Influence of Various Factors on Circulating 25(OH) Vitamin D Concentrations in Dogs with Cancer and Healthy Dogs.

BACKGROUND: Low blood 25-hydroxyvitamin D (25(OH)D) concentrations have been associated with cancer in dogs. Little research has examined what other factors may affect 25(OH)D concentrations. OBJECTIVES: (1) To determine whether the presence of cancer (lymphoma, osteosarcoma, or mast cell tumor [MCT]) in dogs is associated with plasma 25(OH)D concentrations and (2) identify other factors related to plasma 25(OH)D concentrations in dogs. ANIMALS: Dogs newly diagnosed with osteosarcoma (n = 21), lymphoma (n = 27), and MCT (n = 21) presented to a tertiary referral oncology center, and healthy, client-owned dogs (n = 23). METHODS: An observational study design was used. Dietary vitamin D intake, sex, age, body condition score (BCS), muscle condition score (MCS), and plasma concentrations of 25(OH)D, 24,25-dihydroxyvitamin D (24,25(OH)2 D) (a marker of CYP24A1 activity), as well as ionized calcium (ICa), parathyroid hormone, and parathyroid hormone-related protein concentrations were measured. An analysis of covariance was used to model plasma 25(OH)D concentrations. RESULTS: Cancer type (P = 0.004), plasma 24,25(OH)2 D concentrations (P < 0.001), and plasma ICa concentrations (P = 0.047) had significant effects on plasma 25(OH)D concentrations. Effects of age, sex, body weight, BCS, MCS, and plasma PTH concentrations were not identified. A significant interaction between ICa and cancer was found (P = 0.005). Plasma 25(OH)D concentrations increased as ICa concentrations increased in dogs with cancer, whereas plasma 25(OH)D concentrations decreased as ICa concentrations increased in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Results support a relationship between cancer and altered vitamin D metabolism in dogs, mediated by plasma ICa concentrations. The CYP24A1 activity and plasma ICa should be measured in studies examining plasma 25(OH)D concentrations in dogs.

Evaluation of Weight Change During Carboplatin Therapy in Dogs With Appendicular Osteosarcoma.

BACKGROUND: The prevalence of cancer cachexia in veterinary medicine has not been studied widely, and as of yet, no definitive diagnostic criteria effectively assess this syndrome in veterinary patients. OBJECTIVES: (1) To determine the patterns of weight change in dogs with appendicular osteosarcoma treated with amputation and single-agent carboplatin during the course of adjuvant chemotherapy; and (2) to determine whether postoperative weight change is a negative prognostic indicator for survival time in dogs with osteosarcoma. ANIMALS: Eighty-eight dogs diagnosed with appendicular osteosarcoma. Animals were accrued from 3 veterinary teaching hospitals. METHODS: Retrospective, multi-institutional study. Dogs diagnosed with appendicular osteosarcoma and treated with limb amputation followed by a minimum of 4 doses of single-agent carboplatin were included. Data analyzed in each patient included signalment, tumor site, preoperative serum alkaline phosphatase activity (ALP), and body weight (kg) at each carboplatin treatment. RESULTS: A slight increase in weight occurred over the course of chemotherapy, but this change was not statistically significant. Weight change did not have a significant effect on survival. Institution, patient sex, and serum ALP activity did not have a significant effect on survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Weight change was not a prognostic factor in these dogs, and weight loss alone may not be a suitable method of determining cancer cachexia in dogs with appendicular osteosarcoma.

Owners' attitudes and practices regarding nutrition of dogs diagnosed with cancer presenting at a referral oncology service in Ontario, Canada.

OBJECTIVES: To investigate owner attitudes and dietary practices following cancer diagnosis in a dog. METHODS: A retrospective cross-sectional survey of 75 dog owners presenting with their dogs to a tertiary referral oncology service through a demographic questionnaire and in-person or telephone interviews regarding the dog's nutrition. RESULTS: Conventional diets (71%) were most commonly fed as a single diet to canine cancer patients followed by homemade cooked (7%) and homemade raw (4%). Several owners (18%) provided combinations of these diets. Owners reported some distrust towards conventional diets (51%). Appetite loss occurred in 35% of dogs and diet changes reported for 25% of dogs in the study involved exclusion of a conventional (63%) and/or inclusion of a homemade (54%) component. 90% of owners noted the diet change was associated with the cancer diagnosis. Supplements were given by 39% of owners. 85% of owners highly valued veterinary nutritional advice. CLINICAL RELEVANCE: Following a cancer diagnosis, dog owners appear to change their approach to managing their dog's nutrition. Given the value owners place on veterinary nutritional advice, veterinarians have a key role in guiding nutritional management of the canine cancer patient.

