Automated hematologic analysis of bone marrow aspirate samples from healthy Beagle dogs.

BACKGROUND: Interpretation of bone marrow (BM) smears typically is comprised of qualitative assessment and differential counting of cells. Analysis of BM fluid with automated hematology analyzers may provide rapid characterization of cells to supplement microscopic interpretation. OBJECTIVES: The purpose of the study was to examine the practicality and utility of analyzing BM samples in the Advia 2120 hematology analyzer; to determine if results correlate with smear assessment; and to establish descriptive statistics from hematologically normal and clinically healthy Beagle dogs. METHODS: Anticoagulated BM aspirates from 3 different sites of 26 adult Beagle dogs were collected. BM samples were analyzed in the Advia 2120, and numerical results were correlated with microscopic assessment of corresponding BM smears. Results from automated analyses and manual 500-cell differential counts were statistically analyzed. RESULTS: Forty-six samples were suitable for complete analysis. Results were available in approximately 2 (Advia) and 30 (stained and cover-slipped smear) minutes. Advia nucleated cell concentration was significantly correlated with microscopic assessment of smear particle number and smear cellularity. Significant correlations were also identified for Advia percent neutrophils with segmented, band and metamylocyte neutrophils, Advia percent lymphocytes with rubricytes, and Advia percent large unstained cells (LUC) with myeloblasts and promyelocytes. CONCLUSIONS: Automated analysis of BM aspirates was practicable, although techniques to obtain cellular samples and avoid clot formation could be improved. Automated analysis may provide rapid and useful preliminary information regarding sample cellularity, and granulocytic and erythrocytic components. Automated analysis should not supplant microscopic assessment, but may be a useful adjunct.

Comparison of manual and suction pump aspiration techniques for performing bronchoalveolar lavage in 18 dogs with respiratory tract disease.

BACKGROUND: Different aspiration techniques to retrieve bronchoalveolar lavage fluid (BALF) affect sample quality in healthy dogs. Studies evaluating these techniques in dogs with respiratory disease are lacking. OBJECTIVES: To compare sample quality of BALF acquired by manual aspiration (MA) and suction pump aspiration (SPA). ANIMALS: Eighteen client-owned dogs with respiratory disease. METHODS: Randomized, blinded prospective clinical trial. Manual aspiration was performed with a 35-mL syringe attached directly to the bronchoscope biopsy channel and SPA was performed with a maximum of 50 mmHg negative pressure applied to the bronchoscope suction valve using the suction trap connection. Both aspiration techniques were performed in each dog on contralateral lung lobes, utilizing 2 mL/kg lavage volumes per site. Samples of BALF were analyzed by percentage of retrieved infusate, total nucleated cell count (TNCC), differential cell count, semiquantitative assessment of slide quality, and diagnosis score. Data were compared by paired Student's t-test, Wilcoxon signed-rank test, chi-squared test, and ANOVA. Cohen's kappa coefficient was used to assess agreement. RESULTS: The percentage of retrieved BALF (P = .001) was significantly higher for SPA than MA. Substantial agreement was found between cytologic classification of BALF obtained with MA and SPA (kappa = 0.615). There was no significant difference in rate of definitive diagnosis achieved with cytologic assessment between techniques (P = .78). CONCLUSIONS AND CLINICAL IMPORTANCE: Suction pump aspiration, compared to MA, improved BALF retrieval, but did not significantly affect the rate of diagnostic success of bronchoalveolar lavage (BAL) in dogs with pulmonary disease.

N-terminal pro-C-natriuretic peptide and cytokine kinetics in dogs with endotoxemia.

