Effects of Buprenorphine and Estrous Cycle in a Murine Model of Cecal Ligation and Puncture

The effect of opioids on the immunopathology of sepsis models in mice has been controversial. In previous work, we showed that mortality and various inflammatory parameters did not differ between female mice given saline or buprenorphine after cecal ligation and puncture. To investigate further, we hypothesized that buprenorphine would not affect outcomes of sepsis at any stage of estrous. Female mice were allocated into 4 groups (n = 20 per group) according to stage of estrous. Mice then underwent cecal ligation and puncture and received either buprenorphine or saline. In 3-wk survival studies, overall survival did not differ between buprenorphine- and saline-treated mice. When mice were stratified according to stage of estrous, survival did not vary among saline-treated groups but was lower in buprenorphine-treated mice in metestrus compared with proestrus. To investigate inflammation as a potential mechanism for survival, we measured cell counts and cytokine levels in the peripheral blood and peritoneal lavage fluid at 12 and 24 h after cecal ligation and puncture. At 24 h, buprenorphine-treated mice in proestrus had more circulating neutrophils and monocytes than did saline-treated mice in proestrus and more circulating WBC than did mice in any other stage with or without buprenorphine. Our current results suggest that the effects of buprenorphine on a 50% survival model of sepsis in BALB/c female mice are minimal overall but that the stage of estrous has various effects in this model. Investigators should consider the effects of buprenorphine and estrous cycle when using female mice in sepsis research.Abbreviations: CLP, cecal ligation and punctureSepsis is a complex, multifactorial disease that remains a common cause of morbidity and mortality after events such as surgery, trauma, and burns.^2,27 Sepsis is difficult to investigate with a single animal model, but many researchers consider cecal ligation and puncture (CLP) in rodents the ‘gold standard’ in creating polymicrobial peritonitis.^5,18 However, several innate and environmental factors cause variability in CLP studies.One variable is the use of analgesia in rodents undergoing CLP. The use of perioperative analgesia for studies using major surgery is the standard of veterinary care. The 2011 Guide also reminds us that the appropriate use of analgesics in research animals is an “ethical and moral imperative.”²⁰ However, sepsis research focuses on the immune response during the course of the disease, and some analgesics, including nonsteroidal antiinflammatory drugs and opioids such as morphine and fentanyl, are known to have immunomodulatory effects.^31,37Buprenorphine is a safe and effective opioid analgesic¹⁶ for rodents and is considered to be minimally immunosuppressive.³¹ Our laboratory has shown that a specific dosing regimen of buprenorphine did not significantly affect survival or immune parameters in female ICR and C57BL/6 mice undergoing CLP,^7,19 whereas male C57BL/6 mice showed a dose-responsive decrease in survival. There were no obvious changes in immune parameters to explain the differences between male and female mice. However, there are known sex-associated differences in responses to opioids. Several reports show that opioids have greater potency, efficacy, and overall analgesia in male rodents than in female, although results looking specifically at buprenorphine are mixed.^8,35In addition to differences in response to opioids, gender may also play a role in the response to sepsis. In humans, several studies have shown that women are less susceptible to sepsis than men.^24,38 In contrast, a different group showed that women with sepsis have a worse survival rate than do men.²⁶ Similarly, contradictory literature in mice has shown either a higher or lower survival in female mice as compared with male in sepsis studies.^23,41 One possible factor for these contradictory results in both rodent and human studies is that female subjects were not grouped by phase of the estrous or menstrual cycle. In both mice and humans, fluctuations of estrogen and progesterone occur regularly through their respective cycles. Other hormonal factors such as prolactin, follicle stimulating hormone, and luteinizing hormone vary as well.¹⁴ Studies show that estrogen, prolactin, and other hormones can affect the immune system with and without sepsis,^1,22,42 leading to the concern that the changing hormonal status of female subjects may affect the responses to opioids and to sepsis. Although several sepsis and trauma studies in rodents look at specific stages of estrous^34,41 or the effects of exogenous estrogen,^9,10,33,39 few target CLP specifically, and none look at this issue in conjunction with buprenorphine.The purpose of our study was to investigate the effects of buprenorphine and the estrous cycle on a CLP model of sepsis. For these investigations, we chose to use female BALB/c mice to assess survival and immune responses. In addition, we performed survival experiments in male mice for comparison. Coupled with our previous studies, these findings will give a comprehensive profile of the major mouse strains used in sepsis research.     

