Bcl-2 subfamily of proteins, BH3-only molecules (BH3s), promotes apoptosis by either activating Bax/Bak or inactivating Bcl-2/Bcl-xL/Mcl-1.^{8, 9, 10, 11, 12} Upon apoptosis, the ‘activator'' BH3s, including truncated Bid (tBid), Bim, and Puma, activate Bax and Bak to mediate cytochrome c efflux, leading to caspase activation.^{8, 11, 12} Conversely, antiapoptotic Bcl-2, Bcl-xL, and Mcl-1 sequester activator BH3s into inert complexes, which prevents Bax/Bak activation.^{8, 9} Although it has been proposed that Bax and Bak activation occurs by default as long as all of the anti-apoptotic Bcl-2 proteins are neutralized by BH3s,¹³ liposome studies clearly recapitulate the direct activation model in which tBid or BH3 domain peptides derived from Bid or Bim induce Bax or Bak oligomerization and membrane permeabilization.^{12, 14, 15}Numerous studies have demonstrated a critical role for Bax in determining tumor cell sensitivity to drug induction and in tumor development. Bax has been reported to be mutated in colon^{16, 17} and prostate cancers,^{18, 19} contributing to tumor cell survival and promoting clonal expansion. Bax has been shown to restrain tumorigenesis²⁰ and is necessary for tBid-induced cancer cell apoptosis.²¹ Loss of Bax has been reported to promote tumor development in animal models.²² Bax knockout (KO) renders HCT116 cells resistant to a series of apoptosis inducers.^{23, 24, 25} p53 has been reported to be a tumor suppressor,²⁶ and its mutant can cause chemoresistance in cancer cells.^{27, 28, 29} Moreover, p53 is often inactivated in solid tumors via deletions or point mutations.^{30, 31} Thus, it is necessary to find an efficient approach or agent to overcome chemoresistance caused by Bax and/or p53 mutants.Few studies have focused on the role of Bak in tumor cell apoptosis and cancer development. Bak mutations have only been shown in gastric and colon cancer cells.³² Some studies have revealed that Bak is a determinant of cancer cell apoptosis.^{33, 34} Some studies have even demonstrated that Bak renders Bax KO cells sensitive to drug induction.^{33, 35} In this study, we are the first group to show that tBid induces Bak activation and the release of AIF and endoG in colon cancer cells, which causes cellular apoptosis independent of Bax/p53. We also found that caspase-3 is activated in apoptosis. Interestingly, downstream caspase-3 can strengthen Bak activation and the release of AIF and endoG during apoptosis via a feedback loop. Furthermore, we reveal that Akt upregulates apoptosis progression. These results will help us to better understand the function of mitochondrial apoptotic protein members in apoptosis and cancer therapies. Furthermore, our experiments may provide a theoretical basis for overcoming chemoresistance in cancer cells.     

A novel androstenedione derivative induces ROS-mediated autophagy and attenuates drug resistance in osteosarcoma by inhibiting macrophage migration inhibitory factor (MIF)

Osteosarcoma is a common primary bone tumor in children and adolescents. The drug resistance of osteosarcoma leads to high lethality. Macrophage migration inhibitory factor (MIF) is an inflammation-related cytokine implicated in the chemoresistance of breast cancer. In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. The inhibition of MIF by DSTD promoted autophagy by inducing Bcl-2 downregulation and the translocation of HMGB1. N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced ROS-mediated autophagy to rescue cell death. However, in the presence of chemotherapy drugs, DSTD enhanced the chemosensitivity by decreasing the HMGB1 level. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.Osteosarcoma, a common primary bone tumor in children and adolescents, is prone to early metastasis through blood.¹ Treatment with a combination of surgery and aggressive adjuvant chemotherapy has improved the survival rate of osteosarcoma patients. The 5-year-survival rates of non-metastatic patients have reached a plateau of approximately 70%.^{2, 3} However, patients with poor responses to chemotherapeutics will undergo local recurrence and metastasis, which reduce the 5-year-survival rates to only 20% despite additional doses or drugs.