CD8+ T Cell Response to Gammaherpesvirus Infection Mediates Inflammation and Fibrosis in Interferon Gamma Receptor-Deficient Mice.

Idiopathic pulmonary fibrosis (IPF), one of the most severe interstitial lung diseases, is a progressive fibrotic disorder of unknown etiology. However, there is growing appreciation for the role of viral infection in disease induction and/or progression. A small animal model of multi-organ fibrosis, which involves murine gammaherpesvirus (MHV68) infection of interferon gamma receptor deficient (IFNγR-/-) mice, has been utilized to model the association of gammaherpesvirus infections and lung fibrosis. Notably, several MHV68 mutants which fail to induce fibrosis have been identified. Our current study aimed to better define the role of the unique MHV68 gene, M1, in development of pulmonary fibrosis. We have previously shown that the M1 gene encodes a secreted protein which possesses superantigen-like function to drive the expansion and activation of Vß4+ CD8+ T cells. Here we show that M1-dependent fibrosis is correlated with heightened levels of inflammation in the lung. We observe an M1-dependent cellular infiltrate of innate immune cells with most striking differences at 28 days-post infection. Furthermore, in the absence of M1 protein expression we observed reduced CD8+ T cells and MHV68 epitope specific CD8+ T cells to the lungs-despite equivalent levels of viral replication between M1 null and wild type MHV68. Notably, backcrossing the IFNγR-/- onto the Balb/c background, which has previously been shown to exhibit weak MHV68-driven Vß4+ CD8+ T cell expansion, eliminated MHV68-induced fibrosis-further implicating the activated Vß4+ CD8+ T cell population in the induction of fibrosis. We further addressed the role that CD8+ T cells play in the induction of fibrosis by depleting CD8+ T cells, which protected the mice from fibrotic disease. Taken together these findings are consistent with the hypothesized role of Vß4+ CD8+ T cells as mediators of fibrotic disease in IFNγR-/- mice.

Massive occlusive thrombosis of the pulmonary artery in pigtailed macaques chronically infected with R5-tropic simian-human immunodeficiency virus.

BACKGROUND: Pulmonary arterial hypertension (PAH) has been identified as a serious complication of HIV infection. METHODS AND RESULTS: Here, we report sudden death in two pigtailed macaques (Macaca nemestrina) chronically infected (~1-2 years post-infection) with an R5 SHIV strain. At necropsy, total occlusion of the pulmonary artery by a large fibrin thrombus was present in both animals. CONCLUSION: This report describes pulmonary vascular lesions similar to PAH in R5 SHIV-infected pigtail macaques.

Early lymphoid responses and germinal center formation correlate with lower viral load set points and better prognosis of simian immunodeficiency virus infection.

We have investigated the dynamics of germinal center (GC) formation in lymphoid tissues following acute SIV infection. SIV induces a marked follicular hyperplasia, associated with an aberrant accumulation of nonproliferating T follicular helper cells within GCs, but with an abundance of cells producing IL-21, demonstrating that the mechanisms involved for these two events appear independent. IL-21-stimulated T follicular helper cells are considered a critical element for GC formation, a physiological process that seems dysregulated and excessive during HIV/SIV infection, contributing to lymphoid pathogenesis. However, the data suggest that the kinetics by which such GCs are formed may be an important predictor of the host-pathogen equilibrium, as early GC hyperplasia was associated with better control of viral replication. In contrast, monkeys undergoing fast disease progression upon infection exhibited an involution of GCs without local IL-21 production in GCs. These results provide important clues regarding GC-related hyperimmune responses in the context of disease progression within various individuals during HIV/SIV infection and may open novel therapeutic avenues to limit lymphoid dysfunction, postinfection.

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.

PAS-positive extracellular deposits within germinal centers of hyperplastic follicles during SIV infection in a rhesus macaque.