Immune Response to and Histopathology of Campylobacter jejuni Infection in Ferrets (Mustela putorius furo)

Campylobacter jejuni is 1 of the most common enteric bacterial pathogens worldwide. The mechanisms of pathogenesis remain obscure, in part because of limitations of small animal models. Young ferrets develop diarrhea when fed C. jejuni, but their pathology and the immune response after infection have not been examined in detail. In the present study, we examined the pathogenesis of C. jejuni CG8421 and associated immune responses in ferrets. After oral infection with C. jejuni CG8421, 86.7% of the animals developed diarrhea and inflammatory responses that were similar to those seen in human infection. Pronounced histopathologic changes in the colonic mucosa of infected animals were observed during the acute phase (days 1 through 3) of infection. Electron micrographs of colonic epithelium revealed disruption of the villi and internalized bacteria that were not within membrane vacuoles. During the acute phase, C. jejuni was isolated from the livers of 7 of 9 (78%) animals, and bacteria were visualized immunohistochemically in the livers from 5 of the 7 animals (71%) from which C. jejuni was isolated. A vigorous systemic and mucosal immune response to Campylobacter antigens was elicited after infection of ferrets. The data presented contribute to the current knowledge of the pathogenicity of and immunologic response to C. jejuni CG8421 in ferrets and better understanding of this model.Abbreviation: ASC, antibody-secreting cellsCampylobacter jejuni are gram-negative, spiral, microaerophilic, motile bacteria and 1 of the most common enteric bacterial pathogens globally.^15,20 The annual incidence of C. jejuni diarrheal disease varies geographically, ranging from sporadic infections in young adults in developed countries²⁸ to as frequent as 40,000 per 100,000 among children living in hyperendemic regions.¹⁵ In addition, C. jejuni is the primary cause of ‘traveler''s diarrhea’ in various regions of the world.^39,41,44 The clinical disease of C. jejuni infection has a wide spectrum of symptoms, varying from a mild, transient watery diarrhea to a bloody diarrhea with severe abdominal cramps and fever. C. jejuni infection also is associated with development of Guillain–Barré syndrome.⁵¹ Most strains of C. jejuni contain lipooligosaccharides that mimic human gangliosides structurally. Antibodies against these lipooligosaccharide structures lead to an autoimmune response, resulting in Guillain–Barré syndrome.⁵²Despite extensive study, little is understood about the mechanism by which C. jejuni causes diarrheal disease. Various Campylobacter small animal models have been reported, but these require surgical manipulation,^13,46 administration of chemicals or antibiotics (or both),^17,26,30 or inoculation by an unnatural route.⁵ None of these published models incorporate the natural route of infection. Although several immunodeficient mouse models have been reported,^35,36,49 the applicability of these models for studying the pathogenesis and immune response of natural infection is limited. An adult mouse lung model has been described that may be useful for studying pathogenesis and inflammatory response and acquired immunity, but the route of infection is not the same as that in humans.^1,5 Unlike other small animals, naïve ferrets (younger than 11 wk) intragastrically inoculated with C. jejuni developed mild to moderate diarrhea with mucus, fecal leukocytes or frank blood that lasted for as long as 3 d and remained colonized for as long as 8 d.^9,10,18 Moreover, on rechallenge with the homologous bacterial strain, ferrets asymptomatically excreted C. jejuni in their stools.¹⁰ The observation of resistance to disease, but not to infection, is similar to findings from human studies, in which protection against disease developed before colonization resistance.¹¹ Ferrets have been used to evaluate vaccines against C. jejuni¹⁴ and in molecular pathogenesis studies using strain 81-176 to confirm a role in virulence for capsular polysaccharide and flagellar glycans.^3,21 However, we evaluated the histopathology and immune response after C. jejuni infection in ferrets in more detail in the present study.C. jejuni CG8421 was isolated from a case of dysentery in Thailand. It has an lipooligosaccharide core that, as shown by chemical analyses, lacks all ganglioside mimicry.³⁸ The absence of ganglioside mimicry is predicted to reduce the risk of Guillain–Barré syndrome. We selected strain CG8421 for the current studies as part of an evaluation of a potential strain for use in future human vaccine challenge studies. Here we report the pathogenesis, histopathology, inflammatory, and immune responses after oral infection of ferrets with C. jejuni strain CG8421.     