^{4, 5} Drug resistance is responsible for the poor prognosis. Attenuating chemoresistance facilitates better treatment of osteosarcoma.^{6, 7} Novel treatment strategies that combine anticancer drugs with adjuvant agents could improve the antitumor effects.^{8, 9}In the 1960s, macrophage migration inhibitory factor (MIF) was identified as a pluripotent protein that modulates inflammation.¹⁰ Increasing evidence suggests that inflammation is closely related to tumorigenesis.¹¹ MIF plays a bridging role between inflammation and tumorigenesis.^{12, 13, 14} MIF triggers the activation of the MAPK and PI3K pathways by binding its membrane receptor CD74, resulting in the inhibition of cell apoptosis.¹⁵ Recently, MIF was demonstrated to be involved in cell proliferation, differentiation, angiogenesis and tumorigenesis.^{16, 17, 18} Some evidence has indicated that MIF is abundantly expressed in various cancers and is significantly associated with tumor invasion and metastasis.^{19, 20, 21} MIF has been well established to be involved in the development of glioblastoma,²² breast cancer,²³ bladder cancer²⁴ and colon cancer.^{20, 25} MIF was also upregulated in osteosarcoma.^{26, 27} The knockdown of MIF blocked osteosarcoma cell proliferation and invasion.²⁶ However, the effect of MIF on drug resistance in osteosarcoma has not yet been investigated. Wu et al. ²³ have revealed that MIF knockdown promoted chemosensitivity by inducing autophagy in breast cancer. In contrast, autophagy reportedly contributed to chemoresistance in osteosarcoma.⁶ These controversial results prompted us to confirm the role of MIF in drug resistance in osteosarcoma.In this study, we isolated a novel androstenedione derivative identified as 3,4-dihydroxy-9,10-secoandrosta-1,3,5,7-tetraene-9,17-dione (DSTD). DSTD could inhibit MIF expression in MG-63 and U2OS cells. Both N-acetyl-L-cysteine (NAC) and 3-methyladenine (3-MA) attenuated DSTD-induced autophagy but promoted cell death, suggesting that DSTD induced reactive oxygen species (ROS)-mediated autophagy to rescue cell death. Furthermore, MIF inhibition by DSTD enhances chemosensitivity by downregulating HMGB1 in osteosarcoma cells. Our data suggest MIF inhibition as a therapeutic strategy for overcoming drug resistance in osteosarcoma.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death,Phytoalexin Production,and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases,OsCIPK14/15, in Rice Cultured Cells

Although cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca²⁺ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca²⁺ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn²⁺-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.Calcium ions regulate diverse cellular processes in plants as a ubiquitous internal second messenger, conveying signals received at the cell surface to the inside of the cell through spatial and temporal concentration changes that are decoded by an array of Ca²⁺ sensors (Reddy, 2001; Sanders et al., 2002; Yang and Poovaiah, 2003). Several families of Ca²⁺ sensors have been identified in higher plants. The best known are calmodulins (CaMs) and CaM-related proteins, which typically contain four EF-hand domains for Ca²⁺ binding (Zielinski, 1998). Unlike mammals, which possess single molecular species of CaM, plants have at least three distinct molecular species of CaM playing diverse physiological functions and whose expression is differently regulated (Yamakawa et al., 2001; Luan et al., 2002; Karita et al., 2004; Takabatake et al., 2007). The second major class is exemplified by the Ca²⁺-dependent protein kinases, which contain CaM-like Ca²⁺-binding domains and a kinase domain in a single protein (Harmon et al., 2000). In addition, a new family of Ca²⁺ sensors was identified as calcineurin B-like (CBL) proteins, which consists of proteins similar to both the regulatory β-subunit of calcineurin and the neuronal Ca²⁺ sensor in animals (Liu and Zhu, 1998; Kudla et al., 1999).Unlike CaMs, which interact with a large variety of target proteins, CBLs