BACKGROUND: Serum N-terminal pro-C-natriuretic peptide (NT-proCNP) concentration at hospital admission has sufficient sensitivity and specificity to differentiate naturally occurring sepsis from nonseptic systemic inflammatory response syndrome (SIRS). However, little is known about serum NT-proCNP concentrations in dogs during the course of sepsis. OBJECTIVE: To determine serum NT-proCNP and cytokine kinetics in dogs with endotoxemia, a model of canine sepsis. SAMPLES: Eighty canine serum samples. METHODS: Eight healthy adult Beagles were randomized to receive Escherichia coli lipopolysaccharide (LPS, 5 µg/kg) or placebo (0.9% NaCl) as a single IV dose in a randomized crossover study. Serum collected at 0, 1, 2, 4, and 24 hours was stored at -80°C for batch analysis. Serum NT-proCNP was measured by ELISA and 13 cytokines and chemokines by multiplex magnetic bead-based assay. RESULTS: Serum NT-proCNP concentrations did not differ significantly between LPS- and placebo-treated dogs at any time. When comparing serum cytokine concentrations, LPS-treated dogs had higher interleukin-6 (IL-6), IL-10, TNF-α and KC-like at 1, 2, and 4 hours; higher CCL2 at 1, 2, 4, and 24 hours; and higher IL-8 and CXCL10 at 4 hours compared to placebo-treated dogs. There were no differences in serum GM-CSF, IFN-γ, IL-2, IL-7, IL-15 or IL-18 between LPS- and placebo-treated dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum NT-proCNP concentration does not change significantly in response to LPS administration in healthy dogs. Certain serum cytokine and chemokine concentrations are significantly increased within 1-4 hours after LPS administration and warrant further investigation as tools for the detection and management of sepsis in dogs.

Automated analysis of bone marrow aspirates from dogs with haematological disorders.

Automated analysis of bone marrow (BM) aspirates is a useful 'pre-microscopical' screen to identify hypocellular samples and those with potentially abnormal cells. In order to determine whether automated analysis could also be used to identify haemopoietic abnormalities, EDTA-anticoagulated BM aspirates from 43 dogs were analysed using the Advia 2120 instrument. Corresponding Wright-stained BM smears were evaluated microscopically to determine smear quality, cell composition and 500-cell differential counts, and correlation to automated analysis parameters was computed. Leucocyte cytograms generated by the automated analyzer were scrutinized and compared with those of 'normal' BM. Twenty-three neoplastic and 20 non-neoplastic samples were analysed, including samples from 10 cases of acute myeloid leukaemia, four cases of acute lymphocytic leukaemia, four cases of chronic lymphocytic leukaemia, one case of chronic neutrophilic leukaemia, three cases of multiple myeloma, one case of myelodysplastic syndrome, five cases of non-regenerative immune-mediated haemolytic anaemia, one case of immune-mediated neutropenia, three cases of immune-mediated thrombocytopenia, six cases of inflammatory disease, three samples with myelotoxicity and two samples analysed for staging of neoplasia. Automated white blood cell (WBC) counts correlated significantly with smear cellularity, particle cellularity and particle number. There was a significant difference in WBC counts of samples with insufficient versus sufficient particles. Significant correlations between Advia percent neutrophils and microscopical determination of marrow segmented neutrophils/neutrophilic granulocyte reserve, Advia percent lymphocytes and microscopical determination of lymphocytes/rubricytes, Advia percent large unstained cells and microscopical determination of myeloblasts/promyelocytes and between Advia percent eosinophils and manual determination of eosinophils were identified. This suggested that Advia WBC counts may be used to approximate BM sample quality and that Advia differential counts may predict marrow granulocyte reserve and lymphocyte/rubricyte stores. Distinct and consistent alterations in cytogram patterns were observed in cases of acute leukaemia, but were less obvious in chronic leukaemia. Complete automated BM analysis was performed in approximately 2 min, while staining and coverslipping of BM slides required approximately 30 min. Hence, although automated analysis should not supplant microscopical evaluation of BM, it can provide useful ancillary information in a short time and flag potentially inadequate or abnormal samples.

Investigation of a commercial ELISA for the detection of canine procalcitonin.

BACKGROUND: Rapid identification of sepsis enables prompt administration of antibiotics and is essential to improve patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people. Commercial assays to measure canine PCT peptide have not been validated. OBJECTIVE: To investigate the validity of a commercially available enzyme-linked immunosorbent assay (ELISA) marketed for the measurement of canine PCT. ANIMALS: Three dogs with sepsis, 1 healthy dog, 1 dog with thyroid carcinoma. METHODS: Experimental study. The ELISA's ability to detect recombinant and native canine PCT was investigated and intra-assay and interassay coefficients of variability were calculated. Assay validation including mass spectrometry of the kit standard solution was performed. RESULTS: The ELISA did not consistently detect recombinant canine PCT. Thyroid lysate yielded a positive ELISA signal. Intra-assay variability ranged from 18.9 to 77.4%, while interassay variability ranged from 56.1 to 79.5%. Mass spectrometry of the standard solution provided with the evaluated ELISA kit did not indicate presence of PCT. CONCLUSIONS AND CLINICAL IMPORTANCE: The results of this investigation do not support the use of this ELISA for the detection of PCT in dogs.