Mouse Models for the Study of SARS-CoV-2 Infection

Mice are an invaluable resource for studying virus-induced disease. They are a small, genetically modifiable animal for which a large arsenal of genetic and immunologic tools is available for evaluation of pathogenesis and potential vaccines and therapeutics. SARS-CoV-2, the betacoronavirus responsible for the COVID-19 pandemic, does not naturally replicate in wild-type mice, due to structural differences between human and mouse ACE2, the primary receptor for SARS-CoV-2 entry into cells. However, several mouse strains have been developed that allow for SARS-CoV-2 replication and clinical disease. Two broad strategies have primarily been deployed for developing mouse strains susceptible to COVID-19-like disease: adding in the human ACE2 gene and adapting the virus to the mouse ACE2 receptor. Both approaches result in mice that develop several of the clinical and pathologic hallmarks of COVID-19, including acute respiratory distress syndrome and acute lung injury. In this review, we describe key acute pulmonary and extrapulmonary pathologic changes seen in COVID-19 patients that mouse models of SARS-CoV-2 infection ideally replicate, the essential development of mouse models for the study of Severe Acute Respiratory Syndrome and Middle Eastern Respiratory Syndrome and the basis of many of the models of COVID-19, and key clinical and pathologic features of currently available mouse models of SARS-CoV-2 infection.

Coronaviruses are widespread and infect several different species. In humans, coronaviruses historically cause primarily mild respiratory diseases, such as the common cold. However, in 2003 a novel coronavirus emerged, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), which caused severe respiratory disease with high mortality.¹⁸ Since then, 2 other highly pathogenic coronaviruses from the same betacoronavirus genus have emerged: Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) in 2012, and most recently, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).^18,133,134 Spread of SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19), has resulted in a once-in-a-lifetime pandemic, and as of May 2021, more than 160 million cases and more than 3.3 million deaths have been reported worldwide.¹²³Much like SARS-CoV, SARS-CoV-2 has 4 structural proteins—spike (S), envelope (E), membrane (M), and nucleocapsid (N)—and 8 accessory proteins. The S protein includes a receptor-binding domain, which is essential for the virus to bind to and subsequently infect a host cell.¹²⁴ SARS-CoV and SARS-CoV-2 both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor and transmembrane protease serine 2 as a cofactor,^42,56,60 In contrast, the receptor for MERS-CoV is dipeptidyl peptidase 4 (DPP4).⁹¹ Because of amino acid changes in the S protein, SARS-CoV-2 binds ACE2 with a higher affinity than does SARS-CoV,¹²⁴ which may explain the greater human infectivity of SARS-CoV-2.^42,102 ACE2 is expressed throughout the body, allowing SARS-CoV-2 to potentially infect multiple organs, including the lung, heart, kidney, liver, intestines, and brain. Importantly, ACE2 is expressed on the apical surfaces of epithelial cells in these organs, permitting infection from direct viral contact with those cells.³⁷ As its name suggests, ACE2 is a critical component of the renin–angiotensin cascade, limiting vasoconstriction and promoting vasodilation by converting angiotensin II to angiotensin 1–7. ACE2 in the lung is hypothesized to reduce lung inflammation; SARS-CoV and SARS-CoV-2 potentially could exacerbate lung inflammation by altering this pathway.⁹⁸A thorough understanding of disease pathogenesis and any potential vaccines or therapeutics for any emerging pathogen is facilitated by studying animal models. For example, the first FDA-approved treatment for COVID-19 was remdesivir. This drug initially was granted emergency-use authorization for COVID-19 patients in part because of demonstrated therapeutic efficacy against MERS-CoV infection in a mouse model.¹⁰³ An ideal animal model of COVID-19 captures the wide spectrum of disease phenotypes attributed to this multifaceted disease, including organ-specific pathology and systemic changes such as hypercoagulation and cytokine storms. Animal models rarely capture every aspect of human disease, requiring the use of multiple models in order to replicate the numerous features of viral infection and disease and thereby build a comprehensive picture of what happens in human patients.SARS-CoV-2 naturally infects numerous animal species, including mink on farms, big cats in zoos and sanctuaries, and domestic dogs and cats.^{28,36,68,72,81,87,104,107} Although mink are particularly vulnerable to severe SARS-CoV-2 disease and can transmit virus to humans, they are rarely used in research studies.^68,81,87 Mice, hamsters, ferrets, and nonhuman primates are more widely used as experimental models of SARS-CoV-2 infection.⁶⁸ Hamsters replicate human disease well, are small, and are widely available, making them an attractive choice.^12,68,105 However, hamsters are less commonly used in research than are mice, and the availability of strains and reagents necessary for studying genetic and immune drivers of disease is limited for hamsters. Ferrets are frequently used as models for respiratory viruses, particularly influenza, primarily because they disperse and are susceptible to these viruses via airborne droplets, whereas rodents do not. However, ferrets do not develop severe symptoms or generate high virus titers in the lungs after SARS-CoV-2 infection.^52,68,95,104 NHPs have similar immune responses to humans and are invaluable for safety studies for preclinical trials but are costly to use and require significant infrastructure to maintain studies.^66,68,130 Mice provide a valuable model because they are widely available and relatively inexpensive, and a large arsenal of genetic and immunologic tools is available for mice. The use of mice to study SARS-CoV-2 infection provides the potential for a broader understanding of several different aspects of this complex disease.     