BACKGROUND: Lymphoid tissue remodeling is characteristic of chronic simian immunodeficiency virus infection. METHODS: A rhesus macaque infected with SIVmac239 was necropsied and its lymphoid tissues subjected to histopathology characterization. RESULTS: Germinal centers in spleen and lymph nodes contained PAS-positive, non-amyloid extracellular deposits, decreased T follicular helper cells, and normal density of Ki67(+) B cells. CONCLUSIONS: A possible mechanism for PAS-positive deposits includes exaggerated involution of SIV-induced follicular hyperplasia secondary to virus-associated immune reaction.

Behavioral, clinical and pathological effects of multiple daily intraperitoneal injections on female mice.

Pharmacological agents are commonly administered to mice through multiple intraperitoneal (i.p.) injections. The i.p. route of administration is usually considered safe, but questions of animal welfare arise when protocols require that multiple injections be given to the same animal. IACUCs must consider the potential risks associated with multiple i.p. injections in order to determine the maximum number of injections an animal can receive within a study protocol, but there are no published studies of such potential risks. The authors investigated the effects of 30 daily i.p. saline injections on the behavior, body condition, weight, fecal corticosterone levels, hematology and pathology of female adult mice. Results indicate that multiple i.p. injections do not cause any ill effects in mice.

Thyroid autoantibodies are rare in nonhuman great apes and hypothyroidism cannot be attributed to thyroid autoimmunity.

The great apes include, in addition to Homo, the genera Pongo (orangutans), Gorilla (gorillas), and Pan, the latter comprising two species, P. troglodytes (chimpanzees) and P. paniscus (bonobos). Adult-onset hypothyroidism was previously reported in 4 individual nonhuman great apes. However, there is scarce information on normal serum thyroid hormone levels and virtually no data for thyroid autoantibodies in these animals. Therefore, we examined thyroid hormone levels and TSH in all nonhuman great ape genera including adults, adolescents, and infants. Because hypothyroidism in humans is commonly the end result of thyroid autoimmunity, we also tested healthy and hypothyroid nonhuman great apes for antibodies to thyroglobulin (Tg), thyroid peroxidase (TPO), and the TSH receptor (TSHR). We established a thyroid hormone and TSH database in orangutans, gorillas, chimpanzees, and bonobos (447 individuals). The most striking differences are the greatly reduced free-T4 and free-T3 levels in orangutans and gorillas vs chimpanzees and bonobos, and conversely, elevated TSH levels in gorillas vs Pan species. Antibodies to Tg and TPO were detected in only 2.6% of adult animals vs approximately 10% in humans. No animals with Tg, TPO, or TSHR antibodies exhibited thyroid dysfunction. Conversely, hypothyroid nonhuman great apes lacked thyroid autoantibodies. Moreover, thyroid histology in necropsy tissues was similar in euthyroid and hypothyroid individuals, and lymphocytic infiltration was absent in 2 hypothyroid animals. In conclusion, free T4 and free T3 are lower in orangutans and gorillas vs chimpanzees and bonobos, the closest living human relatives. Moreover, thyroid autoantibodies are rare and hypothyroidism is unrelated to thyroid autoimmunity in nonhuman great apes.

Closely related influenza viruses induce contrasting respiratory tract immunopathology.

The swine-origin H1N1 virus which emerged in 2009 resulted in the first influenza pandemic of the 21(st) century. Although the majority of infections were moderate, a significant proportion of infections were severe and characterized by acute respiratory distress syndrome and pulmonary edema. We compared two isolates from the 2009 H1N1 pandemic; A/California/07/09 (CA/07) and A/Netherlands/602/09 (NL/602) viruses that share greater than 99% sequence identity. Though genetically similar, these viruses exhibit contrasting pathological effects. Mice that were infected with 800 plaque forming unit (PFU) of CA/07 virus rapidly lost weight, which was concurrent with detection of high pulmonary concentrations of MCP-1, MIG, IP-10 and TIMP-1. Initially, severe bronchiolar epithelial necrosis and acute respiratory distress was observed, followed by marked bronchiolar epithelial hyperplasia. Mononuclear cell infiltration was initially localized to perivascular and peribronchiolar interstitium and then spread to adjacent alveoli. Infiltrating cells were phenotypically CD11b(hi), F4/80(lo). In contrast, when mice were infected with 800 PFU of NL/602 virus, minimal weight loss was observed, and concentrations of cytokines in the lung were significantly lower. Inflammation was primarily restricted to the bronchioles and perivascular interstitium with minimal spread to alveoli. Infiltrating cells include foamy macrophages and surface markers were characterized as CD11b(lo/-), F4/80(hi). These two genetically similar viruses can be useful strains with which to investigate immune-regulatory determinants of pathogenesis of influenza virus.