specifically target a family of protein kinases referred to as CBL-interacting protein kinases (CIPKs) or SnRK3s (for sucrose nonfermenting 1-related protein kinases type 3), which are most similar to the SNF family protein kinases in yeast (Luan et al., 2002). A database search of the Arabidopsis (Arabidopsis thaliana) genome sequence revealed 10 CBL and 25 CIPK homologues (Luan et al., 2002). Expression patterns of these Ca²⁺ sensors and protein kinases suggest their diverse functions in different signaling processes, including light, hormone, sugar, and stress responses (Batistic and Kudla, 2004). AtCBL4/Salt Overly Sensitive3 (SOS3) and AtCIPK24/SOS2 have been shown to play a key role in Ca²⁺-mediated salt stress adaptation (Zhu, 2002). The CBL-CIPK system has been shown to be involved in signaling pathways of abscisic acid (Kim et al., 2003a), sugar (Gong et al., 2002a), gibberellins (Hwang et al., 2005), salicylic acid (Mahajan et al., 2006), and K⁺ channel regulation (Li et al., 2006; Lee et al., 2007; for review, see Luan, 2009; Batistic and Kudla, 2009). However, physiological functions of most of the family members still remain largely unknown.Plants respond to pathogen attack by activating a variety of defense responses, including the generation of reactive oxygen species (ROS), synthesis of phytoalexins, expression of pathogenesis-related (PR) genes, cell cycle arrest, and mitochondrial dysfunction followed by a form of hypersensitive cell death known as the hypersensitive response (Nürnberger and Scheel, 2001; Greenberg and Yao, 2004; Kadota et al., 2004b). Transient membrane potential changes and Ca²⁺ influx are involved at the initial stage of defense responses (Kuchitsu et al., 1993; Pugin et al., 1997; Blume et al., 2000; Kadota et al., 2004a). Many kinds of defense responses are prevented when Ca²⁺ influx is compromised by Ca²⁺ chelators (Nürnberger and Scheel, 2001; Lecourieux et al., 2002). Since complex spatiotemporal patterns of cytosolic free Ca²⁺ concentration have been suggested to play pivotal roles in defense signaling (Nürnberger and Scheel, 2001; Sanders et al., 2002), multiple Ca²⁺ sensor proteins and their effectors should function in the defense signaling pathways. Although possible involvement of some CaM isoforms (Heo et al., 1999; Yamakawa et al., 2001), Ca²⁺-dependent protein kinases (Romeis et al., 2000, 2001; Ludwig et al., 2005; Kobayashi et al., 2007; Yoshioka et al., 2009), as well as Ca²⁺ regulation of EF-hand-containing enzymes such as ROS-generating NADPH oxidase (Ogasawara et al., 2008) have been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs have so far been implicated as signaling components in defense signaling.N-Acetylchitooligosaccharides, chitin fragments, are microbe-associated molecular patterns (MAMPs) that are recognized by plasma membrane receptors (Kaku et al., 2006; Miya et al., 2007) and induce a variety of defense responses, such as membrane depolarization (Kuchitsu et al., 1993; Kikuyama et al., 1997), ion fluxes (Kuchitsu et al., 1997), ROS production (Kuchitsu et al., 1995), phytoalexin biosynthesis (Yamada et al., 1993), and induction of PR genes (Nishizawa et al., 1999), without hypersensitive cell death in rice (Oryza sativa) cells. In contrast, a fungal proteinaceous elicitor, xylanase from Trichoderma viride (TvX)/ethylene-inducing xylanase (EIX), which is recognized by two putative plasma membrane receptors, LeEix1 and LeEix2 (Ron and Avni, 2004), triggers hypersensitive cell death along with different kinetics of ROS production and activation of a mitogen-activated protein kinase, OsMPK6, previously named as OsMPK2 or OsMAPK6, in rice cells (Kurusu et al., 2005). These two fungal MAMPs thus provide excellent model systems to study innate immunity in rice cells.This study identified two CIPKs involved in various MAMP-induced layers of defense responses, including PR gene expression, phytoalexin biosynthesis, mitochondrial dysfunction, and cell death, in rice. Molecular characterization of these CIPKs, including interaction with the putative Ca²⁺ sensors as well as their physiological functions, is discussed.     