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.     

Evaluation of Buprenorphine in a Postoperative Pain Model in Rats

We evaluated the commonly prescribed analgesic buprenorphine in a postoperative pain model in rats, assessing acute postoperative pain relief, rebound hyperalgesia, and the long-term effects of postoperative opioid treatment on subsequent opioid exposure. Rats received surgery (paw incision under isoflurane anesthesia), sham surgery (anesthesia only), or neither and were treated postoperatively with 1 of several doses of subcutaneous buprenorphine. Pain sensitivity to noxious and nonnoxious mechanical stimuli at the site of injury (primary pain) was assessed at 1, 4, 24, and 72 h after surgery. Pain sensitivity at a site distal to the injury (secondary pain) was assessed at 24 and 72 h after surgery. Rats were tested for their sensitivity to the analgesic and locomotor effects of morphine 9 to 10 d after surgery. Buprenorphine at 0.05 mg/kg SC was determined to be the most effective; this dose induced isoalgesia during the acute postoperative period and the longest period of pain relief, and it did not induce long-term changes in opioid sensitivity in 2 functional measures of the opioid system. A lower dose of buprenorphine (0.01 mg/kg SC) did not meet the criterion for isoalgesia, and a higher dose (0.1 mg/kg SC) was less effective in pain relief at later recovery periods and induced a long-lasting opioid tolerance, indicating greater neural adaptations. These results support the use of 0.05 mg/kg SC buprenorphine as the upper dose limit for effective treatment of postoperative pain in rats and suggest that higher doses produce long-term effects on opioid sensitivity.Relief of postoperative pain is mandated in the Guide for the Care and Use of Animals¹⁸ and the Public Health Service Policy¹⁷ and is a major objective of laboratory animal medicine. Buprenorphine is one of the most commonly used opioid analgesics for postoperative pain in laboratory animals, mainly because of its long duration of action.¹⁰ The typical recommended dose range of buprenorphine in rats is 0.02 to 0.05 mg/kg SC.¹⁰ The upper end of this range, although effective at relieving acute postoperative pain in rats, is associated with side effects such as enhanced postoperative pain after the drug has worn off (rebound hyperalgesia),²³ respiratory depression,²¹ nausea or gastrointestinal distress and pica,²⁵ and neural adaptations (for example, sensitization) that may lead to long-term changes in neural function in the central nervous system and consequent changes in behavior.¹⁴ Central sensitization is a well-studied neural adaptation expressed in the brain and spinal cord and induced by nociceptive stimulation (that is, pain-induced by surgical manipulation) that manifests as hyperalgesia (decreased pain threshold to noxious stimuli) and allodynia (appearance of pain-like responses to nonnoxious tactile stimuli) during the recovery period.^16,29 Central sensitization contributes to persistent pain during the postoperative recovery period (that is, maintenance of increased pain sensitivity during tissue recovery) and chronic pain in some pathologic conditions (that is, persistent pain sensitivity after full tissue recovery). Central sensitization also accounts for the spread of hyperalgesia and allodynia to noninjured areas of the body distal to the injury.³¹ This phenomenon is referred to as ‘secondary pain’ (secondary hyperalgesia and allodynia), because it is not directly associated with the primary injury site.Opioid analgesics inhibit pain by acting on the nervous system to block transduction of pain signals traveling in sensory neurons toward the central nervous system and by facilitating activity of the descending pain inhibition neural pathway.¹⁶ Opioid analgesics also induce neural adaptations in the nervous system, phenomena that underlie the pronounced changes in behavior associated with addiction to narcotics.² Notably, opioid analgesics have been shown to enhance central sensitization initiated by pain transmission.