Paula Grest

Vaccination against IL‐17 suppresses autoimmune arthritis and encephalomyelitis

Interleukin 17 is a T cell‐derived cytokine that induces the release of pro‐inflammatory mediators in a wide range of cell types. Recently, a subset of IL‐17‐producing T helper cells (Th17) distinct from Th1 and Th2 cells has been described, which constitutes a new T cell polarization state. Aberrant Th17 responses and overexpression of IL‐17 have been implicated in a number of autoimmune disorders including rheumatoid arthritis and multiple sclerosis. Molecules blocking IL‐17 such as IL‐17‐specific monoclonal antibodies have proved to be effective in ameliorating disease in animal models. Hitherto, active immunization targeting IL‐17 is an untried approach. Herein we explore the potential of neutralizing IL‐17 by active immunization using virus‐like particles conjugated with recombinant IL‐17 (IL‐17‐VLP). Immunization with IL‐17‐VLP induced high levels of anti‐IL‐17 antibodies thereby overcoming natural tolerance, even in the absence of added adjuvant. Mice immunized with IL‐17‐VLP had lower incidence of disease, slower progression to disease and reduced scores of disease severity in both collagen‐induced arthritis and experimental autoimmune encephalomyelitis. Active immunization against IL‐17 therefore represents a novel therapeutic approach for the treatment of chronic inflammatory diseases.

Active immunization with IL‐1 displayed on virus‐like particles protects from autoimmune arthritis

IL‐1 is an important mediator of inflammation and a major cause of tissue damage in rheumatoid arthritis (RA). Therapeutic administration of recombinant IL‐1 receptor antagonist (IL‐1Ra) is efficacious in reducing clinical symptoms of disease, but suffers from several drawbacks, including the need for frequent administrations of large amounts. Here, we show that immunization of mice with either IL‐1α or IL‐1β chemically cross‐linked to virus‐like particles (VLP) of the bacteriophage Qβ elicited a rapid and long‐lasting autoantibody response. The induced Ab efficiently neutralized the binding of the respective IL‐1 molecules to their receptors in vitro and their pro‐inflammatory activities in vivo. In the collagen‐induced arthritis model, both vaccines strongly protected mice from inflammation and degradation of bone and cartilage. Moreover, immunization with either vaccine showed superior efficacy than daily administrations of high amounts of IL‐1Ra. In the T and B cell‐independent collagen Ab transfer model, immunization with the IL‐1β vaccine strongly protected from arthritis, whereas immunization with the IL‐1α vaccine had no effect. Our results suggest that active immunization with IL‐1α, and especially IL‐1β conjugated to Qβ VLP, might become an efficacious and cost‐effective new treatment option for RA and other systemic IL‐1‐dependent inflammatory disorders.

Contrast-enhanced power and color Doppler ultrasonography of the pancreas in healthy and diseased cats

BACKGROUND The diagnosis of feline pancreatic disease is difficult, because clinical abnormalities and routine noninvasive diagnostic tests are unreliable. OBJECTIVE The purpose of this study was to investigate by Doppler ultrasonography if vascularity and blood volume differs in the otherwise ultrasonographically normal and diseased feline pancreas. ANIMALS Thirty-six client owned cats. METHODS The pancreas was examined with B-mode and contrast-enhanced color and power Doppler ultrasonography. Doppler images were analyzed with a computer program: parameter fractional area represents a vascularity index and color-weighted fractional area assesses blood volume. RESULTS Based on the B-mode findings, the pancreas was considered normal in 11 clinically healthy cats and diseased in 25 cats of which 4 were clinically healthy and 21 had clinical signs consistent with pancreatic disease. Histologic or cytologic samples were taken in all diseased pancreata. Fifteen samples were of diagnostic quality: purulent or mixed cellular inflammation (8), nodular hyperplasia (4), and neoplasia (3) were identified. Vascularity and blood volume for all Doppler methods was significantly higher in cats with pancreatic disease. Significantly higher Doppler values were detected with power Doppler than with color Doppler, and with postcontrast color and power Doppler than with precontrast Doppler technologies. CONCLUSION Contrast-enhanced Doppler ultrasonography appears feasible in the feline pancreas. Significant differences were found between normal cats and those with evidence of pancreatic pathology. Further studies are needed to evaluate its use for the differentiation of pancreatic disorders and in cats suspected to have pancreatic disease but without B-mode ultrasonographic changes of the pancreas.

