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.

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.

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.

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.

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.

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.

Clara cell secretory protein is reduced in equine recurrent airway obstruction.

Horses are prone to recurrent airway obstruction (RAO), an inflammatory lung disease induced by repeated exposure to environmental mold, dust, and bacterial components. Active disease manifests with mucus hyperproduction, neutrophilic inflammation, bronchoconstriction, and coughing. Chronically affected animals have lung remodeling characterized by smooth muscle hyperplasia, collagen deposition, lymphoid hyperplasia, and impaired aerobic performance. Clara cell secretory protein (CCSP) counters inflammation in the lung, hence we hypothesized that CCSP depletion is a key feature of RAO in horses. Recombinant equine CCSP and specific antiserum were produced, and percutaneous lung biopsies were obtained from 3 healthy horses and from 3 RAO-affected horses before and after induction of RAO. CCSP relative gene expression in tissue, as well as protein concentration in lung lavage fluid, was determined. Immunocytochemical analysis, using both light and immunogold ultrastructural methods, demonstrated reduced CCSP staining in lung tissue of animals with RAO. Immunogold label in Clara cell granules was less in animals with chronic RAO than in normal animals, and absent in animals that had active disease. Median lung lavage CCSP concentration was 132 and 129 ng/ml in healthy horses, and 62 and 24 ng/ml in RAO horses before and after challenge, respectively. CCSP lung gene expression was significantly higher in healthy animals than in animals with chronic RAO. Together, these preliminary findings suggest that reduced production of CCSP and subcellular changes in Clara cells are features of chronic environmentally induced lung inflammation in horses.

Alloimmunity does not protect from challenge with the feline immunodeficiency virus.

Immune responses against polymorphic host molecules incorporated into lentiviral envelopes during cell budding have induced protection against primate immunodeficiency virus infection. Dendritic cells (DCs) express high levels of MHC molecules and are infectable by lentiviruses. Therefore, in this pilot study we addressed the hypothesis that immunization of cats with allogeneic DC would induce immune responses that protect against challenge with the feline immunodeficiency virus. Two groups of 3 cats each received 3 subcutaneous injections of allogeneic or autologous DC, and were then challenged with viruses propagated in the immunizing DC. Infection status and lymphocyte parameters of cats were assessed during 6 weeks after challenge. MHC II antigens were incorporated into viral particles as identified by Western blot; and antibodies reactive with MHC class II antigens were detected in the serum of cats immunized with allogeneic but not autologous DC. After challenge, all cats had proviral DNA in blood leukocytes from 2 weeks post-challenge onward and seroconverted. Cats immunized with allogeneic DC maintained higher total and CD21(+) lymphocyte concentrations, and higher CD4(+)/CD8(+) lymphocyte ratios; however, these differences were not significantly different from cats that received autologous DC immunizations. Plasma viral load was not significantly different between groups of cats (p=0.204). These results suggest that immunization of cats with allogeneic DC does not induce protective immunity against FIV infection.

Differentiation of feline immunodeficiency virus vaccination, infection, or vaccination and infection in cats.

BACKGROUND: Serodiagnosis of feline immunodeficiency virus (FIV) is complicated by the use of a formalin-inactivated whole-virus FIV vaccine. Cats respond to immunization with antibodies indistinguishable from those produced during natural infection by currently available diagnostic tests, which are unable to distinguish cats that are vaccinated against FIV, infected with FIV, or both. HYPOTHESIS: An enzyme-linked immunosorbent assay (ELISA) detecting antibodies against formalin-treated FIV whole virus and untreated transmembrane peptide will distinguish uninfected from infected cats, regardless of vaccination status. ANIMALS: Blood samples were evaluated from uninfected unvaccinated cats (n = 73 samples), uninfected FIV-vaccinated cats (n = 89), and FIV-infected cats (n = 102, including 3 from cats that were also vaccinated). METHODS: The true status of each sample was determined by virus isolation. Plasma samples were tested for FIV antibodies by a commercial FIV diagnostic assay and an experimental discriminant ELISA. RESULTS: All samples from uninfected cats were correctly identified by the discriminant ELISA (specificity 100%). Of the samples collected from FIV-infected cats, 99 were correctly identified as FIV-infected (sensitivity 97.1%). CONCLUSIONS AND CLINICAL IMPORTANCE: With the exception of viral isolation, the discriminant ELISA is the most reliable assay for diagnosis of FIV. A practical strategy for the diagnosis of FIV infection would be to use existing commercial FIV antibody assays as screening tests. Negative results with commercial assays are highly reliable predictors for lack of infection. Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is negative, the cat is probably vaccinated against FIV but not infected. Positive results are likely to represent infection.