Safety and efficacy of topical lime sulfur in mice infested with Myocoptes musculinus.

Current treatment options for murine fur mites have limitations in safety and efficacy. This study evaluated whether topical lime sulfur (LS) is an adjunct or alternative to traditional treatment options for Myocoptes musculinus. To evaluate the safety of topical LS, mice were dipped in a 3% LS solution at 34 and 41 d of age. Mice were observed daily for side effects and mortality, with blood work and necropsy at 42 d of age to evaluate for pathologic changes. To determine the efficacy of topical LS, postweanling mice infested with M. musculinus were treated with LS once weekly for 2 wk and then housed with uninfested sentinel mice for 4 wk. Weekly tape tests and postmortem tape tests and skin scrapings were performed on all mice. Treated postweanling mice had significantly lower Hgb levels and higher BUN levels than did control animals. In mite-infested mice, the number of positive cages at euthanasia was the same between treated and control animals. Although topical LS did not cause gross or microscopic changes to organ systems, it may cause clinicopathologic changes, and topical LS is not effective as a sole treatment for M. musculinus infestation of postweanling mice.

Retrograde ejaculation associated spontaneous sperm cystolithiasis in four rhesus macaques (Macaca mulatta).

Retrograde ejaculation (RE) has been reported in humans and animals but RE with subsequent sperm calculi has rarely been reported. This report documents clinical and pathological findings of spontaneous sperm cystolithiasis in four rhesus macaques. While this condition has been associated with repeated electroejaculation, spontaneous sperm cystolithiasis is highly unusual. The animals presented with either stranguria, dysuria, hematuria, distended abdomen or lethargy. Ultrasound examination revealed several hyperechoic masses within the lumen of the urinary bladder. The animals were euthanized due to poor prognosis or study end points. Postmortem examination revealed multiple angular, amorphous, soft to firm, pale yellow to greenish-brown and variably sized calculi in the lumen of the urinary bladder or prostatic/penile urethra. Histologically, the calculi were composed of numerous sperm embedded in abundant brightly eosinophilic matrix. Based on gross and histologic findings, RE associated sperm cystolithiasis was diagnosed, with ulcerative urethritis as the major primary apparent etiology. To the authors' knowledge, this is the first report of four spontaneous cases of sperm cystolithiasis in rhesus macaques.

Post-transplant lymphoproliferative disorder associated with immunosuppressive therapy for renal transplantation in rhesus macaques (Macaca mulatta).

Human post-transplant lymphoproliferative disorder (PTLD) is an abnormal lymphoid proliferation that arises in 1-12% of transplant recipients as a consequence of prolonged immunosuppression and Epstein-Barr viral infection (EBV). Nonhuman primates, primarily rhesus macaques (Macaca mulatta), have been used extensively in research models of solid organ transplantation, as the nonhuman primate immune system closely resembles that of the human. Lymphocryptovirus of rhesus monkeys has been characterized and shown to be very similar to EBV in humans in regards to its cellular tropism, host immune response, and ability to stimulate B lymphocyte proliferation and lymphomagenesis. Thus, it appears that the NHP may be an appropriate animal model for EBV-associated lymphoma development in humans. The clinical management of post-transplant nonhuman primates that are receiving multiple immunosuppressive agents can be complicated by the risk of PTLD and other opportunistic infections. We report 3 cases of PTLD in rhesus macaques that illustrate this risk potential in the setting of potent immunosuppressive therapies for solid organ transplantation.

Osteochondromatosis in a Rhesus macaque (Macaca mulatta).

