Technical note: Assessment of neutrophil endocytosis and proteolytic degradation and its relationship with phagocytosis and oxidative burst in dairy cows.

Conventional assays of polymorphonuclear cell (PMN, neutrophil) function such as oxidative burst (OB) and phagocytosis (PC) are widely used to evaluate innate immunity in the transition period of dairy cows. Oxidative burst is commonly evaluated by measuring PMN median fluorescence intensity (MFI) involving the release of reactive oxygen species after in vitro stimulation. Phagocytosis can be measured by engulfment of fluorescent beads by PMN. DQ-ovalbumin (DQ-OVA) is a molecule suitable for the assessment of intracellular proteolytic degradation, so it might be informative about an additional pathway of pathogen handling by PMN. In this study, we evaluated the use of the DQ-OVA assay for the assessment of PMN function and the relationships among OB, PC, and DQ-OVA results in PMN isolated from blood of dairy cows between 5 and 21 d post partum. Results of the DQ-OVA validation assay were assessed with mixed linear regression models. Pearson correlation tests and kappa values for agreement were used to associate the MFI between each PMN function assay (OB, PC, and DQ-OVA). For the validation assay (9 cows in 3 replicates), PMN incubated with DQ-OVA were stimulated with IFN-γ or inhibited with cytochalasin D, and fluorescence was compared with untreated PMN. Stimulated and inhibited PMN had greater (970 ± 160 units) and lesser (593 ± 55 units) MFI relative to untreated PMN (791 ± 154 units), respectively, indicating that DQ-OVA fluorescence reflected enhanced or reduced endocytic and proteolytic function. To associate the MFI outcomes among OB, PC, and DQ-OVA, 153 samples from 40 cows were analyzed. Results showed significant, although weak association between DQ-OVA and PC MFI (Pearson r = 0.16). When values of MFI were categorized according to the first ("high" PMN functionality), second and third ("moderate" PMN functionality), or fourth ("low" PMN functionality) quartiles, agreement beyond chance (κ) was moderate: κ = 0.38 for DQ-OVA and OB, κ = 0.43 for DQ-OVA and PC, and κ = 0.43 for OB and PC. The DQ-OVA assay may complement traditional PMN functional assays because it provides additional information regarding the combination of endocytosis and proteolytic degradation, but it is not a substitute for assessment of OB or PC.

Expression of Kidney Injury Molecule-1 in Healthy and Diseased Feline Kidney Tissue.

Sensitive markers to detect acute kidney injury (AKI) in cats are lacking. Kidney injury molecule-1 (KIM-1) is a promising marker of acute tubular injury in humans, and sequence and structure of feline KIM-1 have been determined. KIM-1 is shed into urine of cats with natural AKI. The objectives of this study were to characterize temporal and cellular expression of KIM-1 in kidneys from cats without and with experimental and natural AKI using histopathology and immunohistochemistry. Tissue sections from 8 cats without kidney disease, 3 to 4 cats with experimentally induced AKI on each day 1, 3, 6, and 12 after unilateral ischemia/reperfusion, and 9 cats with natural AKI were assessed. In sections from cats without kidney disease, patterns of periodic acid-Schiff and aquaporin-1 staining allowed identification of 3 distinct segments of the proximal tubule. KIM-1 staining was absent in segments 1 (S1) and S2, and faint in S3. Injury of S3 in cats with experimental and natural AKI was characterized by cell loss and necrosis, and remaining intact cells had cytoplasmic blebs and reduced brush borders. In experimental AKI, intensity of KIM-1 expression increased in proportion to the severity of injury and was consistently present in S3 but only transiently in other segments. Vimentin was absent in proximal tubules of healthy cats but expressed in injured S3. These findings indicate that S3 is the proximal tubular segment most susceptible to ischemic injury and that KIM-1 is a sensitive tissue indicator of AKI in cats.

Characterization of kidney injury molecule-1 in cats.

BACKGROUND: Kidney disease (KD) is common in older cats and presumed to arise from subclinical kidney injuries throughout life. Sensitive markers for detecting kidney injury are lacking. Kidney injury molecule 1 (KIM-1) is a useful biomarker of kidney injury in humans and rodents. HYPOTHESIS/OBJECTIVES: Feline KIM-1 is conserved across species, expressed in kidney, and shed into urine of cats with acute kidney injury (AKI). The objectives were to characterize the feline KIM-1 gene and protein, assess available immunoassays for detecting KIM-1 in urine of cats, and identify KIM-1 expression in kidney sections. ANIMALS: Samples from 36 hospitalized and 7 clinically healthy cats were evaluated. Hospitalized cats were divided into 2 groups based on absence (n = 20) or presence (n = 16) of historical KD. METHODS: Feline KIM-1 genomic and complementary DNA sequences were amplified, sequenced and analyzed to determine the presence of isoforms, exon-intron organization and similarity with orthologous sequences. Presence in urine was evaluated by immunoassay and expression in kidney by immunohistochemistry. RESULTS: Three expressed feline KIM-1 transcript variants comprising 894, 810, and 705 bp were identified in renal tissue. KIM-1 immunoassays yielded positive results in urine of cats with conditions associated with AKI, but not chronic KD. Immunohistochemistry of kidney sections identified KIM-1 in proximal tubular cells of cats with positive urine immunoassay results. CONCLUSIONS AND CLINICAL IMPORTANCE: Kidney injury molecule 1 was expressed in specific segments of the nephron and detected in urine of cats at risk of AKI. Urine KIM-1 immunoassay may be a useful indicator of tubular injury.