Primary nasal histiocytic sarcoma of macrophage-myeloid cell type in a cat.

A 16-year-old neutered male Burmese cat was presented with a locally invasive nasal mass. The cytological and histological findings on incisional biopsy of this mass were suggestive of histiocytic sarcoma. Tumour cells expressed CD18, major histocompatibility complex class II, lysozyme and alpha-naphthyl acetate esterase; and lacked expression of CD3, CD79a, CD1a, CD1b, calprotectin, CD11c and E-cadherin. These findings are consistent with a myeloid-macrophage lineage. Metastasis to the bone marrow was present on necropsy examination. Histiocytic sarcoma should be considered in cats presented with primary round cell neoplasia of the nasal cavity.

Flow cytometry in veterinary oncology.

Flow cytometry is a highly sensitive and specific method for simultaneous analysis of multiple parameters of individual cells in a suspension. It has a range of applications in veterinary medicine, and it is increasingly used in veterinary oncology as more species-specific antibodies are generated and cross-reactivity of antibodies is characterized. Two major applications in veterinary oncology are (1) immunophenotyping with a panel of fluorescently labeled antibodies to assess expression of cell markers and (2) determination of the DNA content of cells with fluorescent dyes that bind nucleic acids. The diagnostic and prognostic value of classifying round cell tumors of animals-especially, lymphocyte proliferations-remains to be fully determined, but studies to date have indicated benefit to patient management. Similarly, determining the proliferating fraction of tumors through DNA analysis remains to be standardized and validated in veterinary oncology but shows promise as an adjunct to morphologic tumor classification. This article reviews technical aspects of flow cytometry, availability of antibodies suitable for studies in domestic animals, and applications in veterinary oncology with emphasis on characterization of round cell tumors.

Classification of canine malignant lymphomas according to the World Health Organization criteria.

A study was carried out to test the accuracy and consistency of veterinary pathologists, not specialists in hematopathology, in applying the World Health Organization (WHO) system of classification of canine lymphomas. This study represents an initiative of the ACVP Oncology Committee, and the classification has been endorsed by the World Small Animal Veterinary Association (WASVA). Tissue biopsies from cases of canine lymphoma were received from veterinary oncologists, and a study by pathologists given only signalment was carried out on 300 cases. Twenty pathologists reviewed these 300 cases with each required to choose a diagnosis from a list of 43 B and T cell lymphomas. Three of the 20 were hematopathologists who determined the consensus diagnosis for each case. The 17 who formed the test group were experienced but not specialists in hematopathology, and most were diplomates of the American or European Colleges of Veterinary Pathology. The overall accuracy of the 17 pathologists on the 300 cases was 83%. When the analysis was limited to the 6 most common diagnoses, containing 80% of all cases, accuracy rose to 87%. In a test of reproducibility enabled by reintroducing 5% of cases entered under a different identity, the overall agreement between the first and second diagnosis ranged from 40 to 87%. The statistical review included 43,000 data points for each of the 20 pathologists.

Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

Gelatinous marrow transformation and hematopoietic atrophy in a miniature horse stallion.

Gelatinous marrow transformation, or serous atrophy of bone marrow fat, has been noted in livestock, laboratory animals, and wildlife in association with an inadequate plane of nutrition, inanition, or intoxication. This is a report of gelatinous marrow transformation and hematopoietic marrow atrophy in a 5-year-old miniature horse stallion. The horse had oral malformations leading to poor food assimilation and emaciation. A bone marrow biopsy obtained to investigate persistent anemia and leukopenia showed hematopoietic atrophy and replacement of fat with a granular extracellular substance, which stained with alcian blue, consistent with acidic mucopolysaccharide content. Surgical correction of the dental abnormalities resulted in improved food assimilation, weight gain, and resolution of cytopenias. In humans, gelatinous bone marrow transformation and hematopoietic atrophy are commonly associated with malnutrition from anorexia nervosa and other causes. The cause of hematopoietic atrophy is unknown but may relate to a nonsupportive marrow microenvironment and inadequate hematopoietic substrate availability. Similar pathogenic mechanisms were suspected in this horse.