Double-blind placebo-controlled study with interleukin-18 and interleukin-12-encoding plasmid DNA shows antitumor effect in metastatic melanoma in gray horses

Melanoma is a disease with high incidence in gray horses and has limited therapeutic options in metastatic disease. Gene therapy has shown some success in animal models and human patients. A randomized double-blind, placebo-controlled study was conducted to investigate 2 treatment options using cytokine-encoding plasmid DNA in horses with metastatic melanoma to induce immunologic antitumor effects. Adult gray horses with spontaneously occurring metastatic melanoma (n=26) were included in the study. Treatment of 26 gray horses with metastatic melanoma consisted of interleukin-18-encoding plasmid DNA, interleukin-12-encoding plasmid DNA, or empty plasmid DNA (control group), injected intratumorally, respectively. Tumor response was assessed using ultrasound and caliper measurements and histologic assessment of tumor biopsies. Significant tumor regression could be shown in both the treatment groups receiving IL-18 and IL-12-encoding plasmid DNA whereas placebo-treated control patients showed tumor growth over the course of the treatment. In addition, 7 of 10 tumors from horses treated with IL-18 or IL-12 showed peritumoral and/or intratumoral inflammatory infiltrates after treatment compared with 1 of the 6 in the control group. The treatment as assessed by serial blood draws and clinical investigation, was safe and well tolerated. These data suggest that the intratumoral treatment with IL-18 and IL-12-encoding plasmid DNA has antitumor effects, which is well tolerated and thus holds promise for the treatment of patients with metastatic melanoma.

EcPV2 DNA in equine papillomas and in situ and invasive squamous cell carcinomas supports papillomavirus etiology

Equine penile papillomas, in situ carcinomas, and invasive carcinomas are hypothesized to belong to a continuum of papillomavirus-induced diseases. The former ones clinically present as small grey papules, while the latter 2 lesions are more hyperplasic or alternatively ulcerated. To test the hypothesis that these lesions are papillomavirus-induced, samples of 24 horses with characteristic clinical and histologic findings of penile papillomas or in situ or invasive squamous cell carcinomas were collected. As controls, 11 horses with various lesions—namely, Balanoposthitis (6 cases), melanoma (3 cases), follicular cyst (1 case), and amyloidosis (1 case)—were included. DNA was extracted and polymerase chain reaction applied to amplify papillomavirus DNA. The respective primers were designed to amplify DNA of the recently discovered equine papillomavirus EcPV2. All tested papilloma and squamous cell carcinoma samples were found to contain DNA of either of 2 previously published EcPV2 variants. Among the other samples 6 of 11 were found to contain EcPV2 DNA. To further support the findings and to determine where the papillomavirus DNA was located within the lesions, an in situ hybridization for the detection of EcPV2 DNA was established. The samples tested by this technique were found to clearly contain papillomavirus nucleic acid concentrated in the nucleus of the koilocytes. The findings of this study support previous data and the hypothesis that papillomaviruses induce the described penile lesions in horses.

Urinary catecholamine and metadrenaline to creatinine ratios in dogs with a phaeochromocytoma.

Urinary adrenaline (epinephrine), noradrenaline, dopamine, metadrenaline (metanephrine) and normetadrenaline to creatinine ratios were measured from spot samples of seven client-owned dogs with a histologically confirmed phaeochromocytoma. Urine was collected on day 0 in the hospital in six dogs, and additionally on days 2, 6 and 7 after discharge in two of these dogs. In one dog, urine was sampled on day 7 only. Samples were also collected from 10 healthy control dogs on days 0, 1 and 7. In dogs with phaeochromocytomas, normetadrenaline:creatinine ratios at all time points ranged from 103 to 6430 nmol/mmol. From day 0, ratios of samples taken at the hospital (range 157 to 925 nmol/mmol) were significantly higher (P<0.0012) compared with control samples (range 14 to 91 nmol/mmol). The highest normetadrenaline:creatinine ratios were found in two dogs with bilateral phaeochromocytomas. Adrenaline:creatinine and noradrenaline:creatinine ratios were also significantly increased (P<0.016) in dogs with a phaeochromocytoma at day 0 compared with controls, although the difference was less pronounced than that between controls and dogs with a phaeochromocytoma for the normetadrenaline:creatinine ratio. Urine normetadrenaline:creatinine ratios may be useful in the diagnosis of canine phaeochromocytomas.

POSSIBLE HUMAN-AVIAN TRANSMISSION OF MYCOBACTERIUM TUBERCULOSIS IN A GREEN-WINGED MACAW (ARA CHLOROPTERA)

Abstract This report describes a case of Mycobacterium tuberculosis infection in a green-winged macaw (Ara chloroptera), confirmed by microbiologic and pathologic diagnostics, and notes a possible human–avian transmission. Clinical signs included cutaneous swellings, profound leukocytosis, and signs of osteomyelitis in the long bones. Proliferation consisted of several nodules with small greenish-caseous foci in cross-section and revealed a severe granulomatous inflammation with intralesional acid-fast rods. Mycobacterium tuberculosis was isolated from subcutaneous nodules and biochemically confirmed. The disease in avian species is of zoonotic importance.

Microbiologic evaluation of gallbladder bile of healthy dogs and dogs with iatrogenic hypercortisolism: a pilot study.