Comparison of concurrent imaging modalities for staging of dogs with appendicular primary bone tumours.

This study assessed the use of whole body computed tomography (CT) for the evaluation of metastasis in dogs with primary appendicular bone tumours compared to long bone survey radiography, bone scintigraphy and thoracic radiographs. Fifteen dogs were included in this pilot study. A construct reference standard was used for detection of bone metastasis, and negative thoracic radiographs were compared against CT. Definitive lesions were only identified on bone scintigraphy. Not all lesions agreed with the construct reference standard. No definitive lesions were identified on survey radiographs or CT. Lesions were identified on thoracic CT that were not visible radiographically. Equivocal ground glass pulmonary lesions progressed in three of four cases. Whole body CT was not a suitable alternative to bone scintigraphy; however, it was useful as an adjunctive diagnostic modality. Pulmonary lesions were visible on CT that were not seen radiographically and ground glass pulmonary lesions in dogs should be considered suspicious for metastasis.

The immunophenotype of peripheral blood lymphocytes in clinically healthy dogs and dogs with lymphoma in remission.

Lymphoma is a common cancer of dogs that frequently is treated with chemotherapy or radiation therapy. Response to therapy is variable and currently available diagnostic tests do not reliably predict response to therapy. Treatment for lymphoma often results in lymphopenia, but it is unknown whether the changes in circulating lymphocytes result from generalized or specific reduction of lymphocytes. In this study, blood lymphocytes from 12 clinically healthy dogs, 10 dogs in remission because of treatment for B-cell lymphoma, and 8 dogs in remission from T-cell lymphoma were analyzed by flow cytometry by using a panel of 20 antibodies reactive with canine leukocyte antigens. Results identified similar lymphocyte parameters in treated dogs regardless of the type of lymphoma. Treated dogs had >50% reduction in blood lymphocyte concentration, and an absolute decrease in most subsets of lymphocytes. Both groups of treated dogs had relative increases in the proportion of CD3+, T-cell receptor (TCR)alphabeta+, and CD90+ lymphocytes, and a decreased proportion of CD45RA+ cells. In addition, dogs with T-cell lymphoma in remission had a significant increase in the proportion of CD49d+ lymphocytes. These findings were interpreted as representing likely suppression of lymphocyte regeneration by chemotherapy, with a relative increase in the proportion of memory over naive lymphocytes. Lack of correlation with the T- or B-cell origin of the initial lymphoma suggested that, by using flow cytometric methods, residual circulating neoplastic cells could not be detected. However, the changes in the lymphocyte profile of dogs treated with chemotherapy may have relevance to their immunocompetence.

Flow cytometric immunophenotype of canine lymph node aspirates.

Increasing availability of reagents able to distinguish subtypes of lymphocytes and other leukocytes has enabled greater understanding of lymphocyte biology and pathology in the dog. Lymphocytes in circulation most commonly are subjected to immunophenotypic assessment by flow cytometry, but needle aspirates of lymph nodes can be similarly suitable for immunophenotypic examination. In this investigation, the feasibility of immunophenotyping samples obtained by needle aspiration of lymph nodes from 32 dogs with no physical abnormalities and 6 dogs with lymphoma was determined. In addition, samples from 6 dogs were stored overnight at 4 degrees C and reanalyzed 24 hours later. For each sample, stained smear preparations were examined microscopically for lymphocyte morphology, neoplasia, and the presence of inflammatory cells. Expression of antigens on a corresponding sample of aspirated cells was determined by flow cytometric detection of antibody binding on a minimum of 10,000 events. The distribution of data was determined with Anderson-Darling tests, and reference intervals incorporating the central 95% of values were established. Adequate samples were obtained from 30 of 32 clinically normal dogs. Immunophenotypic results after 24 hours of storage were consistent with those obtained immediately after sampling. Reference intervals for lymphocyte subsets from normal dog lymph nodes were similar to the proportions of CD3+, CD4+, CD8+, and CD21+ lymphocytes found in blood. Aspirates of enlarged lymph nodes from dogs with lymphoma were readily classified by this technique. Aspiration of lymph nodes from dogs for comprehensive analysis by flow cytometry is feasible and applicable to immunophenotyping of lymphoma.