A 5-y-old, male, rhesus macaque (Macaca mulatta) presented with a prominent mass slightly anteriomedial to the right stifle. On exam, multiple radiopaque masses were identified protruding from the mid- and distal femur. Lateral and anteroposterior radiographs of the right stifle region revealed multiple exophytic masses arising from the femur, with mild bony reaction of the proximal tibia. Histologic examination of biopsy tissue revealed woven and lamellar bone with granulation tissue and skeletal muscle. Because the macaque was exhibiting no lameness or signs of pain, we decided to monitor the progression of the masses. Minimal change was noted during the time prior to study termination at 6.5 y of age. Necropsy revealed that the bony masses were cartilage-capped lesions arising near the growth plate of the distal femur and midshaft of the femur and tibia. Histologic examination revealed chondro-osseous exophytic growths that blended imperceptibly with the cortex and spongiosa of the femur, consistent with a final diagnosis of multiple osteochondromas.

Spontaneous gastric carcinomas in sooty mangabeys (Cercocebus atys).

Sooty mangabeys (Cercocebus atys) are native to West Africa and are a natural host of SIV, which is implicated in the origin of HIV2. They have been used in studies of AIDS pathogenesis, leprosy, immune responses, reproductive biology, and behavior. Spontaneous tumors have rarely been reported in this species. However, we noted spontaneous gastric carcinomas in 8 sooty mangabeys. Four male and 4 female mangabeys had mild to severe chronic weight loss, with abdominal distention in 5 of 8 animals. At necropsy, 7 of the 8 mangabeys had prominent large ulcerated masses with severe, diffuse thickening of the pyloric wall at or near the gastric-duodenal junction, which often partially occluded the gastric lumen. Early carcinoma was an incidental finding in one mangabey. Histologically, all of the tumors were classified as adenocarcinomas. Adenocarcinomas were noncircumscribed with infiltrates of neoplastic epithelial cells, often arranged in acini. In 3 mangabeys, these infiltrates were transmural and invaded surrounding tissue locally. The adenocarcinomas were locally invasive, with metastasis to regional lymph nodes in 2 animals, but widespread metastasis was not seen. Anisocytosis, anisokaryosis, and high mitotic rates were seen in all 8 tumors. In the samples available, serology and Steiner stain did not detect Helicobacter, and immunohistochemistry failed to reveal Helicobacter or Epstein-Barr virus, 2 potential causes for human gastric carcinomas.

Toxicity and toxicokinetics of the cyclin-dependent kinase inhibitor AG-024322 in cynomolgus monkeys following intravenous infusion.

PURPOSE: Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects. METHODS: Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred at > or =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose. RESULTS: No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen at > or =6 mg/kg. AG-024322 at > or =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes at > or =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent. CONCLUSION: The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.

Cutaneous lesions in the rat following administration of an irreversible inhibitor of erbB receptors, including the epidermal growth factor receptor.

CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, including the epidermal growth factor (EGF) receptor. Various inhibitors of the EGF receptor, including CI-1033, have resulted in cutaneous toxicity in humans as a common adverse event. In a chronic toxicity study in rats, CI-1033 produced cutaneous lesions with morphologic characteristics similar to that reported in man. Here the authors describe in detail the dermal changes observed, along with other noteworthy findings of that study. Male and female Wistar rats (15/sex/group) were administered CI-1033 for 27 weeks at 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m(2), respectively) by gavage. Control animals (15/sex) received vehicle alone (aqueous 0.5% methylcellulose) in a dose volume of 5 mL/kg. Six animals/sex/dose were included for toxicokinetic evaluations. Skin lesions were the primary drug-related toxicity and occurred at > or = 2.5 mg/kg in a dose-dependent fashion. The major gross lesions were papules that evolved into crusts and scales that were first observed in weeks 1 and 3, respectively. Alopecia developed in conjunction with the papular eruptions. Skin changes were most pronounced in females, possibly due to higher drug levels. In week 13, CI-1033 plasma AUC(0-24) values were 527 to 1980 ng.h/mL in males and 844 to 2920 ng x h/mL in females at 2.5 to 10 mg/kg. Microscopic changes could be described as 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration. Areas of hyperplasia on the tail were often associated with the development of new hair follicles. Pattern 2 was characterized by a suppurative to pyogranulomatous infundibular folliculitis. Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis; this correlated with alopecia. Elevations in bone marrow myeloid counts correlated with a peripheral leukocytosis, consistent with inflammatory changes in the dermis. In addition, hepatic cholestasis and epithelial atrophy in the gastrointestinal tract and vagina occurred at > or = 2.5 mg/kg. In conclusion, CI-1033 produced cutaneous lesions involving the epidermis and hair follicle, and the morphologic characteristics were similar to that reported in clinical studies with various inhibitors of the EGF receptor. These changes are consistent with pharmacologic inhibition of the EGF receptor in these tissues and demonstrate that the rat can serve as an animal model for investigating the mechanisms for this toxicity.