^6,8,14,20 This property means that opiate analgesics facilitate both the inhibition of pain and central sensitization that leads to the enhancement of pain. Because central sensitization is a neural adaptation, the interaction of opiates on this pain mechanism outlasts the presence of the drug; in contrast, opiate effects on pain inhibition are limited to the presence of the drug. This arrangement is thought to account for rebound pain, that is, increased pain sensitivity after the opiate analgesic has worn off. Opiate side effects can compromise the success of recovery by increasing the level of distress experienced during recovery (for example, inducing nausea) and possibly increasing the duration of distress during recovery (for example, allowing for rebound pain). Moreover, and of importance specifically to laboratory animal medicine, the general neural adaptations induced by even a single dose of an opiate analgesic²⁶ may induce changes in the nervous system that alter and therefore compromise the validity of the animal model under study (for example, opioid mechanisms involved in behavioral control).We previously evaluated the feasibility of oral administration of buprenorphine.^15,25 As a basis for comparison, we used the ‘gold-standard’ postoperative buprenorphine dose of 0.05 mg/kg SC. The results of those studies showed that oral administration of buprenorphine was not feasible because the dose necessary to produce analgesia comparable to the standard dose of 0.05 mg/kg SC was 10 times the oral dose recommended in the literature and because the resulting concentration of oral buprenorphine was too bitter for rats to ingest voluntarily in a volume of flavored foodstuff that they could eat in a single meal.^15,25 We also observed that both subcutaneous and oral buprenorphine caused conditioned aversion to flavors,²⁵ suggestive of gastrointestinal distress⁵, with a greater effect for the oral route. Our conclusions and the associated clinical recommendation were limited by our presumption that buprenorphine at 0.05 mg/kg SC was the ideal postsurgical dose.An assessment of the literature that established this dose identified 2 problems. First, little or no research had directly assessed the effect of buprenorphine on pain sensitivity in animals in the hyperalgesic state that characterized the postoperative period,²³ and to our knowledge, no study has directly assessed the dose–response function of postsurgical buprenorphine on hyperalgesia. We hypothesized that endogenous opioids activated during the postoperative period²⁴ might act synergistically with buprenorphine to allow adequate relief of postoperative pain with a lower dose of buprenorphine than is necessary in an algesiometric test, thereby making predictions and extrapolations from algesiometric tests inaccurate. Second, we found that little consideration had been given to the consequences of other physiologic effects of buprenorphine on the recovery process (for example, gastrointestinal distress⁵, rebound hyperalgesia, and allodynia). As stated earlier, recent research on central sensitization has determined that although opioid analgesics inhibit pain sensation acutely, they also enhance neural adaptations that account for rebound pain and other long-term chronic pain conditions.^16,28,29,31 We hypothesized secondarily that a lower dose of buprenorphine, if effective acutely, would result in reduced side effects and be less likely to initiate or enhance neural adaptations, such as rebound hyperalgesia and allodynia.The current study had 2 goals. The first was to establish the minimum dose of buprenorphine needed to relieve acute postoperative pain effectively in rats. As a starting point, we defined effective relief of acute pain as the induction of isoalgesia during the postoperative period; isoalgesia is the normal level of pain sensation, in contrast to analgesia (absence of pain sensation) or hypoalgesia (lower-than-normal pain sensation). The second goal was to evaluate the effect of postoperative buprenorphine on factors that slow recovery (that is, rebound hyperalgesia and allodynia) or create long-term changes (that is, sensitization or tolerance to opiates). We tested our hypothesis by using various doses of buprenorphine in a rat model of incisional pain.^3,4,31 This model was selected because it induces cutaneous and muscular pain common to most surgery and generates mild to moderate persistent pain so that both the acute inhibitory effects of the buprenorphine (that is, pain relief) and the lasting effects of buprenorphine (that is, rebound hyperalgesia) could be studied.     