The effect of lead poisoning on hematologic and biochemical values in trumpeter swans and Canada geese.

BACKGROUND: Lead is a persistent contaminant in the environment, and waterfowl are susceptible to lead toxicity from ingestion of lead pellets and fishing weights. Lead affects numerous physiologic processes through inhibition of enzyme activity and protein function, but its effects on commonly assessed avian blood values are incompletely understood. OBJECTIVES: Our aim was to evaluate hematologic and biochemical changes associated with blood lead concentrations in trumpeter swans and Canada geese. METHODS: Data for CBCs, plasma biochemical profiles (total protein, albumin, glucose, cholesterol, total bilirubin, calcium, phosphorus, gamma-glutamyltransferase [GGT], aspartate aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, amylase, and lipase), and whole blood lead concentrations were retrospectively analyzed for 69 trumpeter swans and 52 Canada geese. Laboratory data obtained prospectively from an additional 20 trumpeter swans also were included. RBC morphology was semiquantitated in blood smears from 70 of the birds. Data were analyzed initially by ANOVA and covariance. A statistical model then was constructed to determine the relationship between each parameter and lead concentration. RESULTS: In both avian species, PCV, hemoglobin concentration, and MCHC decreased significantly (P < .05) with increasing blood lead concentration. Uric acid concentration and GGT activity were increased in trumpeter swans and phosphorus concentration was decreased in Canada geese in association with high blood lead concentration (P < .05). CONCLUSIONS: Lead toxicosis induced significant changes in the values of commonly measured hematologic parameters in waterfowl. These changes may be useful indicators of severe lead intoxication during routine laboratory assessment. Changes in clinical chemistry values, although statistically significant, were too inconsistent to serve as indicators of lead toxicosis.

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.

Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.

Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.

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.

Cytotoxic T-lymphocytes from HIV-infected individuals recognize an activation-dependent, non-polymorphic molecule on uninfected CD4+ lymphocytes.

OBJECTIVES: Correlation of lysis of autologous CD4+ target cells by cytotoxic lymphocytes from HIV-seropositive patients to target activation, viral replication, and major histocompatibility complex (MHC) restriction. DESIGN: Twenty-two HIV-infected patients were evaluated for lysis of activated CD4+ cells, concurrent with measurement of proliferation of the target cells, and with viral replication. METHODS: Titrated standard 51Cr-release assays for specific effector-to-target cell recognition, blocking antibodies and cell depletion for cell characterization, incorporation of [3H]-thymidine for proliferation, and p24 antigen capture assays for viral replication. RESULTS: HIV-infected patients had cytotoxic lymphocytes capable of recognizing activated CD4+ target cells in a non-MHC-restricted manner. The lysis depended on the degree of target activation, and was independent of viral replication. CONCLUSIONS: This cytolytic activity is unique to HIV-infected patients, and is suggestive of activation-induced cell death that may contribute to the progressive depletion of CD4+ lymphocytes during HIV pathogenesis.

T cell-derived suppressive activity: evidence of autocrine noncytolytic control of HIV type 1 transcription and replication.