Development of a mouse model of induced Staphylococcus aureus infective endocarditis.

We developed a mouse model of Staphylococcus aureus infective endocarditis to evaluate the efficacy of experimental antibacterial compounds for this disease. Experimental infective endocarditis was produced in CD1 mice by intravenous challenge with approximately 6 log10 colony-forming units (CFU) of methicillin-sensitive (MSSA) SA-3529 or -resistant (MRSA) SA-2015 S. aureus 1 d after aortic valve trauma. Valve trauma was produced by introduction of an indwelling 32-gauge polyurethane catheter into the aortic valve via the left carotid artery. Histologic examination of MSSA- and MRSA-infected and catheterized aortic valve sections revealed neutrophilic inflammation and vegetative bacterial colonies encapsulated within fibrin along the aortic valves 1 d after infection. The MSSA or MRSA endocarditis was determined to be catheter-dependent based on catheterized mice exhibiting heart bacterial counts 4 orders of magnitude greater than those seen for noncatheterized mice. The model was validated by using a 3-d regimen of vancomycin at exposures comparable to human dosing (500 microg x h/ml). Vancomycin treatment produced statistically significant reductions of 3.4 and 3.1 log10 CFU/heart for MSSA and MRSA, respectively, relative to controls. This mouse model of endocarditis shows promise in evaluating the predictive efficacy of antibiotics for S. aureus infective endocarditis.

Cartilage dysplasia and tissue mineralization in the rat following administration of a FGF receptor tyrosine kinase inhibitor.

PD176067 is a reversible and selective inhibitor of fibroblast growth factor receptor tyrosine kinase, and was in preclinical development as an angiogenesis inhibitor for the treatment of solid tumors. A 14-day oral toxicity study of PD176067 in young female rats (7 weeks old) was conducted at doses of 2.5, 5, and 10 mg/kg/day (15, 30, and 60 mg/m(2), respectively). Skeletal changes, and vascular and soft tissue mineralization were observed as primary drug-related toxicities. To determine if these changes are specific to young, rapidly growing animals with increased vascular and osseous development, PD176067 was administered to mature (11 months old) rats. Female rats received PD176067 by gavage for 14 days at doses of 2.5, 5, and 10 mg/kg/day and necropsied on day 15. Clinical signs of toxicity were seen at > or =5 mg/kg and one death occurred at 10 mg/kg. Physeal dysplasia (distal femur, proximal tibia, sternum) occurred in all drug-treated animals and was characterized by dose-related increased thickness of the zones of chondrocyte proliferation and hypertrophy, and marked thickening of the zone of ossification. Cartilage hyperplasia was characterized by proliferation of chondrocytes along margins of the synchondrosis and subperiosteum of sternebrae. Serum phosphorus levels increased 47% and 166% at 5 and 10 mg/kg, respectively. Mineralization of cardiac myocytes, aorta, various arteries, renal tubules, and gastric mucosa and muscularis was seen at 10 mg/kg, and consistent with the presence of calcium-phosphorus deposition. Physeal changes occurred at similar plasma PD176067 exposures in young and mature rats (AUC > or = 4.83 microg.hr/mL). PD176067 produced morphologically similar lesions in young and adult rats.