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.     

Infection of Cesarean-Derived Colostrum-Deprived Pigs with Porcine Circovirus Type 2 and Swine Influenza Virus

Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important pathogens for porcine respiratory disease complex, which is economically significant worldwide. The pathogenesis of PCV2–SIV coinfection is unknown. In this study, we focused on establishing a challenge model for PCV2 to determine whether SIV influences PCV2 replication and increases the severity of PCV2-associated disease. Cesarean-derived colostrum-deprived pigs were inoculated intratracheally with cell culture medium only (negative control group), PCV2 only, or PCV2 followed 1 wk later with SIV H1N1. Two pigs from each group were necropsied at 12, 21, 28, and 35 d after inoculation. Coinfection with SIV did not increase the number of PCV2 genomic copies in serum or target tissues or the severity of microscopic lesions associated with PCV2 in lung or lymph node. The antibody titer to PCV2 did not differ significantly between PCV2–SIV- and PCV2-infected groups. In conclusion, SIV H1N1 did not influence PCV2 replication in dually infected pigs in this study.Abbreviations: PCV2, porcine circovirus type 2; PRDC, Porcine respiratory disease complex; SIV, Swine influenza virusPorcine respiratory disease complex (PRDC) is an economically significant problem characterized by slow growth, poor food utilization, lethargy, anorexia, fever, cough, and dyspnea in pigs 16 to 22 wk of age.^14,38 PRDC is associated with complex sequential or concurrent infections with multiple viral or bacterial respiratory pathogens.^6,8,17,29 Field investigations and case-trend analyses demonstrate that porcine circovirus type 2 (PCV2) plays a role in PRDC.^12,17,24PCV2 belongs to the family Circoviridae, which contains the smallest nonenveloped, single-stranded, circular DNA viruses.^21,39 In the late 1990s, a pathogenic circovirus designated PCV2 was isolated, which differed from the nonpathogenic PCV1.^2,21 PCV2 is considered ubiquitous and can be detected in both diseased and clinically healthy pigs.¹ Infection induces various degrees of lymphoid depletion and immune suppression, demonstrated by experimentally infecting pigs with PCV2 infectious DNA clones.¹⁰ However, the factors that contribute to the pathogenicity of PCV2 remain unknown.²⁴ Generally, infection with PCV2 alone is limited in its ability to induce the full spectrum of symptoms associated with PRDC; the role of PCV2 in PRDC always involves interaction or synergism with other respiratory pathogens.^4,17Swine influenza virus (SIV) is a common pathogen associated with PRDC.^6,8 SIV is an enveloped, negative-sense, segmented RNA virus belonging to the family Orthomyxoviridae.³⁶ SIV infects the epithelium of the respiratory tract of pigs, causing an acute infection with clinical signs of cough, fever, lethargy, and anorexia beginning 1 to 2 d after experimental infection and lasting for 3 to 4 d.^14,44 High morbidity and low mortality are quite common in uncomplicated disease, but mortality usually is high when other infectious agents are present along with SIV.³⁶ Together with PCV2, SIV frequently is found in pigs with clinical signs of PRDC. At one farm, mortality reached as high as 10% in pigs coinfected with PCV2 and SIV, and 5% of the coinfected pigs failed to reach market weight.¹⁵ In a cross-sectional study, SIV infection was 11 times more likely to occur in PCV2-positive pigs compared with PCV2-negative pigs.⁸ Field studies on pigs with PRDC conducted in different years showed a 1.9% to 13% rate of coinfection with PCV2 and SIV.^{6,9,15,17,28,29} Clinical evidence suggests that SIV acts synergistically with PCV2 to cause PRDC. However, the pathogenesis of PCV2–SIV coinfection is unknown.In this study, our goal was to establish a challenge model for PCV2 and PCV2–SIV and to determine whether SIV influences PCV2 replication and increases the severity of PRDC. Throughout the study, microscopic lesions attributable to PCV2 and PCV2 viral load in serum, nasal swab, lung, and lymph node did not differ between PCV2- and PCV2–SIV-inoculated pigs. On the basis of these findings, we conclude that SIV H1N1 did not influence PCV2 replication in dually infected animals.     

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.     

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.     

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.     

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.     

Overview of Nonhuman Primate Models of SARS-CoV-2

COVID-19, the disease caused by the SARS-CoV-2 betacoronavirus, was declared a pandemic by the World Health Organization on March 11, 2020. Since then, SARS-CoV-2 has triggered a devastating global health and economic emergency. In response, a broad range of preclinical animal models have been used to identify effective therapies and vaccines. Current animal models do not express the full spectrum of human COVID-19 disease and pathology, with most exhibiting mild to moderate disease without mortality. NHPs are physiologically, genetically, and immunologically more closely related to humans than other animal species; thus, they provide a relevant model for SARS-CoV-2 investigations. This overview summarizes NHP models of SARS-CoV-2 and their role in vaccine and therapeutic development.