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.

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.

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.

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.

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.

Clara cell secretory protein increases phagocytic and decreases oxidative activity of neutrophils.

Horses suffer from recurrent airway obstruction, an asthma-like condition induced by repeat inhalation of environmental substances present in barn air. Clara cell secretory protein (CCSP) is much reduced during active inflammation when neutrophils predominate in the airways, and in chronic asthmatics. We sought to investigate morphologic and functional interactions of CCSP with neutrophils. Bronchoalveolar and blood neutrophils from healthy control animals, and from animals with recurrent airway obstruction in remission and exacerbation, were evaluated by immuno-cytochemistry and immuno-electron microscopy for presence of CCSP. Blood neutrophil oxidative burst and phagocytic activities were determined in the presence of different concentrations of recombinant equine CCSP. Bronchoalveolar lavage neutrophils from horses with exacerbated lung inflammation, but not from control horses, and not blood neutrophils from either group of animal, contained abundant immunoreactive CCSP. On immuno-electron microscopy, CCSP localized to the cytoplasm and nucleus. Incubation of blood neutrophils with CCSP significantly reduced oxidative burst activity (P<0.0001) and increased phagocytosis (P<0.001) of neutrophils. These findings indicate that CCSP enters neutrophils in horses with active neutrophilic lung inflammation and alters the function of neutrophils in blood. Presence in the nucleus suggests a potential transcriptional role of CCSP in neutrophils.

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.

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.

Immunohistochemical identification of collagen in the equine lung.

Recurrent airway obstruction (RAO) in the horse is a disease characterized by reversible bronchoconstriction and by mucus and neutrophil accumulation in the airways. It has been hypothesized that in horses with RAO, remodeling changes occur that are similar to those described in humans with asthma. Although collagen fibrils are present surrounding normal airways, they are a prominent feature of airway remodeling in human asthma with evidence of enhanced collagen III and I fibril deposition. An immunolabeling method was developed to identify collagen I and III in equine lung and to describe the collagen fiber type and distribution within the walls of the noncartilagenous bronchioles. The health status of 14 horses was characterized by clinical respiratory exam, bronchoalveolar lavage cytology, and pulmonary function tests. Following postmortem examination and histological assessment, horses were divided into RAO-affected (n = 4) and nonaffected (n = 10) groups. Eight sections per horse from all lung regions were evaluated histologically. Results of the study showed that collagens I and III were present in the lamina propria and adventitial area of the noncartilaginous bronchioles. There was clear staining differentiation between collagen I or III, airway smooth muscle, and the airway epithelium. Collagen I and III were present in the lamina propria and adventitial areas of the noncartilaginous bronchioles of horses, and there was no significant difference in the relative amount of collagen I and III between this group of RAO-affected and nonaffected horses.

Feline programmed death and its ligand: characterization and changes with feline immunodeficiency virus infection.

Programmed death (PD)-1 and its ligand, PD-L1, are co-stimulatory molecules expressed on T cells and antigen-presenting cells, respectively, that modulate T cell receptor signals. Altered PD expression or signalling contributes to pathogen persistence in chronic infections. The sequence of the feline PD genes was derived from gene amplification with primers conserved across human and canine homologs, and by sequence extension through rapid amplification of cDNA ends. Feline PD-1 was similar to that of other mammalian species and consisted of extracellular, transmembrane and cytoplasmic regions. Functional motif analysis of the translated amino acid sequence predicted immunoreceptor tyrosine-based inhibitory and switch motifs, and a SH3-binding region, in the cytoplasmic tail. PD-1 and PD-L1 were expressed in resting lymphocytes and dendritic cells, and up-regulated on mitogen-activated or irradiated lymphocytes of both CD4 and CD8-positive subsets. In vitro infection with the feline immunodeficiency virus (FIV) significantly decreased PD-1, but not PD-L1, gene expression in lymphocytes at 24h, and decreased expression of both genes at 168h. No significant changes in gene or protein expression from FIV infection were noted in dendritic cells. Blood lymphocytes from cats chronically FIV-infected expressed significantly higher PD protein than lymphocytes from FIV-negative cats. These findings indicate that both feline PD-1 and PD-L1 are expressed by resting lymphocytes and dendritic cells. Apoptosis and cell activation increased protein expression on lymphocytes, while in vitro acute FIV infection decreased PD-1 gene expression. Increased PD levels in lymphocytes from chronically FIV-infected cats suggests that alterations in T cell co-signalling may contribute to immune dysfunction in lentiviral infection.

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.