Prognostic markers for myeloid neoplasms: a comparative review of the literature and goals for future investigation.

Myeloid neoplasms include cancers associated with both rapid (acute myeloid leukemias) and gradual (myelodysplastic syndromes and myeloproliferative neoplasms) disease progression. Percentage of blast cells in marrow is used to separate acute (rapid) from chronic (gradual) and is the most consistently applied prognostic marker in veterinary medicine. However, since there is marked variation in tumor progression within groups, there is a need for more complex schemes to stratify animals into specific risk groups. In people with acute myeloid leukemia (AML), pretreatment karyotyping and molecular genetic analysis have greater utility as prognostic markers than morphologic and immunologic phenotypes. Karyotyping is not available as a prognostic marker for AML in dogs and cats, but progress in molecular genetics has created optimism about the eventual ability of veterinarians to discern conditions potentially responsive to medical intervention. In people with myelodysplastic syndromes (MDS), detailed prognostic scoring systems have been devised that use various combinations of blast cell percentage, hematocrit, platelet counts, unilineal versus multilineal cytopenias and dysplasia, karyotype, gender, age, immunophenotype, transfusion dependence, and colony-forming assays. Predictors of outcome for animals with MDS have been limited to blast cell percentage, anemia versus multilineal cytopenias, and morphologic phenotype. Prognostic markers for myeloproliferative neoplasms (eg, polycythemia vera, essential thrombocythemia) include clinical and hematological factors and in people also include cytogenetics and molecular genetics. Validation of prognostic markers for myeloid neoplasms in animals has been thwarted by the lack of a large case series that requires cooperation across institutions and veterinary specialties. Future progress requires overcoming these barriers.

Recommended guidelines for submission, trimming, margin evaluation, and reporting of tumor biopsy specimens in veterinary surgical pathology.

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Structure and function of programmed death (PD) molecules.

Programmed death (PD) molecules belong to the B7 family of co-stimulatory proteins and function in adaptive immunity. PD-1 (CD279) is expressed on lymphocytes and macrophages, and its ligand (PD-L1, CD274) on immune cells and non-hematopoietic cells. Ligation of PD-1 on lymphocytes inhibits T-cell proliferation, cytokine production, and cytolytic function by phosphorylation of immunoreceptor tyrosine-based switch motifs and blockade of T cell receptor signaling. PD-1 and PD-L1 interactions are essential to maintain peripheral immune tolerance and to modulate activation of naïve T cells. Decreased expression results in autoimmunity in mouse models, and increased expression is a key feature of chronic viral infections in humans.

Structure and sequence variation of the canine perforin gene.

Lymphocyte-mediated cytotoxicity is essential to control viral infections, limit lymphocyte expansion and activation, and survey for malignant cells. Humans with defects in lymphocyte cytotoxicity have reduced perforin function resulting in uncontrolled lymphocyte expansion, leading to excessive histiocyte activation and a hemophagocytic disorder. Dog breeds such as Bernese mountain dogs (BMD) have a high incidence of reactive and malignant diseases affecting histiocytes. This study addressed the hypothesis that changes in the perforin gene contribute to the development of hemophagocytic histiocytic sarcoma (HHS) in BMD. Canine perforin DNA was amplified and sequenced through multiple PCR assays from healthy and diseased dogs, and the gene structure determined by rapid amplification of cDNA ends. The coding component of the gene consists of 1679bp, with two exons of 536bp and 1143bp separated by an intron of 865bp. Gene configuration and location differ from that in other species although the coding sequence is highly conserved. Three silent single nucleotide polymorphisms (SNP) were identified. Analysis of their distribution indicated a consistent genotype among 6 middle-aged to older BMD without histiocytic diseases. Among samples from 10 dogs with HHS and 10 without histiocytic diseases SNP occurred with variable frequency. It was concluded that changes in the amino acid sequence of perforin were not associated with HHS but that a constellation of SNP may characterize BMD without histiocytic disease. Investigation of more dogs is required to confirm a specific genotype. Future studies should focus on the potential contribution of reduced perforin expression and/or function to HHS in dogs.

Laboratory findings, histopathology, and immunophenotype of lymphoma in domestic ferrets.