Coronaviruses are enveloped, single-stranded, positive-sense, RNA viruses in the subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales. There are 4 coronavirus genera, that is, Alphacoronavirus and Betacoronavirus, which infect mammals; and Gammacoronavirus and Deltacoronavirus, which primarily infect birds, with some able to infect mammals.¹³³ From these natural reservoirs, coronaviruses may infect other animals and humans. Human transmission typically requires an intermediate host.Prior to the 2002 SARS-CoV epidemic, only 2 human coronaviruses (HCoVs) had been identified - an alphacoronavirus (HCoV-229E) transmitted from bats to humans by alpacas, and a betacoronavirus (HCoV-OC43) transmitted from rodents to humans by cattle.^16,18 In 2004, HCoV-NL63 (alphacoronavirus, bat reservoir) and in 2005, HCoV-HKU1 (betacoronavirus, rodent reservoir) were identified.^39,132 Together, these 4 HCoVs cause an estimated 15% to 30% of common cold cases in humans, but can cause severe infections in infants, juvenile children, and the elderly.^23,64 However, in 2002, a new betacoronavirus caused an epidemic that originated in China, resulting in 8,000 confirmed cases with a mortality rate of 9.6%. The virus was named SARS-CoV and was transmitted from bats to humans by a palm civet intermediate host.^59,63,83 Ten years later in June 2012, MERS-CoV, a novel betacoronavirus transmitted from bats to humans by dromedary camels, emerged in Saudi Arabia.^17,25 MERS-CoV was also responsible for a 2015 outbreak in South Korea. Although human-to-human transmission of MERS-CoV was limited, the virus resulted in more than 2,000 confirmed cases and a mortality rate of approximately 35%.⁹ Elderly people and those with comorbidities were more likely to develop severe disease.⁴³Seven y later, in December 2019, another novel betacoronavirus named SARS-CoV-2, emerged in Wuhan City, Hubei Province China.^19,26 The animal reservoir responsible for transmission to humans has not been definitively identified but has been reported to be bats.^4,143 In February 2020, the World Health Organization named the disease associated with SARS-CoV-2, Corona virus disease 19 (COVID-19) and declared it a pandemic on March 11, 2020.^22,62,95 COVID-19 causes fever and pneumonia that can progress to acute respiratory distress syndrome (ARDS), multiple organ dysfunction and failure, coagulopathy, and death.³¹ Common gross findings in human autopsy specimens include lung consolidation, pulmonary edema, increased lung weight, pleurisy, white mucous and pink froth in airways, and hemorrhage. Histopathologic changes of human COVID-19 follow a timeline relative to the onset of symptoms.⁸⁶ During early infection, microvascular damage, thrombi, exudate formation, and intra-alveolar fibrin deposits occur. Epithelial changes can be present at all stages of disease, specifically diffuse alveolar damage (DAD), which includes hyaline membrane formation, epithelial denudation and pneumocyte hyperplasia. Finally, interstitial fibrosis develops about 3 wk after symptom onset.¹¹⁰ The clinical presentation of those infected with SARS-CoV-2 ranges from mild to severe to critical in 81%, 14%, and 5% of cases, respectively.^135,145 Similar to SARS-CoV and MERS-CoV, severe disease from SARS-CoV-2 is more likely in elderly individuals or in those with comorbidities.^12,72,127 In a New York City hospital study, deaths among hospital patients at the study endpoint were 3.3% or lower in patients in their 40s or younger, 4.8% among those in their 50s, 6.4% in their 60s, 12.6% in their 70s, and 25.9% in their 80s or above. Age related death rates reported by China, Italy and France are similar to the United States. Reported rates of asymptomatic infection range from 4% to 32%; however,¹²⁷ a systematic review concluded that true asymptomatic infection could be uncommon.^8,82,111,127The contagiousness of an infectious disease is referred to as the R₀, or reproduction number, and indicates the average number of people who will contract a contagious disease from someone infected with that disease. SARS-CoV (R₀ of 1.5 to 1.9)^12,72,127 and MERS-CoV (R₀ of less than 1) have R₀ values lower than SARS-CoV-2 (initial R₀ was calculated to be 2.0 to 2.5, now revised upward to 5.7) and a lower fatality rate (2.3%).^84,97 As of December 26, 2020, 78,604,532 confirmed SARS-CoV-2 cases and 1,744,235 COVID-19 related deaths have been reported worldwide.¹²⁹ The global impact of COVID-19 has been catastrophic, with adverse effects on physical and mental health, an overwhelming need for health care resources, and increased poverty and economic insecurity.⁴⁷ Effective vaccines and therapeutics are key to controlling the SARS-CoV-2 pandemic. The success of these efforts depends in part on animal models that replicate human COVID-19 disease.^52,81,105The ideal animal model for SARS-CoV-2 should be permissive to infection, have the same receptors for viral entry as in humans, and replicate the full spectrum of human COVID-19 disease and pathology.¹⁰⁹ Several publications review and compare common animal models for SARS-CoV-2 and conclude that current models simulate mild infection with full recovery, but not severe COVID-19 disease.^{13,31,52,78,81,100,105} Disease features not expressed in current animal models include ARDS, coagulopathy, systemic sequelae, and mortality.³¹This review will focus on why NHPs provide a valuable model for SARS-CoV-2 research. Using NHPs has several drawbacks as compared with small animal models, including higher purchase cost, limited availability, higher housing cost, larger space requirement, and need for specialized staff. In addition, NHPs are outbred, leading to greater variation in results among individual animals, sometimes making data analysis and interpretation difficult.^40,68 The preexisting shortage of NHPs available for biomedical research, combined with the high demand for COVID-19 research and China’s ban on the sale, transport, and export of NHPs to curtail the spread of COVID-19 (instituted January 26, 2020) has affected NHP research globally.^{3,116,138,141} Nevertheless, the scientific benefits of using NHPs for SARS-CoV-2 research outweigh these drawbacks. NHPs are physiologically, genetically, and immunologically more closely related to humans than are small animals.⁶⁸ Furthermore, the main receptor for SAR-CoV-2 binding, angiotensin l converting enzyme 2 (ACE2), in catarrhines (apes, Asian monkeys, and African monkeys) is identical to the human ACE2 receptor.^24,74 Moreover, like most humans, macaques infected with SARS-CoV-2 develop mild to moderate respiratory disease. Thus, macaques offer a relevant model to study SARS-CoV-2 pathogenesis, therapeutics, vaccines, and the impact of age and other comorbidities on disease outcome.     