BACKGROUND In people, hypercortisolism (HC) has been associated with acalculous cholecystitis and biliary dyskinesia, which may potentiate ascending biliary infections. In dogs, an association between HC and gallbladder disease recently has been documented, although the role of bacteria remains controversial. Furthermore, there is no information on the gallbladder bile microbial flora in healthy dogs. OBJECTIVES To investigate the microbial flora in gallbladder bile in healthy dogs, the relationship between iatrogenic hyperadrenocorticism and bactibilia and possible changes in biliary microbial flora after cortisol withdrawal in dogs. ANIMALS Six control dogs and 6 dogs treated with hydrocortisone. METHODS Gallbladder bile obtained by percutaneous ultrasound-guided cholecystocentesis was cultured aerobically and anaerobically and examined cytologically before (d0), during (d28, d56, d84), and after (d28p, d56p, d84p) administration of hydrocortisone (8 mg/kg PO q12h). RESULTS In the control group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp.; Escherichia coli on d0) and 1/42 bile smears had cytological evidence of bacteria (d28). In the HC group, 2/42 bile cultures yielded bacterial growth (Enterococcus sp. on d28; Bacillus sp. on d28p) and 3/42 bile smears had cytological evidence of bacteria (d84, d84, d28p). All dogs remained healthy throughout the study period (168d). CONCLUSIONS AND CLINICAL IMPORTANCE Based on the results of conventional bacterial culture techniques, gallbladder bile of healthy dogs periodically may harbor bacteria, which do not appear to be clinically relevant. A 3-month period of iatrogenic HC was not associated with bactibilia. A higher prevalence of bactibilia may be detected with micromolecular techniques.

A Virus-Like Particle-Based Anti-Nerve Growth Factor Vaccine Reduces Inflammatory Hyperalgesia: Potential Long-Term Therapy for Chronic Pain

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. For a substantial proportion of patients, conventional drug treatments do not provide adequate pain relief. Consequently, novel approaches to pain management, involving alternative targets and new therapeutic modalities compatible with chronic use, are being sought. Nerve growth factor (NGF) is a major mediator of chronic pain. Clinical testing of NGF antagonists is ongoing, and clinical proof of concept has been established with a neutralizing mAb. Active immunization, with the goal of inducing therapeutically effective neutralizing autoreactive Abs, is recognized as a potential treatment option for chronic diseases. We have sought to determine if such a strategy could be applied to chronic pain by targeting NGF with a virus-like particle (VLP)-based vaccine. A vaccine comprising recombinant murine NGF conjugated to VLPs from the bacteriophage Qβ (NGFQβ) was produced. Immunization of mice with NGFQβ induced anti-NGF–specific IgG Abs capable of neutralizing NGF. Titers could be sustained over 1 y by periodic immunization but declined in the absence of boosting. Vaccination with NGFQβ substantially reduced hyperalgesia in collagen-induced arthritis or postinjection of zymosan A, two models of inflammatory pain. Long-term NGFQβ immunization did not change sensory or sympathetic innervation patterns or induce cholinergic deficits in the forebrain, nor did it interfere with blood-brain barrier integrity. Thus, autovaccination targeting NGF using a VLP-based approach may represent a novel modality for the treatment of chronic pain.

Long-term follow up of feline leukemia virus infection and characterization of viral RNA loads using molecular methods in tissues of cats with different infection outcomes.

It is a remarkable feature for a retrovirus that an infection with feline leukemia virus (FeLV) can result in various outcomes. Whereas some cats contain the infection and show a regressive course, others stay viremic and succumb to the infection within a few years. We hypothesized, that differences in the infection outcome might be causally linked to the viral RNA and provirus loads within the host and these loads therefore may give additional insight into the pathogenesis of the virus. Thus, the goals of the present study were to follow-up on experimentally infected cats and investigate tissues from cats with different infection outcomes using sensitive, specific TaqMan real-time PCR and reverse transcriptase (RT)-PCR. Nineteen experimentally FeLV-A/Glasgow-1-infected cats were categorized into having regressive, progressive or reactivated FeLV infection according to follow-up of FeLV p27 antigen detection in the blood. Remarkably, regressively infected cats showed detectable provirus and viral RNA loads in almost all of the 27 tested tissues, even many years after virus exposure. Moreover, some regressively infected cats reactivated the infection, and these cats had intermediate to high viral RNA and provirus tissue loads. The highest loads were found in viremic cats, independent of their health status. Tissues that represented sites of virus replication and shedding revealed the highest viral RNA and provirus loads, while the lowest loads were present in muscle and nerve tissues. A supplementary analysis of 20 experimentally infected cats with progressive infection revealed a median survival time of 3.1 years (range from 0.6 to 6.5 years); ∼70% (n=14) of these cats developed lymphoma, while leukemia and non-regenerative anemia were observed less frequently. Our results demonstrate that the different infection outcomes are associated with differences in viral RNA and provirus tissue loads. Remarkably, no complete clearance of FeLV viral RNA or provirus was detected in cats with regressive infection, even up to 12 years after exposure. In several cases FeLV reactivation could be observed. Thus, retroviruses integrated as provirus into the hosts genome, could not be eliminated completely by the host and maintained their full potential for replication and reactivation.