Isolation and characterization of pediatric canine bone marrow CD34+ cells

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.     

Marking of peripheral T-lymphocytes by retroviral transduction and transplantation of CD34+ cells in a canine X-linked severe combined immunodeficiency model

A retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) was used to mark and dynamically follow vector-expressing cells in the peripheral blood of bone marrow transplanted X-linked severe combined immunodeficient dogs. CD34(+) cells isolated from young normal dogs were transduced, using a 2 day protocol, with an amphotropic retroviral vector that expressed enhanced green fluorescent protein (EGFP) and the canine common gamma chain (gammac) cDNAs. Following transplantation of the transduced cells, normal donor peripheral blood lymphocytes (PBL) appeared by 1 month post-bone marrow transplant (BMT) and rescued three of five treated dogs from their lethal immunodeficiency. PCR and flow cytometric analysis of post-BMT PBL documented the peripheral EGFP expressing cells as CD3(+) T cells, which varied from 0% to 28%. Sorting of EGFP(+) and EGFP(-) peripheral blood T cells from two dogs, followed by vector PCR analysis, showed no evidence of vector shutdown. EGFP expression in B cells or monocytes was not detected. These marking experiments demonstrate that the transduction protocol did not abolish the lymphoid engraftment capability of ex vivo transduced canine CD34(+) cells and supports the potential utility of the MSCV retroviral vector for gene transfer to XSCID affected canine hematopoietic progenitor cells (HPC).     

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.     

Collection of peripheral blood CD34+ progenitor cells from healthy dogs and dogs diagnosed with lymphoproliferative diseases using a Baxter-Fenwal CS-3000 Plus blood cell separator

Background

Canine peripheral blood mononuclear cell (PBMC) apheresis using a Baxter‐Fenwal CS‐3000 Plus automated blood cell separator has not been reported.

Objective

To determine the feasibility and safety of using a CS‐3000 Plus blood cell separator with a small volume separation container holder (SVSCH) and small volume collection chamber (SVCC) to harvest canine PBMCs from dogs weighing <50 kg.

Animals

Eight healthy mongrel dogs and 11 client‐owned dogs in clinical remission for lymphoproliferative diseases (LPD).

Methods

In this prospective study, aphereses were performed using a Baxter‐Fenwal CS‐3000 Plus blood cell separator, with or without recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) treatment.

Results

Aphereses from 6 healthy dogs given rhG‐CSF yielded an average of 1.1 × 10⁷ ± 8.2 × 10⁶ CD34+ cells/kg. Aphereses from LPD dogs given rhG‐CSF yielded an average of 5.4 × 10⁶ ± 3.25 × 10⁶ CD34+ cells/kg (P = .17). Higher hematocrit in both groups of dogs receiving rhG‐CSF correlated with an increased number of CD34+ cells/kg harvested (healthy, P = .04; LPD, P = .05). Apheresis was well tolerated by all dogs.

Conclusions and Clinical Importance

Canine PBMC apheresis using the Baxter‐Fenwal CS‐3000 Plus cell separator with an SVSCH and SVCC is a feasible and safe option for harvesting an adequate number of CD34+ peripheral blood progenitor cells from dogs weighing ≥17 kg for hematopoietic cell transplantation.     

A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma

Background: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor‐initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B‐cell lymphoma have not been conclusively identified. Hypothesis/Objectives: Tumor cells with a progenitor phenotype exist in B‐cell lymphoma, reflecting a hierarchical organization. Animals: Twenty‐eight client‐owned dogs with previously untreated B‐cell lymphoma and 6 healthy dogs. Methods: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B‐cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL‐2Rγ^?/? mice was adapted to expand and serially transplant primary canine B‐cell lymphoma. Results: LPCs were expanded in lymph nodes from 28 dogs with B‐cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B‐cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. Conclusions and Clinical Importance: These results suggest the presence of a hierarchy of tumor cells in B‐cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.     

Cytometric evaluation of peripheral blood lymphocytes in dogs with lymphoma during chemotherapy

Twenty dogs with clinically diagnosed multicentric lymphoma were evaluated for percentages of peripheral blood lymphocyte subpopulations. Cytometric analysis was performed before and during chemotherapy. The results were compared to those obtained from a control group of healthy dogs. The percentages of CD5+, CD4+ and CD8+ cells were markedly decreased and CD21-like+ cells markedly increased in dogs with lymphoma in comparison with the control group. During the course of chemotherapy these values returned to ranges observed in healthy animals.     

Changes in Regulatory T Cells in Dogs with Cancer and Associations with Tumor Type

Background: Regulatory T cells (Treg) have been shown to suppress antitumor immunity and often are increased in humans and rodents with cancer. However, Tregs have not been well studied in dogs with cancer and it is not known if certain tumor types are associated with increased Tregs.
Hypothesis: We hypothesized that Treg percentages would be increased in dogs with cancer and that Treg percentages would be higher in dogs with certain types of cancer.
Animals: The percentages and numbers of Tregs and nonregulatory T cells and B cells were assessed in 34 dogs with cancer and 9 age-matched control dogs. Dogs evaluated included 14 dogs with sarcoma, 7 dogs with carcinoma, 7 dogs with lymphoma, and 6 dogs with mast cell tumor.
Methods: Numbers and percentages of Tregs, CD4⁺, and CD8⁺ T cells and B cells were determined using flow cytometry and compared between control dogs and dogs with cancer.
Results: The percentage of Tregs was significantly increased overall in dogs with cancer compared with control dogs. When tumor types were compared, Treg percentages were significantly increased in dogs with carcinoma. The Treg/CD8 T cell ratio was significantly higher in dogs with cancer compared with control dogs and was also significantly increased in 2 dogs with T-cell lymphoma.
Conclusions: Treg percentages in blood were increased in dogs with cancer, particularly in dogs with carcinoma. The Treg/CD8 ratio also identified tumor-specific abnormalities in dogs with cancer. These findings indicate that tumor-specific factors may affect Tregs in dogs.     

