Christopher Premanandan

Rabbit and Nonhuman Primate Models of Toxin-Targeting Human Anthrax Vaccines

SUMMARY The intentional use of Bacillus anthracis, the etiological agent of anthrax, as a bioterrorist weapon in late 2001 made our society acutely aware of the importance of developing, testing, and stockpiling adequate countermeasures against biological attacks. Biodefense vaccines are an important component of our arsenal to be used during a biological attack. However, most of the agents considered significant threats either have been eradicated or rarely infect humans alive today. As such, vaccine efficacy cannot be determined in human clinical trials but must be extrapolated from experimental animal models. This article reviews the efficacy and immunogenicity of human anthrax vaccines in well-defined animal models and the progress toward developing a rugged immunologic correlate of protection. The ongoing evaluation of human anthrax vaccines will be dependent on animal efficacy data in the absence of human efficacy data for licensure by the U.S. Food and Drug Administration.

Effects of disinfection on the molecular detection of porcine epidemic diarrhea virus.

Abstract Routine detection of porcine epidemic diarrhea virus (PEDV) is currently limited to RT-PCR but this test cannot distinguish between viable and inactivated virus. We evaluated the capability of disinfectants to both inactivate PEDV and sufficiently damage viral RNA beyond RT-PCR detection. Five classes of disinfectants (phenol, quaternary ammonium compound, sodium hypochlorite, oxidizing agent, and quaternary ammonium/glutaraldehyde combination) were evaluated in vitro at varying concentrations, both in the presence and absence of swine feces, and at three different temperatures. No infectious PEDV was recovered after treatment with evaluated disinfectants. Additionally, all tested disinfectants except for 0.17% sodium hypochlorite dramatically reduced qRT-PCR values. However, no disinfectants eliminated RT-PCR detection of PEDV across all replicates; although, 0.52%, 1.03% and 2.06% solutions of sodium hypochlorite and 0.5% oxidizing agent did intermittently produce RT-PCR negatives. To simulate field conditions in a second aim, PEDV was applied to pitted aluminum coupons, which were then treated with either 2.06% sodium hypochlorite or 0.5% oxidizing agent. Post-treatment surface swabs of the coupons tested RT-PCR positive but were not infectious to cultured cells or naïve pigs. Ultimately, viable PEDV was not detected following application of each of the tested disinfectants, however in most cases RT-PCR detection of viral RNA remained. RT-PCR detection of PEDV is likely even after disinfection with many commercially available disinfectants.

Serum haptoglobin–matrix metalloproteinase 9 (Hp–MMP 9) complex as a biomarker of systemic inflammation in cattle

A reliable and specific test that discriminates between acute neutrophil activation and chronic inflammatory disease may be useful in clinical decision making in a variety of conditions encountered in veterinary medical practice. An ELISA specific for neutrophil-derived haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complexes was used to determine serum concentrations of Hp-MMP 9 and was compared to ELISA assays for Haptoglobin (Hp) and matrix metalloproteinase 9 (MMP 9) in 15 animals with acute sepsis, 10 animals with chronic inflammatory or metabolic disease and 10 healthy cows. Animal disease classifications were completed prior to the determination of serum concentrations of the 3 proteins. Duration of illness, disease process and lesions observed at necropsy were used to place animals into a specific classification. The serum MMP 9 concentrations in healthy cows differed significantly from those measured in sera of acutely septic and chronically ill animals. Serum haptoglobin concentrations in healthy cows were negligible when compared to animals with acute septic or chronic diseases. There was substantial overlap in MMP 9 and Hp concentrations between acute and chronic disease animals. In contrast, serum concentrations of Hp-MMP 9 complexes found almost exclusively in sera from acutely septic animals but not in chronically ill and normal cattle. The Hp-MMP 9 ELISA may be the serological test of choice in the determination of systemic inflammation associated with bacterial sepsis.

Cyclosporine-induced immune suppression alters establishment of HTLV-1 infection in a rabbit model

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia and several lymphocyte-mediated inflammatory diseases. Persistent HTLV-1 infection is determined by a balance between host immune responses and virus spread. Immunomodulatory therapy involving HTLV-1-infected patients occurs in a variety of clinical settings. Knowledge of how these treatments influence host-virus relationships is not understood. In this study, we examined the effects of cyclosporine A (CsA)-induced immune suppression during early infection of HTLV-1. Twenty-four New Zealand white rabbits were split into 4 groups. Three groups were treated with either 10 or 20 mg/kg CsA or saline before infection. The fourth group was treated with 20 mg/kg CsA 1 week after infection. Immune suppression, plasma CsA concentration, ex vivo lymphocyte HTLV-1 p19 production, anti-HTLV-1 serologic responses, and proviral load levels were measured during infection. Our data indicated that CsA treatment before HTLV-1 infection enhanced early viral expression compared with untreated HTLV-1-infected rabbits, and altered long-term viral expression parameters. However, CsA treatment 1 week after infection diminished HTLV-1 expression throughout the 10-week study course. Collectively, these data indicate immunologic control is a key determinant of early HTLV-1 spread and have important implications for therapeutic intervention during HTLV-1-associated diseases.

Complement protein C3 binding to Bacillus anthracis spores enhances phagocytosis by human macrophages.