Effects of Buprenorphine Treatment on Influenza Pathogenesis in the Ferret (Mustela putorius furo)

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups (n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

Despite decades of international research and the availability of public health countermeasures, including vaccines and antivirals, influenza viruses remain a persistent threat to human and animal health.^26,35 Influenza A viruses (IAV) exhibit a diverse range of virulence, exist in several host reservoirs, and can show rapid rates of antigenic change.²⁶ As a result, IAV are associated with both seasonal epidemics and occasional pandemics in humans,³⁵ and animal infections with IAV have become key for understanding multifactorial traits that include pathogenicity, transmissibility, and vaccine efficacy. Due to their relatively small size, adaptability to the research setting, and similarities to human lung anatomy and physiology, ferrets provide an excellent model for respiratory diseases in humans and are a valuable small-animal model for such studies.^8,30 Data generated from ferrets are included in numerous risk-assessment rubrics evaluating the pandemic potential of novel and emerging influenza viruses, including those established by the Centers for Disease Control and Prevention and the World Health Organization.^14,51The study of influenza virus in ferrets dates back to the early 1930s, when this species was first found to be susceptible to influenza virus.⁴⁴ Ferrets are naturally susceptible to both human and zoonotic IAV.⁴⁷ After infection, ferrets present with clinical signs like those of humans; these signs are often not recapitulated in other species, such as mice and guinea pigs.^28,39,46 The severity and spectrum of clinical signs associated with influenza virus–inoculated ferrets can vary, depending on the virus strain, route and dose of inoculation, and various host parameters.⁵ Whereas influenza viruses with low virulence in ferrets may cause only acute pyrexia and mild to moderate weight loss, isolates with high virulence can cause severe, systemic illness with gastrointestinal and neurologic symptoms.⁴The 3Rs, replace, reduce, refine, encourage investigation of how research involving animals can be conducted in more humane ways.^2,13,37,41 Analgesia for symptoms of influenza in ferrets represents an opportunity for refinement, but this intervention could confound research assessing disease progression. NSAID and corticosteroids are often prescribed to treat the clinical signs associated with influenza in humans.⁴³ These interventions could alter the inflammatory cascade and subsequent pathophysiology of the disease, thus reducing the validity of studies designed to characterize and compare influenza viruses.^6,43 NSAID reportedly inhibit nuclear factor κB, a regulator of inflammatory processes that is involved in viral RNA synthesis.^25,27 In addition, NSAID have been found to increase survival rates in influenza virus-infected mice.⁵³ Therefore, the use of NSAID may be problematic in studies investigating the pathogenesis of influenza viruses.Buprenorphine, an opioid, is an established analgesic in ferrets that can be administered either intravascularly, intramuscularly, or subcutaneously at 0.01 to 0.05 mg/kg with an analgesic duration of 6 to 12 h.^{11,16,24,38,52} Historically buprenorphine has been described as a partial µ receptor agonist and κ and γ receptor antagonist,^22,29,40,48 but the drug recently was described to behave as a full µ agonist.³⁶ The ceiling effect of analgesia and the immunosuppressive effects reported with other opioids have not been documented to occur with buprenorphine.^15,36,42 However, the use of buprenorphine does have the possibility of adverse effects, including sedation, weight loss, constipation, and respiratory depression.^{10,15,16,22,23,34,42} Nonetheless, buprenorphine is a commonly prescribed analgesic for numerous small mammalian species used in research settings.^20,22,40Given that influenza is an ongoing threat to human and animal health and because no replacement is available for data gained with the ferret model, pain mitigation options for research conducted in this species must be addressed. To date, concerns about altering the course of the disease have precluded the evaluation of refinements options in IAV-infected ferrets. The goal of the current study was to assess the effects of buprenorphine treatments on the pathogenesis of a seasonal IAV in ferrets; this assessment was achieved by comparing virus-inoculated ferrets that were either sham-treated or that received buprenorphine according to 2 different dosing schedules. We hypothesized that buprenorphine treatments would not affect experimental readouts, including morbidity, viral shedding, lymphopenia, and seroconversion in convalescent serum; these parameters are commonly measured during IAV research. Study results indicate that buprenorphine did not uniformly or significantly modulate disease progression, peak viral titers in the upper respiratory tract, or clinical responses used to characterize viral pathogenicity in ferrets.     