Lymphoma is a common tumor in ferrets, but anatomic distribution, histomorphology, immunophenotype, laboratory abnormalities, and response to chemotherapy are incompletely defined. In this study, lymphoma was diagnosed by histopathology of tumor tissue in 29 ferrets ranging in age from 0.8 to 8.5 years, including 12 males and 17 females. Tumors involved the viscera of the abdominal cavity (n = 11), thoracic cavity (n = 1), or abdominal and thoracic cavities (n = 7); the skin (n = 2); or the viscera of both body cavities plus other sites (n = 8). Microscopically, all tumors had diffuse architecture. Assessment by histomorphology and immunophenotype classified tumors as peripheral T-cell lymphoma (n = 17), anaplastic large T-cell lymphoma (n = 5), anaplastic large B-cell lymphoma (n = 4), diffuse large B-cell lymphoma (n = 1), and Hodgkin-like lymphoma (n = 2). Cytologic evaluation of tumor tissue was diagnostic in 11 of 13 cases. Twenty-two of 27 ferrets had anemia, 2 had leukemia, and 5 were neutropenic. Common comorbid disorders were adrenal disease (n = 27) and insulinoma (n = 6). Tumors most frequently involved mesenteric lymph nodes, while enlargement of peripheral lymph nodes was uncommon (n = 3). Ferrets with Hodgkin-like lymphoma had massive enlargement of single lymph nodes. Mean survival of ferrets not immediately euthanized was 5.0 months (T-cell lymphoma) and 8.4 months (B-cell lymphoma). Ferrets treated with chemotherapy survived an average of 4.3 months (T-cell lymphoma, n = 9) or 8.8 months (B-cell lymphoma, n = 4). Results indicate that lymphomas in ferrets most commonly affect abdominal viscera, may be amenable to cytologic diagnosis, are frequently associated with anemia and, in some cases, may be chemosensitive, resulting in relatively long survival times.

CD134 and CXCR4 expression corresponds to feline immunodeficiency virus infection of lymphocytes, macrophages and dendritic cells.

The lymphotropic lentiviruses feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) enter cells by sequential interaction with primary receptors CD134 or CD4, respectively, and subsequently with chemokine receptors. The host-cell range for FIV is broader than that for HIV, but whether this is a function of receptor expression is unknown. Lack of reagents specific to feline molecules has limited detection and analysis of receptors and their interaction with viral components. Here, the expression of CD134 and CXCR4 on feline T and B lymphocytes, dendritic cells (DCs) and macrophages was examined and the kinetics of FIV replication were assessed. Quantification of CD134 mRNA by real-time PCR indicated expression in all leukocytes, with significantly more transcripts in CD4(+) lymphocytes than in other leukocytes. Antibodies against human CD134 bound inconsistently to feline leukocytes. CXCR4 was detected with antibody clone 12G5 on the surface of monocyte-derived cells only, but gene transcripts were present in all cells, with the highest copy number in lymphocytes. CXCR4 expression decreased and CD134 expression increased with cell activation in lymphocytes. A subtype B biological isolate of FIV infected DCs, macrophages and lymphocytes, with the highest replication in CD4(+) lymphocytes, whilst cloned FIV P14 infected all cells, but replicated less efficiently. Although viral replication was lower in DCs and macrophages than in lymphocytes, DCs expressed specific receptors and were infected productively with FIV, as indicated by viral ultrastructure and DNA detection. These results may implicate altered function of DCs in the induction of specific immunity against FIV.

Clinical, laboratory, and histopathologic features of equine lymphoma.

Clinical, laboratory and tissue findings from 37 horses with lymphoma were investigated. Horses ranged in age from 0.3 to 20.5 years (median 5.0 years) and included 18 females and 19 males. Weight loss (n = 25) and ventral edema (n = 21) were the most common historical and physical abnormalities. The most common laboratory abnormalities were hyperfibrinogenemia (n = 26), hypoalbuminemia (n = 19), anemia (n = 19), leukemia (n = 14), hyperglobulinemia (n = 13), and thrombocytopenia (n = 13). Thirty-four tumors involved multiple lymphoid tissues and abdominal or thoracic organs, and 3 tumors were restricted to cutaneous and subcutaneous sites. Histopathologically, all tumors diffusely effaced normal lymph node architecture. Tumor cell morphology was heterogeneous in 17 tumors, and 8 tumors had marked histiocytic and multinucleated giant cell infiltrates. Extensive necrosis or focal fibrosis was present in 22 and 4 lymphomas, respectively. Staining of tumor sections with antibodies against CD3 and CD79alpha molecules resulted in classification of T-cell (n = 26) or B-cell (n = 7) origin. Four tumors could not be classified. Most T-cell tumors comprised small to medium CD3(+) lymphocytes, whereas 5 of 7 B-cell tumors were infiltrated by numerous small T lymphocytes and classified as T-cell-rich B-cell lymphoma. Neither estrogen nor progesterone receptor expression was consistently identified by immunochemical assessment of tumor tissues. Fresh tumor cells from 6 horses bound antibodies reactive with equine CD4, CD5, CD8, CD21, or major histocompatibility class II molecules, confirming T-cell (n = 5) or B-cell origin (n = 1). These findings suggest that T-cell lymphoma is more common than B-cell lymphoma in horses and that inflammation, possibly from tumor cytokine production, is frequent.