Myasthenia gravis and masticatory muscle myositis in a dog.

A 21-month-old, castrated male Vizsla was presented for pelvic limb weakness, difficulty opening his mouth, ptyalism, voice change, and urinary incontinence. Myasthenia gravis and masticatory myositis were diagnosed. The unusual clinical findings, diagnosis, treatment, and case outcome are described, followed by a brief discussion of myasthenia gravis and masticatory myositis.

Potential role for extracellular glutathione-dependent ferric reductase in utilization of environmental and host ferric compounds by Histoplasma capsulatum.

The mammalian host specifically limits iron during Histoplasma capsulatum infection, and fungal acquisition of iron is essential for productive infection. H. capsulatum expresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. The H. capsulatum siderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).

Quantification of hepatic and renal cortical echogenicity in clinically normal cats.

OBJECTIVE: To quantitatively determine echogenicity of the liver and renal cortex in clinically normal cats. ANIMALS: 17 clinically normal adult cats. PROCEDURE: 3 ultrasonographic images of the liver and the right kidney were digitized from video output from each cat. Without changing the ultrasound machine settings, an image of a tissue-equivalent phantom was digitized. Biopsy specimens of the right renal cortex and liver were obtained for histologic examination. Mean pixel intensities within the region of interest (ROI) on hepatic, renal cortical, and tissue-equivalent phantom ultrasonographic images were determined by histogram analysis. From ultrasonographic images, mean pixel intensities for hepatic and renal cortical ROI were standardized by dividing each mean value by the mean pixel intensity from the tissue-equivalent phantom. RESULTS: The mean (+/- SD) standardized hepatic echogenicity value was 1.06 +/- 0.02 (95% confidence interval, 1.02 to 1.10). The mean standardized right renal cortical echogenicity value was 1.04 +/- 0.02 (95% confidence interval, 1.01 to 1.08). The mean combined standardized hepatic and renal cortical echogenicity value was 1.02 +/- 0.05 (95% confidence interval, 0.99 to 1.04). CONCLUSIONS AND CLINICAL RELEVANCE: Quantitative determination of hepatic and renal cortical echogenicity in cats is feasible, using histogram analysis, and may be useful for early detection of diffuse parenchymal disease and for serially evaluating disease progression.

Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay.

We have developed a fluorometric microtiter plate assay to quantify the internalization of Histoplasma capsulatum yeasts by macrophages. The assay utilizes the fluorescent dye Calcofluor White to label the yeast cell wall and the vital dye trypan blue, which does not enter viable macrophages, to quench fluorescence of extracellular labeled yeasts. Murine RAW 264.7 cells showed more efficient internalization of strain G217B yeasts than human U937 cells. Both cell lines exhibited a dependence upon actin, and, to a lesser degree, microtubules, in G217B uptake.

The effects of a unilateral ultrasound-guided renal biopsy on renal function in healthy sedated cats.

Complications of renal biopsies are well documented except for the change in renal function after a biopsy. Eighteen healthy, adult cats were divided into two groups (n = 9 cats/group). For the measurement of global and split renal function, Group 1 used the renal uptake of 99mTc-DTPA and Group 2 used the renal uptake of 99mTc-MAG3. Scintigraphic data were collected on days (-4), (-3), 0, 1, 2, and 4 post renal biopsy. Using ultrasound guidance, biopsies were taken from the right renal cortex on dO, before acquiring scintigraphic images. P - values less than 0.10 were considered significant due to the limited number of observations. The only statistically significant change (p = 0.08) in global renal function detected was by day following a unilateral renal biopsy. Cats imaged using 99mTc-MAG3 had discernible liver activity. A unilateral, ultrasound guided renal biopsy has minimal effect on renal function in normal, healthy sedated cats.

Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum.

Understanding how pathogens survive within the host cell is of paramount importance in the development of vaccines and therapeutic agents. This task has been particularly daunting in the study of fungal pathogens due to the lack of easily manipulated genetic systems. In recent years several molecular genetic reporter systems have been developed to identify genes expressed during the infection process and potential virulence determinants. The development of one method in particular, in vivo expression technology (IVET), has led to the discovery of several genes from various bacterial pathogens necessary for survival during infection. The recent development of molecular genetic tools for Histoplasma capsulatum has enabled us to adapt the IVET technology for this pathogenic fungus utilizing the URA5 gene, which is essential for H. capsulatum survival in mice and in cultured macrophages, as a reporter of in vivo gene expression. We report the first successful application of IVET screening of a fungal pathogen for genes expressed exclusively during infection.