Comparison of Biomarkers of Oxidative Stress and Cardiovascular Disease in Humans and Chimpanzees (Pan troglodytes)

In the oxidative stress hypothesis of aging, the aging process is the result of cumulative damage by reactive oxygen species. Humans and chimpanzees are remarkably similar; but humans live twice as long as chimpanzees and therefore are believed to age at a slower rate. The purpose of this study was to compare biomarkers for cardiovascular disease, oxidative stress, and aging between male chimpanzees and humans. Compared with men, male chimpanzees were at increased risk for cardiovascular disease because of their significantly higher levels of fibrinogen, IGF1, insulin, lipoprotein a, and large high-density lipoproteins. Chimpanzees showed increased oxidative stress, measured as significantly higher levels of 5-hydroxymethyl-2-deoxyuridine and 8-iso-prostaglandin F_2α, a higher peroxidizability index, and higher levels of the prooxidants ceruloplasmin and copper. In addition, chimpanzees had decreased levels of antioxidants, including α- and β-carotene, β-cryptoxanthin, lycopene, and tocopherols, as well as decreased levels of the cardiovascular protection factors albumin and bilirubin. As predicted by the oxidative stress hypothesis of aging, male chimpanzees exhibit higher levels of oxidative stress and a much higher risk for cardiovascular disease, particularly cardiomyopathy, compared with men of equivalent age. Given these results, we hypothesize that the longer lifespan of humans is at least in part the result of greater antioxidant capacity and lower risk of cardiovascular disease associated with lower oxidative stress.Abbreviations: 5OHmU, 5-hydroxymethyl-2-deoxyuridine; 8isoPGF_2α, 8-iso-prostaglandin F_2α; HDL, high-density lipoprotein; IGF1, insulin-like growth factor 1; LDL, low-density lipoprotein; ROS, reactive oxygen speciesAging is characterized as a progressive reduction in the capacity to withstand the stresses of everyday life and a corresponding increase in risk of mortality. According to the oxidative stress hypothesis of aging, much of the aging process can be accounted for as the result of cumulative damage produced by reactive oxygen species (ROS).^{6,21,28,41,97} Endogenous oxygen radicals (that is, ROS) are generated as a byproduct of normal metabolic reactions in the body and subsequently can cause extensive damage to proteins, lipids, and DNA.^6,41 Various prooxidant elements, in particular free transition metals, can catalyze these destructive reactions.⁶ The damage caused by ROS can be counteracted by antioxidant defense systems, but the imbalance between production of ROS and antioxidant defenses, over time, leads to oxidative stress and may contribute to the rate of aging.^28,97Oxidative stress has been linked to several age-related diseases including neurodegenerative diseases, ophthalmologic diseases, cancer, and cardiovascular disease.^21,28,97 Of these, cardiovascular disease remains the leading cause of adult death in the United States and Europe.⁷¹ In terms of cardiovascular disease, oxidative stress has been linked to atherosclerosis, hypertension, cardiomyopathy, and chronic heart failure in humans.^55,78,84 Increases in oxidant catalysts (prooxidants)—such as copper, iron, and cadmium—have been associated with hypertension, coronary artery disease, atherosclerosis, and sudden cardiac death.^98,102,106 Finally, both endogenous and exogenous antioxidants have been linked to decreased risk of cardiovascular disease, although the mechanisms behind this relationship are unclear.^11,52,53 However, the oxidative stress hypothesis of aging aims to explain not only the mechanism of aging and age-related diseases (such as cardiovascular disease) in humans but also the differences between aging rates and the manifestations of age-related diseases across species.The differences in antioxidant and ROS levels between animals and humans offer promise for increasing our understanding of human aging. Additional evidence supporting the oxidative stress hypothesis of aging has come from comparative studies linking differences in aging rates across taxa with both antioxidant and ROS levels.^{4,17-21,58,71,86,105} In mammals, maximum lifespan potential is positively correlated with both serum and tissue antioxidant levels.^{17,18,21,71,105} Research has consistently demonstrated that the rate of oxidative damage varies across species and is negatively correlated with maximum lifespan potential.