Sharon M. Dial

Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories.

This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Short-term dietary administration of celecoxib enhances the efficacy of tumor lysate-pulsed dendritic cell vaccines in treating murine breast cancer.

Cyclooxygenase‐2 (COX‐2) is a rate‐limiting enzyme in the synthesis of prostaglandins. It is over‐expressed in multiple cancers and has been associated with diminished tumor immunity. Dendritic cells (DCs) are considered candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC‐based vaccines have exhibited minimal effectiveness against established tumors. In this study, we evaluated the effect of short‐term administration of the selective COX‐2 inhibitor celecoxib on the efficacy of DC‐based vaccines in preventing and treating established 4T1 murine mammary tumors. We show that dietary celecoxib alone significantly suppresses the growth of primary tumors and the incidence of lung metastases in the prophylactic setting but is less effective against pre‐established tumors. However, we demonstrate that celecoxib administered after primary tumor establishment synergizes with tumor lysate‐pulsed DC and the adjuvant, GM‐CSF, to improve the antitumor immune response by suppressing primary tumor growth and markedly reducing the occurrence of lung metastases. This triple combination therapy elicits a tumor‐specific immune response evidenced by elevated IFN‐γ and IL‐4 secretion by CD4+ T cells and results in increased infiltration of CD4+ and CD8+ T cells to the tumor site. In addition, dietary celecoxib inhibits angiogenesis evidenced by decreased vascular proliferation within the tumor and serum vascular endothelial growth factor levels. These studies suggest that short‐term celecoxib therapy in combination with DC vaccines may be safely used for treating metastatic breast cancer.

Coccidioidomycosis in dogs and cats: A review

The dimorphic fungi Coccidioides immitis and Coccidioides posadasii are the causative agents of coccidioidomycosis. Dogs and cats residing in and visiting endemic areas are at risk of exposure to infectious arthrospores. The primary infection is pulmonary and frequently results in chronic cough. Disseminated disease is common and causes cutaneous, osseous, cardiac, ocular, nervous system, or other organ disease. Radiographic changes include a variable degree of interstitial pulmonary infiltration, hilar lymphadenopathy, and osseous lesions. Serological titers support the diagnosis, but definitive diagnosis relies on identification of Coccidioides in cytological or tissue samples. Coccidioidomycosis should be considered in any dog or cat that has been potentially exposed during the previous 3 years and is presented with chronic illness, respiratory signs, lameness, lymphadenopathy, nonhealing cutaneous lesions, or neurological, ocular, or cardiac abnormalities.

Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux–Lamy) disease

Mucopolysaccharidosis (MPS) type VI, also known as Maroteaux–Lamy disease, is an inherited disorder of glycosaminoglycan  catabolism  caused  by  deficient  activity of the lysosomal hydrolase, N‐acetylgalactosamine 4‐sulphatase (4S). A variety of prominent visceral and skeletal defects are characteristic, but primary neurological involvement has generally been considered absent. We report here that the feline model of MPS VI exhibits abnormal lysosomal storage in occasional neurones and glia distributed throughout the cerebral cortex. Abnormal lysosomal inclusions were pleiomorphic with some resembling zebra bodies and dense core inclusions typical of other MPS diseases or the membranous storage bodies characteristic of the gangliosidoses. Pyramidal neurones were shown to contain abnormal amounts of GM2 and GM3 gangliosides by immunocytochemical staining and unesterified cholesterol by histochemical (filipin) staining. Further, Golgi staining of pyramidal neurones revealed that some possessed ectopic axon hillock neurites and meganeurites similar to those described in Tay–Sachs and other neuronal storage diseases with ganglioside storage. Some animals evaluated in this study also received allogeneic bone marrow transplants, but no significant differences in neuronal storage were noted between treated and untreated individuals. These studies demonstrate that deficiency of 4S activity can lead to metabolic abnormalities in the neurones of central nervous system in cats, and that these changes may not be readily amenable to correction by bone marrow transplantation. Given the close pathological and biochemical similarities between feline and human MPS VI, it is conceivable that children with this disease have similar neuronal involvement.

Equine Colitis X Associated with Infection by Clostridium Difficile NAP1/027

A 14-year-old Quarter Horse with a 48-hr history of colic was euthanized after failure to respond to treatment. At necropsy, cecal and colonic mucosae were congested throughout, and there was segmental edema and significant thickening of the intestinal wall. Excessive numbers of mononuclear cells were found in mucosal lamina propria. Submucosal hemorrhage was diffuse and extensive, and Clostridium difficile toxins A and B were detected. Large numbers of C. difficile were isolated, and genetic characterization revealed them to be North American pulsed-field gel electrophoresis type 1, polymerase chain reaction ribotype 027, and toxinotype III. Genes for the binary toxin were present, and toxin negative–regulator tcdC contained an 18-bp deletion. This genotype comprises the current human “epidemic strain,” which is associated with human C. difficile–associated disease of greater than historical severity. The diagnosis was peracute typhlocolitis, with lesions and history typical of those attributed to colitis X.