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.     

Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2

Biology and tropism of MuAstV2Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains—a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD-Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Astroviruses are nonenveloped, positive-sense, single-stranded RNA viruses with a star-like appearance—from which the name derives—when examined by transmission electron microscopy. First identified in 1975, astroviruses are commonly associated with gastrointestinal illness in children.^1,23 They demonstrate considerable diversity, and unique strains have been identified in numerous species through advances in molecular diagnostics.^{2,4-6,11-13,18,19,21,25,27,29,30,32,35-40} This broad distribution likely resulted from cross-species transmission and subsequent adaptation to the novel host.¹¹ Clinical presentation varies among species, although most infections are asymptomatic or limited to mild gastrointestinal illness.^4,9,11 Extraintestinal disease resulting in fatal encephalitis has been described in several species (including cows, mink, and immunocompromised people).^{3,19,22,26,35}Astroviral infection of mice was first described in 1985, when an unknown astrovirus was identified by electron microscopy in the feces of nude mice.¹⁷ Since then, astroviruses have been detected in many wild and laboratory mouse populations.^12,29,30,34 Despite their prevalence, studies have been limited and their effects on host biology remains largely unknown. Murine astrovirus (MuAstV) was identified through molecular sequencing in 2012 and has since been discovered to be enzootic in numerous research and production mouse colonies.^12,29,34 Whether the strain described in 1985 was MuAstV is unknown. Immunocompetent and immunodeficient mouse strains are both susceptible to MuAstV infection, although no clinical disease and only minimal pathology are observed.^7,47 Similar to astroviruses infecting other species, MuAstV infection is frequently localized to the gastrointestinal tract.⁴⁷ A recent study demonstrated MuAstV replication in goblet cells and altered mucus production within the gastrointestinal tract, highlighting the potential effect of the virus on select research studies despite the lack of clinical disease and pathology.⁸Our group previously reported the detection of a novel murine astrovirus, murine astrovirus 2 (MuAstV2), in a laboratory mouse colony.³¹ MuAstV2 is genetically distinct from MuAstV but is closely related to a strain recently reported in wild mice.^31,43 The MuAstV2 strain identified in the laboratory mouse colony shares 89.2% nucleotide identity to a strain detected in wild mice in New York City but less than 50% nucleotide identity to MuAstV, the strain commonly isolated from laboratory mice. In addition, MuAstV2 was found to share as much as 80.8% nucleotide similarity to an astrovirus strain isolated from urban brown rats (Rattus norvegicus) in Hong Kong.^5,31 Antibodies to MuAstV2 were inadvertently detected in laboratory colony mice when a serologic immunoassay for mouse thymic virus prepared from a murine T-cell line tested positive. Further analysis showed that the mice were negative for mouse thymic virus and that the T-cell line was contaminated with a novel astrovirus strain similar to MuAstV2, resulting in the positive test. The observation that MuAstV2 did not appear to infect highly immunocompromised mice via natural exposure or experimental inoculation was highly unusual.³¹ This finding is distinct from other murine viruses, including MuAstV, given that infection of immunocompromised mice leads to persistent infection and chronic virus shedding.^12,15,16,47We sought to further understand the biology of MuAstV2 by evaluating viral shedding kinetics and tissue tropism in immunocompetent mice and to further characterize the presumptive resistance to infection observed in highly immunocompromised mice. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA after oral inoculation. Tissue and cell tropism were characterized using in-situ hybridization (ISH) and immunohistochemistry during the course of infection. We hypothesized that MuAstV2 initially infects the gastrointestinal tract, as occurs with other astroviruses, but speculated that components of the immune system were required to support infection or replication or both. Furthermore, we sought to characterize the extraintestinal spread of MuAstV2.