The effect of glucocorticoids on canine lymphocyte marker expression and apoptosis.

BACKGROUND: Glucocorticoids are commonly administered to dogs for the treatment of inflammatory disorders, autoimmunity and cancers such as lymphoma. Despite evidence of clinical efficacy, understanding of the effects of glucocorticoids on cells of the canine immune system is limited. HYPOTHESIS: Glucocorticoids affect the expression of phenotypic markers on canine lymphocytes and induce apoptosis. ANIMALS: Fifteen healthy mixed breed dogs. METHODS: Prospective randomized study. Prednisone was administered orally for 3 days, and cells aspirated from the popliteal lymph node before prednisone administration, and on days 1, 3, 10, 17, 24, and 38, were labeled with antibodies against canine CD3, CD4, CD8alpha, CD18, CD21, CD45, CD45RA, and CD90 molecules, and analyzed by flow cytometry. Additional samples were cultured in media with prednisolone for 24 hours and analyzed by cytometry for marker expression, and by gel electrophoresis for DNA fragmentation. RESULTS: Treatment of dogs with glucocorticoids resulted in reduced (p < or = .05) proportions of CD3 (days 1, 3, 17, and 24), CD4 (days 3 and 10), CD21 (day 1, 3, and 38), CD45RA (day 17) and CD90 (days 1, 10, and 17) expressing lymphocytes, and reduced intensity of CD18 (day 17) and CD45 (day 17 and 24) molecules on nodal lymphocytes. Culture oflymphocytes with prednisolone for 24 hours caused a significant reduction in the expression of all markers (p < or = .05) and DNA fragmentation. CONCLUSIONS AND CLINICAL IMPORTANCE: Glucocorticoids significantly alter the expression of phenotypic markers on canine lymphocytes, and in vitro induce apoptosis. These findings identify potential mechanisms for clinical immunosuppression from glucocorticoid treatment.

Gene-expression changes induced by Feline immunodeficiency virus infection differ in epithelial cells and lymphocytes.

Infection of cats with Feline immunodeficiency virus (FIV) is an important model for understanding comparative lentivirus biology. In vivo, FIV infects lymphocytes and monocyte/macrophages, but in vitro infection is commonly investigated in epithelial Crandell-Reese Feline Kidney (CRFK) cells. In this study, the transcriptional responses of CRFK cells and primary lymphocytes to infection with FIV 34TF, a cloned subtype A virus, and FIV USgaB01, a biological subtype B isolate, were determined. Reverse-transcribed mRNA from both cell types was hybridized to microarrays containing 1700 human expressed sequence tags in duplicate and data were analysed with Significance Analysis of Microarrays (sam) software. Results from six experiments assessing homeostatic cross-species hybridization excluded 3.48 % inconsistently detected transcripts. Analysis of data from five time points over 48 h after infection identified 132 and 24 differentially expressed genes in epithelial cells and lymphocytes, respectively. Genes involved in protein synthesis, the cell cycle, structure and metabolism were affected. The magnitude of gene-expression changes ranged from 0.62 to 1.62 and early gene induction was followed by downregulation after 4 h. Transcriptional changes in CRFK cells were distinct from those in lymphocytes, except for heat-shock cognate protein 71, which was induced at multiple time points in both cell types. These findings indicate that FIV infection induces transcriptional changes of a modest magnitude in a wide range of genes, which is probably reflective of the relatively non-cytopathic nature of virus infection.

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.