Ferric reduction is a potential iron acquisition mechanism for Histoplasma capsulatum.

For the fungus Histoplasma capsulatum, and for other microbial pathogens, iron is an essential nutrient. Iron sequestration in response to infection is a demonstrated host defense mechanism; thus, iron acquisition may be considered an important pathogenic determinant. H. capsulatum is known to secrete Fe(III)-binding hydroxamate siderophores, which is one common microbial process for acquiring iron. Here, we report H. capsulatum ferric reduction activities in whole yeast cells and in both high- and low-molecular-weight fractions of culture supernatants. Each of these activities was induced or derepressed by growth under iron-limiting conditions, a phenomenon often associated with specific iron acquisition mechanisms. The high-molecular-weight culture supernatant activity was enhanced by the addition of reduced glutathione, was proteinase K sensitive and heat labile, and could utilize ferric chloride, ferric citrate, and human holotransferrin as substrates. The low-molecular-weight culture supernatant activity was resistant to proteinase K digestion. These results are consistent with the expression by H. capsulatum of both enzymatic ferric reductase and nonproteinaceous ferric reductant, both of which are regulated by iron availability. Such components could be involved in fungal acquisition of iron from inorganic or organic ferric salts, from H. capsulatum hydroxamate siderophores, or from host Fe(III)-binding proteins, such as transferrin.

Canine hepatozoonosis: comparison of lesions and parasites in skeletal muscle of dogs experimentally or naturally infected with Hepatozoon americanum.

We report previously undescribed, early lesions in skeletal muscle of dogs experimentally infected with Hepatozoon americanum by ingestion of laboratory-reared, infected Amblyomma maculatum. The earliest muscle lesion was recognized at the first interval of examination 3 weeks following exposure. The lesion consisted of a large, modified host cell whose cytoplasm frequently contained a demonstrable parasite. In skeletal muscle, the cell was consistently located between muscle fibers or in loose connective tissue adjacent to those fibers. Evidence suggesting that the parasite arrives in muscle and other tissue within the host cell cytoplasm is presented. Mucopolysaccharide encystment of the host cell, absent at this early stage, was acquired gradually and approached maximal development 26 weeks post exposure. Completion of the asexual cycle as evidenced by the presence of parasites entering vascular lumens within granulomas and also by the presence of gamonts in peripheral blood leukocytes, occurred within 28-32 days postexposure. Progression of the parasite cycle from meront to passage of zoites into vessel lumens of granulomas can occur in 11 or fewer days. The density with which parasitic lesions occur in one named skeletal muscle compared to other named muscles, although somewhat variable, was not significantly different in either experimentally induced or natural infections. The distribution of developmental stages of the parasite/lesion in four experimental infections (969 lesions) is compared with those in eight dogs with natural infections (557 lesions).

Experimental transmission of Hepatozoon americanum Vincent-Johnson et al., 1997 to dogs by the Gulf Coast tick, Amblyomma maculatum Koch.

Experimental transmission of Hepatozoon americanum to dogs was attempted with four ixodid ticks, viz., Rhipicephalus sanguineus, Amblyomma americanum, Amblyomma maculatum and Dermacentor variabilis. Ticks that dogs ingested included some that were laboratory-reared and experimentally fed as nymphs on a dog with naturally occurring hepatozoonosis; other ticks were collected as replete or partially engorged larvae, nymphs and adults from dogs that had hepatozoonosis and natural infestations of ticks. Whole ticks used to expose susceptible dogs orally were partially dissected to help release oocysts. Among eight dogs exposed, only the three that were fed A. maculatum adults experimentally acquisition fed as nymphs became infected. Dogs developed elevated body temperature and other evidence of clinical disease starting 4 weeks after exposure. 'Cysts' typical of H. americanum were found in skeletal muscle when samples were first examined 5 weeks after dogs ingested ticks, and parasites were also observed in peripheral blood smears at approximately the same time. Our study demonstrates that A. maculatum nymphs can acquire H. americanum by feeding on a parasitemic dog and that transstadial transmission of the protozoan occurs, with dogs acquiring infection when they ingest newly molted adult ticks.

Concurrent lymphangioma, immune-mediated thrombocytopenia, and von Willebrand's disease in a dog.