^{4,19,20,58,71,86} However, few studies involved detailed comparisons of hypothesized biochemical indicators of aging and oxidative stress between humans and animals.⁶ This type of interspecies comparison has great potential for directly testing the oxidative stress hypothesis of aging.Much evolutionary and genetic evidence supports remarkable similarity between humans and chimpanzees.^95,100 Despite this similarity, humans have a lifespan of almost twice that of chimpanzees.^3,16,47 Most comparative primate aging research has focused on the use of a macaque model,^62,81,88 and several biochemical markers of age-related diseases have been identified in both humans and macaque monkeys.^{9,22,28,81,93,97} Several other species of monkeys have also been used in research addressing oxidative stress, antioxidant defenses, and maximum lifespan potential.^18,21,58,105 However, no study to date has examined biochemical indicators of oxidative stress and aging in chimpanzees and humans as a test of the oxidative stress hypothesis for aging. The purpose of this study is to compare biochemical markers for cardiovascular disease, oxidative stress, and aging directly between male chimpanzees and humans. Given the oxidative stress hypothesis for aging and the known role of oxidative stress in cardiovascular disease, we predict that chimpanzees will show higher levels of cardiovascular risk and oxidative stress than humans.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

CPK13, a Noncanonical Ca2+-Dependent Protein Kinase,Specifically Inhibits KAT2 and KAT1 Shaker K+ Channels and Reduces Stomatal Opening

Ca²⁺-dependent protein kinases (CPKs) form a large family of 34 genes in Arabidopsis (Arabidopsis thaliana). Based on their dependence on Ca²⁺, CPKs can be sorted into three types: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially calcium-insensitive CPKs. Here, we report on the third type of CPK, CPK13, which is expressed in guard cells but whose role is still unknown. We confirm the expression of CPK13 in Arabidopsis guard cells, and we show that its overexpression inhibits light-induced stomatal opening. We combine several approaches to identify a guard cell-expressed target. We provide evidence that CPK13 (1) specifically phosphorylates peptide arrays featuring Arabidopsis K⁺ Channel KAT2 and KAT1 polypeptides, (2) inhibits KAT2 and/or KAT1 when expressed in Xenopus laevis oocytes, and (3) closely interacts in plant cells with KAT2 channels (Förster resonance energy transfer-fluorescence lifetime imaging microscopy). We propose that CPK13 reduces stomatal aperture through its inhibition of the guard cell-expressed KAT2 and KAT1 channels.Stomata are microscopic organs at the leaf surface, each made of two so-called guard cells forming a pore. Opening or closing these pores is the way through which plants control their gas exchanges with the atmosphere (i.e. carbon dioxide uptake to feed the photosynthetic process and transpirational loss of water vapor). Stomatal movements result from osmotically driven fluxes of water, which follow massive exchanges of solutes, including K⁺ ions, between the guard cells and the surrounding tissues (Hetherington, 2001; Nilson and Assmann, 2007).Both Ca²⁺-dependent and Ca²⁺-independent signaling pathways are known to control stomatal movements (MacRobbie, 1993, 1998; Blatt, 2000; Webb et al., 2001; Mustilli et al., 2002; Israelsson et al., 2006; Marten et al., 2007; Laanemets et al., 2013). In particular, Ca²⁺ signals have been reported to promote stomatal closure through the inhibition of inward K⁺ channels and the activation of anion channels (Blatt, 1991, 1992, 2000; Thiel et al., 1992; Grabov and Blatt, 1999; Schroeder et al., 2001; Hetherington and Brownlee, 2004; Mori et al., 2006; Marten et al., 2007; Geiger et al., 2010; Brandt et al., 2012; Scherzer et al., 2012). However, little is known about the molecular identity of the links between Ca²⁺ events and Shaker K⁺ channel activity. Several kinases and phosphatases are believed to be involved in both the Ca²⁺-dependent and Ca²⁺-independent signaling pathways. Plants express two large kinase families whose activity is related to Ca²⁺ signaling. Firstly, CBL-interacting protein kinases (CIPKs; 25 genes in Arabidopsis [Arabidopsis thaliana]) are indirectly controlled by their interaction with a set of calcium sensors, the calcineurin B-like proteins (CBLs; 10 genes in Arabidopsis). This complex forms a fascinating network of potential Ca²⁺ signaling decoders (Luan, 2009; Weinl and