Alveolar macrophages are thought to play a central role in the pathogenesis of inhalational anthrax. Receptors present on macrophages that mediate phagocytosis of Bacillus anthracis spores have yet to be completely defined. To begin to determine if soluble factors that are present in the lung such as immunoglobulin and complement are involved, we characterized the binding of human IgG and C3 to the surface of B. anthracis spores at different concentrations of nonimmune human serum. Furthermore we investigated the uptake of B. anthracis spores by human monocyte-derived macrophages in the presence of nonimmune human serum. Here we show that C3b is bound to B. anthracis spores and is activated through the classical pathway by IgG bound to the spore surface. Furthermore, we show that C3 serves as an opsonin for B. anthracis spores resulting in enhanced phagocytosis by human macrophages. These studies provide evidence that nonimmune serum contains IgG which binds to B. anthracis spores but is not sufficient to initiate phagocytosis. However, surface-bound IgG does initiate the classical pathway of complement activation, which is active in the lung, resulting in deposition of the opsonin C3b on the spore surface.

Mycoplasma felis-associated meningoencephalomyelitis in a cat

Mycoplasmas are frequently isolated from many animal species. In domestic cats, mycoplasmas may be isolated from respiratory and ocular mucosae, but other sites are also occasionally colonized by these organisms. No cases of Mycoplasma species-associated neurologic disease have been reported in cats. We describe a case of Mycoplasma felis-associated meningoencephalitis in a 10-month-old domestic shorthair cat.

Multicentric mast cell tumors in a horse

A 6-year-old female Rocky Mountain horse was presented for evaluation of draining tracts and distal limb subcutaneous edema on the left front and left hind limbs that had been present for 2 weeks. Direct smears of fluid collected by fine-needle aspiration of subcutaneous fluid from both limbs were highly cellular with a predominance of eosinophils accompanied by numerous, moderately atypical, variably granulated mast cells. The cytologic diagnosis was mast cell tumor (MCT) with prominent eosinophilic infiltration with a differential diagnosis of eosinophilic granuloma. Histologic evaluation of surgical biopsies of lesions from both limbs was performed on sections stained with H&E, toluidine blue, and Luna stains. The histologic diagnosis was MCT, and staining with toluidine blue and Luna stains confirmed the presence of mast cells and eosinophils, respectively. In addition, the mast cells strongly expressed CD117. This is the first reported case of cutaneous mast cell neoplasia in a horse in which primary presenting complaints were draining tracts and distal limb subcutaneous edema involving multiple limbs. This case illustrates the utility of staining for CD117 expression in combination with traditional stains, such as toluidine blue and Luna, in differentiating MCTs from other eosinophilic lesions in horses.

MRI FEATURES OF GLIOMATOSIS CEREBRI IN A DOG

The features of gliomatosis cerebri involving the brainstem and cerebellum in a 3-year-old dog are described. In magnetic resonance (MR) images, there was diffuse loss of the cerebellar folia and cerebellar gray and white matter contrast. Multiple illdefined T2-hyperintensities were present in the cerebellar parenchyma. A poorly defined, T2-hyperintense mass effect was present ventral to the pons and rostral medulla. No contrast enhancement was noted. Cerebrospinal fluid (CSF) was normal. Postmortem examination was consistent with gliomatosis cerebri, based on compatible histopathology and immunohistochemical findings. Although rare, gliomatosis cerebri should be included as a differential for diffuse infiltrative central nervous system (CNS) lesions.

Canine pulmonary adenocarcinoma tyrosine kinase receptor expression and phosphorylation

BackgroundThis study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18–21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence.ResultsThe pro-angiogenic growth factor receptor, PDGFRα, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the corresponding normal lung tissue. The EGFR mRNA, protein expression and phosphorylation were not increased compared to the normal lung and no activating mutations were identified in exons 18–21.ConclusionsCanine pulmonary adenocarcinoma TKRs are detected at both the mRNA and protein levels and are activated. Further investigation into the contribution of TKR activation in cpAC tumorigenesis is warranted.

Early spatial and temporal events of human T-lymphotropic virus type 1 spread following blood-borne transmission in a rabbit model of infection.

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell leukemia/lymphoma (ATL) and is associated with a variety of lymphocyte-mediated disorders. HTLV-1 transmission occurs by transmission of infected cells via breast-feeding by infected mothers, sexual intercourse, and contaminated blood products. The route of exposure and early virus replication events are believed to be key determinants of virus-associated spread, antiviral immune responses, and ultimately disease outcomes. The lack of knowledge of early events of HTLV-1 spread following blood-borne transmission of the virus in vivo hinders a more complete understanding of the immunopathogenesis of HTLV-1 infections. Herein, we have used an established animal model of HTLV-1 infection to study early spatial and temporal events of the viral infection. Twelve-week-old rabbits were injected intravenously with cell-associated HTLV-1 (ACH-transformed R49). Blood and tissues were collected at defined intervals throughout the study to test the early spread of the infection. Antibody and hematologic responses were monitored throughout the infection. HTLV-1 intracellular Tax and soluble p19 matrix were tested from ex vivo cultured lymphocytes. Proviral copy numbers were measured by real-time PCR from blood and tissue mononuclear leukocytes. Our data indicate that intravenous infection with cell-associated HTLV-1 targets lymphocytes located in both primary lymphoid and gut-associated lymphoid compartments. A transient lymphocytosis that correlated with peak virus detection parameters was observed by 1 week postinfection before returning to baseline levels. Our data support emerging evidence that HTLV-1 promotes lymphocyte proliferation preceding early viral spread in lymphoid compartments to establish and maintain persistent infection.