Lymphangioma, immune-mediated thrombocytopenia (IMT), and von Willebrand's disease (vWD) were diagnosed by histology, hematology, and a coagulation profile in a 14-month-old, female dachshund. Clinical and laboratory findings included ecchymotic inguinolabial swelling, thrombocytopenia, positive platelet factor-3 assay, prolonged buccal mucosal bleeding time, and subnormal von Willebrand factor antigen concentration and factor VIII activity. The IMT resolved with immunosuppressive glucocorticoid therapy. Histologic examination identified lymphangioma which was too extensive for surgical excision. The history and the clinical and laboratory findings were consistent with congenital vWD, although acquired vWD secondary to lymphangioma could not be ruled out.

Canine bone marrow as a potential thermal sanctuary during the plateau phase of 41.8 degrees C whole body hyperthermia.

Whole body hyperthermia (WBH) is currently being evaluated as an adjunct to various forms of antineoplastic therapy. In this regard, the uniformity of temperature in an individual subject, induced by any given WBH system, is a significant factor. Preliminary animal investigations suggested that the bone marrow temperature may differ from core temperature during 41.8 degrees C WBH. To quantitatively evaluate this possible phenomena, dogs were utilized in conjunction with a radiant heat WBH system. It was found that mean bone marrow temperature was significantly less than core (i.e. rectal) temperature (p < 0.001), i.e. 0.27 degree C for the ilium 0.40 degree C for the humerus and 0.95 degree C for the tibia. The implications of these results to current clinical trials are discussed.

A new technological approach to radiant heat whole body hyperthermia.

A new methodology for administering radiant heat whole body hyperthermia (WBH) in humans is described. The technology utilized circulates hot water in a cylinder constructed from copper tubing; the design incorporates a counter current distribution system to maintain thermal constancy. The tubing is coated with a temperature resistant high emissivity finish. Other features include a humidification system to eliminate evaporative heat losses. Data accrued from initial evaluation of this apparatus with a canine model shows that there was no detectable WBH-related hematological, biochemical or physiological toxicity. The perceived advantages of this WBH-system are discussed.

Infection of P388D1 macrophages and respiratory epithelial cells by Histoplasma capsulatum: selection of avirulent variants and their potential role in persistent histoplasmosis.

We evaluated P388D1 macrophagelike cells as model host cells for studying the intracellular survival and strain-specific virulence of Histoplasma capsulatum. Previously characterized strains which were virulent for mice destroyed monolayers of these cells within a few days. In contrast, related avirulent "smooth" variants failed to do so even after 20 days, although they persisted within P388D1 cells for at least 7 days. On the basis of this observation, we developed a quantitative radiolabel assay to use as an initial screen for virulence. Another cell type lining the respiratory tract was then examined as a potential host for H. capsulatum. Hamster trachea epithelial (HTE) cells readily internalized a variety of strains lacking alpha-(1,3)-glucan in their cell walls; however, the tracheal cells were only rarely infected by organisms possessing this polysaccharide. We subsequently inoculated HTE cells with alpha-(1,3)-glucan-positive strains and enriched for the few yeasts infecting these cells. The progeny resembled smooth variants in terms of colony morphology, the absence of alpha-(1,3)-glucan in their cell walls, and their inability to kill macrophages. Did the HTE cells select for these variant yeasts from the parent inoculum or instigate a change from the parental phenotype? Following a 3-h uptake period, only 2% of the ingested yeasts lacked alpha-(1,3)-glucan. One day later, nearly half of the intracellular organisms lacked this polymer. This rapid conversion of a large proportion of the inoculum suggests some type of environmentally triggered change, perhaps analogous to phase variation seen in many other pathogens. Infection of epithelial cells or some other nonprofessional phagocyte during natural histoplasmosis might give rise to similar variants, thus establishing a reservoir of organisms capable of causing chronic or latent infections.

Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis.

The bactericidal action of the antibiotic streptonigrin is enhanced by large intracellular iron pools. Using this observation, we have utilized a simple enrichment protocol to aid in the isolation of iron uptake mutants of N. meningitidis, based on the relative resistance of iron-starved meningococci to streptonigrin. One such mutant, FAM29, was impaired in its use of transferrin-bound iron; transferrin is the principal iron-binding protein in human plasma. FAM29 retained wild-type ability to utilize iron bound to lactoferrin, heme, or ferric citrate. FAM29 did not produce two iron-repressible outer membrane proteins, of 85,000 and 95,000 daltons, made by the parent strain. However, genetic transformation experiments indicated that the outer membrane protein alterations were not necessary for the transferrin-deficient phenotype.