Kudla, 2009), which have been addressed in numerous reports (Xu et al., 2006; Hu et al., 2009; Batistic et al., 2010; Held et al., 2011; Chen et al., 2013). In particular, some CBL-CIPK pairs have been shown to regulate Shaker channels such as Arabidopsis K⁺ Transporter1 (AKT1; Xu et al., 2006; Lan et al., 2011) or AKT2 (Held et al., 2011). Second, Ca²⁺-dependent protein kinases (CPKs) form an even larger family (34 genes in Arabidopsis) of proteins combining a kinase domain with the ability to bind Ca²⁺, thanks to the so-called EF hands (Harmon et al., 2000; Harper et al., 2004). CPKs, which, interestingly, are not found in animal cells, exhibit different calcium dependencies (Boudsocq et al., 2012). With respect to this, three types of CPKs can be considered: strictly Ca²⁺-dependent CPKs, Ca²⁺-stimulated CPKs (with a significant basal activity in the absence of Ca²⁺), and essentially Ca²⁺-insensitive CPKs (however, structurally close to kinases of groups 1 and 2).Pioneering work by Luan et al. (1993) demonstrated in Vicia faba guard cells that inward K⁺ channels were regulated by some Ca²⁺-dependent kinases. Then, such a Ca²⁺-dependent kinase was purified from guard cell protoplasts of V. faba and shown to actually phosphorylate the in vitro-translated KAT1 protein, a Shaker channel subunit natively expressed in Arabidopsis guard cells (Li et al., 1998). KAT1 regulation by CPK was shown by the inhibition of KAT1 currents after the coexpression of KAT1 and CDPK from soybean (Glycine max) in oocytes (Berkowitz et al., 2000). Since then, several cpk mutant lines of Arabidopsis have been shown to be impaired in stomatal movements, for example cpk10 (Ca²⁺ insensitive), cpk4/cpk11 (Ca²⁺ dependent), and cpk3/cpk6/cpk23 (Ca²⁺ dependent; Mori et al., 2006; Geiger et al., 2010; Munemasa et al., 2011; Hubbard et al., 2012).Of the nine genes encoding voltage-dependent K⁺ channels (Shaker) in Arabidopsis (Véry and Sentenac, 2002, 2003; Lebaudy et al., 2007; Hedrich, 2012), six are expressed in guard cells and play a role in stomatal movements: the Gated Outwardly-Rectifying K⁺ (GORK) gene, encoding an outward K⁺ channel subunit, and the AKT1, AKT2, Arabidopsis K⁺ Rectifying Channel1 (AtKC1), KAT1, and KAT2 genes, encoding inward K⁺ channel subunits (Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Pandey et al., 2007; Lebaudy et al., 2008a). Shaker channels result from the assembly of four subunits, and it has been shown that inward subunits tend to heterotetramerize, thus potentially widening the functional and regulatory scope of inward K⁺ conductance in guard cells (Xicluna et al., 2007; Jeanguenin et al., 2008; Lebaudy et al., 2008a, 2010). Inhibition of inward K⁺ channels has been shown to reduce stomatal opening (Liu et al., 2000; Kwak et al., 2001). This has grounded a strategy for disrupting inward K⁺ channel conductance in guard cells by expressing a nonfunctional KAT2 subunit (dominant negative mutation) in a kat2 knockout Arabidopsis line. The resulting Arabidopsis lines, named kincless, have no functional inward K⁺ channels and exhibit delayed stomatal opening (Lebaudy et al., 2008b) with, in the long term, a biomass reduction compared with the Arabidopsis wild-type line.Among the CPKs presumably expressed in Arabidopsis guard cells (Leonhardt et al., 2004), we looked for CPK13, which belongs to the atypical Ca²⁺-insensitive type of CPKs (Kanchiswamy et al., 2010; Boudsocq et al., 2012; Liese and Romeis, 2013) and whose role remains unknown in stomatal movements. Here, we confirm first that CPK13 kinase activity is independent of Ca²⁺ and show that CPK13 expression is predominant in Arabidopsis guard cells using CPK13-GUS lines. We then report that overexpression of CPK13 in Arabidopsis induces a dramatic default in stomatal aperture. Based on the previously reported kincless phenotype (Lebaudy et al., 2008b), we propose that CPK13 could reduce the activity of inward K⁺ channels in guard cells, particularly that of KAT2. We confirm this hypothesis by voltage-clamp experiments and show an inhibition of KAT2 and KAT1 activity by CPK13 (but not that of AKT2). In addition, we present peptide array phosphorylation assays showing that CPK13 targets, with some specificity, several KAT2 and KAT1 polypeptides. Finally, we demonstrate that KAT2 and CPK13 interact in planta using Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM).     

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.     

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.