Clinicopathologic Evaluation of the Liver

Clinicopathologic data are important tools in the evaluation of a patient with liver dysfunction. It is imperative, however, that the data not be looked at with a dogmatic approach. Many of the laboratory tests are sensitive indicators of hepatic dysfunction; however, they seldom, if ever, indicate a specific cause of hepatic dysfunction. History, physical, radiographic, and ultrasonographic examination findings, and laboratory data must be used together to fully evaluate a patient with biochemical abnormalities related to hepatic function. The prevalence of secondary hepatic disease and the difficulty of differentiating it from primary hepatic disease based on laboratory data must always be kept in mind.

Cellular immune suppressor activity resides in lymphocyte cell clusters adjacent to granulomata in human coccidioidomycosis

ABSTRACT The in situ immunologic response in human coccidioidomycosis remains undefined. To explore this further, pulmonary necrotizing coccidioidal granulomata were examined using immunohistochemical staining for lymphocyte subsets and for the cytokines interleukin-10 (IL-10) and gamma interferon (IFN-γ). Discrete perigranulomatous lymphocytic clusters were seen in eight of nine tissues examined. In these tissues, T lymphocytes (CD3+) significantly outnumbered B lymphocytes (CD20+) in the mantle area of the granulomata (P = 0.028), whereas the clusters were composed of roughly equal numbers of T and B lymphocytes. While the number of cells in the mantle expressing IL-10 was similar to those in the perigranulomatous clusters, there were significantly more cells expressing IFN-γ in the mantle than in the clusters (P = 0.037). Confocal microscopy revealed that CD4+ T lymphocytes and B lymphocytes are associated with IL-10 production. CD4+CD25+ T lymphocytes were identified in the perigranulomatous clusters but were not associated with IL-10 production. This is the first report noting perigranulomatous lymphocyte clusters and IL-10 in association with human coccidioidal granulomata and suggests that down-regulation of the cellular immune response is occurring within coccidioidal granulomata.

Vaccine-Induced Cellular Immune Responses Differ from Innate Responses in Susceptible and Resistant Strains of Mice Infected with Coccidioides posadasii†

ABSTRACT Susceptibility to Coccidioides spp. varies widely in humans and other mammals and also among individuals within a species. Among strains of mice with various susceptibilities, immunohistopathology revealed that C57BL/6 mice were highly susceptible to the disease following intranasal infection, DBA/2n mice were intermediate, and Swiss-Webster mice were innately resistant. Resistant Swiss-Webster mice developed prominent perivascular/peribronchiolar lymphocytic cuffing and well-formed granulomas with few fungal elements and debris in the necrotic center, surrounded by a mantle of macrophages, lymphocytes, and fibrocytes. Susceptible C57BL/6 mice became moribund between 14 and 18 days postinfection, with overwhelming numbers of neutrophils and spherules and very few T cells, the drastic reduction of which was associated with failure and death, while intermediate DBA/2n mice controlled the fungal burden but demonstrated progressive lung inflammation with prominent suppuration, and they deteriorated clinically. Vaccinated C57BL/6 mice had an early and robust lymphocyte response, which included significantly higher Mac2+, CD3+, and CD4+ cell scores on day 18 than those of innately resistant SW mice and DBA/2n mice; they also had prominent perivascular/peribronchiolar lymphocytic infiltrates not present in their unvaccinated counterparts, and they appeared to be resolving lesions by day 56 compared to the other two strains, based on significantly lower disease scores and observably smaller and fewer lesions with few spherules and neutrophils.

Identification of Calcium Oxalate Monohydrate Crystals by X-ray Diffraction in Urine of Ethylene Glycol-Intoxicated Dogs

Urine sediments of dogs with experimentally induced ethylene glycol poisoning were examined by light microscopy and X-ray diffraction. Massive calcium oxalate crystalluria was observed in all poisoned dogs. By light microscopy, the frequency with which six-sided hippurate-like prisms and envelope forms of calcium oxalate dihydrate occurred was approximately equal. The hippurate-like crystals were shown to be calcium oxalate monohydrate by X-ray diffractometry.

Repair pathways evident in human liver organ slices

The extension of human liver slice culture viability for several days broadens the potential of this ex vivo model for characterizing pathways of organ injury and repair, and allows for the multiple dosing of compounds. Extended viability is demonstrated by continued synthesis of GSH and ATP, and maintenance of intracellular K(+) levels. Gene expression profiling revealed the activation of regeneration pathways via increased expression of collagens (I, IV, and V), laminins, ninjurin, growth factors (EGF, epiregulin, and TGF-β1), matrix metalloproteinase-7, and insulin like growth factor 5. Collagen IV protein levels began to increase by day 4 of culture. Some markers of hepatic stellate cells, detected by RT-PCR, were up-regulated (HSP47, αSMA, pro-collagen 1a1, PDGF receptor, thrombospondin-2) with time in culture, while other markers exhibited no change or were down-regulated (αB-crystallin, synaptophysin), suggesting that the induction of regenerative pathways may in part be the role of the stellate cells as well as resident fibroblasts. Complimentary to the gene expression was evidence of regeneration in the human liver slices, as evaluated by histopathology. Improvements in organ acquisition, organ slice preparation and culture methods demonstrates that organ slice viability, integrity and morphology can be extended reproducibly for several days in culture which allows for the investigation of injury and repair processes.