The ability of CD8+ T lymphocytes to suppress the transcription and replication of HIV-1 is well documented. We have demonstrated that the factor(s) responsible for the suppression of HIV-1 LTR-mediated gene expression are not the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. Interestingly, these and other chemokines and cytokines are produced by both CD8+ and CD4+ T lymphocytes. On the presumption that CD4+ T lymphocytes may also be able to modulate HIV-1 expression in vitro we assessed the LTR-modulatory effects of a panel of culture supernatants derived from stimulated CD4+ T lymphocytes from HIV-positive patients and uninfected controls. Supernatants of both CD4+ and CD8+ T cells mediated a suppression of LTR-driven gene expression in Jurkat T cells and an enhancement of gene expression in U38 monocytic cells. On the basis of these results, and using a herpesvirus saimiri (HVS)-transformed CD4+ T lymphocyte clone (HVSCD4), we demonstrate that both suppressive and enhancing effects are dose dependent. Furthermore, we have shown that supernatants of both HVSCD4 and HVSCD8 cells suppress LTR-mediated gene expression and HIV-1 replication in transfected/infected T cells. In U1 monocytic cells, supernatants of both CD4+ and CD8+ lymphocytes from an HIV-1-infected individual enhanced LTR-mediated gene expression, HIV-1 replication, and TNF-alpha production. However, only these effects as induced by CD8+ T cells were sensitive to the G protein inhibitor pertussis toxin. These results indicate that factors produced by both CD4+ and CD8+ T cells exert dichotomous effects on HIV-1 gene expression and replication in T cells and monocytes.

Immunophenotype and functional properties of feline dendritic cells derived from blood and bone marrow.

Dendritic cells (DCs) are a heterogeneous population of cells of fundamental importance in initiating innate as well as specific immune responses. The identity and function of DCs in the cat are unknown, although they are likely pivotal in the response to infection. In this study, feline DCs were derived by 3-10-day culture of adherent blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) in the presence of IL 4 and GM-CSF. BMMC consistently yielded a greater number of DCs than PBMC, and there were fewer macrophages than DC from both compartments. DCs expressed a distinct constellation of surface molecules, which included CD1a, CD1b, and CD1c, CD11b, CD14, and 2-3-fold higher levels of MHC class I and II molecules than co-cultured macrophages or fresh blood monocytes. DCs displayed typical cytoplasmic processes, limited non-specific esterase activity, and acquired antigen by phagocytosis, pinocytosis, and binding to specific receptors. Cytokine-exposed cells induced proliferation of allogeneic lymphocytes. Thus, the cells derived by these culture conditions had markers and functions analogous to immature myeloid DCs. Availability of feline DCs will enable investigation of their role in infectious disease and their potential therapeutic application.

The immunophenotype of blood and cerebrospinal fluid mononuclear cells in dogs.

Inflammatory neurologic diseases are common in dogs, but establishing a definitive diagnosis often is difficult. Nucleated cell number and type in cerebrospinal fluid (CSF) rarely are suggestive of an etiologic agent. We speculated that CSF leukocyte immunophenotyping would be a useful adjunct in the investigation of canine inflammatory neurologic diseases by yielding more specific etiologic information. The goals of this study were to establish the feasibility of flow cytometric evaluation of individual canine CSF samples and to identify the cell distribution in healthy dogs. The mononuclear cell populations of paired blood and CSF samples from 23 healthy dogs were characterized by labeling of cells with antibodies against CD4, CD8alpha, CD21, and CD14 molecules and by flow cytometric analysis of their expression. The mean proportion of CD4+ and CD21+ cells was significantly higher in blood than in the CSF (P < .002 and P < .001, respectively). In contrast, the mean proportion of CD14+ and CD8a+ cells was not significantly different between blood and CSF (P = .5 and p = .9, respectively). These findings demonstrate differences in the distribution and function of mononuclear cells in the circulating venous and subarachnoid compartments in the dog.