CD56 is expressed exclusively on CD3+ T lymphocytes in canine peripheral blood

CD56+ cells in canine blood leukocytes were characterized by flow-cytometric analysis of peripheral blood of 30 healthy adult beagle-dogs (15 males and 15 non-pregnant females). In 19 of the 30 dogs, anti human CD56 antibody, Leu-19, reacted with 8.8-21.7% of peripheral blood lymphocytes. All CD56+ cells simultaneously expressed CD3 molecules on their surface. Further phenotypic analysis revealed that 50.6+/-13.1% of the CD56+ cells showed CD4-CD8+ phenotype and 43.7+/-10.1% showed CD4+CD8- phenotype. Expression intensity of CD56 on the CD4-CD8+CD56+ cells was significantly higher than that on CD4+CD8-CD56+ cells (P<0.001). These findings indicate that CD56, which is a neural cell adhesion molecule, is uniquely expressed on subsets of T lymphocytes in canine peripheral blood.     

Induction of remission results in spontaneous enhancement of anti-tumor cytotoxic T-lymphocyte activity in dogs with B cell lymphoma

Characterization of the tumor microenvironment, particularly the immune cells that infiltrate tumors, provides important predictive and prognostic information in humans with lymphoma and other types of cancer. Tumor associated T lymphocytes have not been previously described in dogs with lymphoma. Therefore, we investigated the phenotype and function of T cells in the lymph nodes of dogs with B cell Non-Hodgkin's lymphoma (NHL), as well as the function of T cells in circulation of these dogs. We found that CD4+ and CD8+ T lymphocytes were few in number and minimally responsive to mitogenic stimuli compared to T cells in lymph nodes of normal dogs. Additionally, regulatory T cells (Treg) were significantly increased in tumor tissues compared to lymph nodes of healthy dogs. To better understand cell mediated antitumor immune responses we developed a non-radioactive assay to measure cytotoxic T lymphocyte (CTL) mediated killing of autologous tumor cells. Using this assay, we found that spontaneous CTL activity in the blood of dogs with lymphoma improved significantly following induction of tumor remission using doxorubicin. Coincident with the improvement in CTL activity, circulating Treg numbers were significantly decreased compared to pretreatment levels. We conclude from these studies that CTL activity in dogs with lymphoma can be significantly improved following induction of tumor remission using chemotherapy, as assessed using a new non-radioactive CTL assay.     

Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis

BACKGROUND: Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS: Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS: Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS: Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS: Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.     

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)αβ+, 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 naïve 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.     

Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs

Peripheral blood stem cell (PBSC) transplantation following consolidation therapy is a feasible treatment option for canine haematological malignancies. In veterinary medicine, haematopoietic stem cells are generally mobilized into peripheral circulation using a granulocyte colony‐stimulating factor (G‐CSF). This pilot study aimed to evaluate the haematopoietic stem cell mobilization effect of three different regimens for PBSC apheresis with Spectra Optia continuous mononuclear cell (CMNC) protocol in healthy dogs. Stem cell mobilization was performed using high‐dose plerixafor (CXCR‐4 inhibitor) alone, a G‐CSF alone, or a combination of the low‐dose plerixafor and G‐CSF. Three dogs were assigned to each mobilization protocol. Regardless of the mobilization protocol, the total blood volume processed was uniformly set as 270 mL/kg and many PBSCs, defined as CD34+/CD45^dim cells, within the apheresis product were compared. Changes in complete blood count, PBSC counts, and blood chemistry analysis were monitored before, during, and after apheresis. All dogs tolerated the apheresis procedure using the Spectra Optia system with minimal adverse effects. The mean PBSC counts of the apheresis products for plerixafor, G‐CSF, and the combination groups were 1.3 ± 0.24, 4.2 ± 0.47, and 6.4 ± 0.9 × 10⁶ cells/kg, respectively. The apheresis procedure using Spectra Optia CMNC protocol in dogs is safe and feasible. Furthermore, PBSC mobilization with a combination of G‐CSF and plerixafor appeared more effective than either compound alone in mobilizing PBSC to the peripheral blood in dogs.     

Immunophenotypic comparison of blood and lymph node from dogs with lymphoma

Peripheral blood and lymph node tissue from 12 dogs with lymphoma was immunophenotyped. Additionally, the bone marrow was immunophenotyped in 6 dogs. The lymphomas were characterized as B-cell in 11 dogs and T-cell in 1 dog. Immunophenotypic patterns in the peripheral blood and bone marrow were variable. The trend in dogs with B-cell lymphoma was normal to increased percentage of IgG-positive cells, decreased percentage of pan-T-positive cells, decreased percentage of CD4-positive cells, and decreased CD4/CD8 ratio. Simultaneous immunophenotyping of lymph node, blood and bone marrow cannot be recommended routinely without further studies to document its value as an independent prognostic indicator. However, it is potentially useful for tumor staging and monitoring remission, especially in lymphoma patients with a leukemic phase.     

RNA‐transfected CD40‐activated B cells as a vaccine strategy in canine malignancies

Introduction: Cell‐based vaccine strategies using dendritic cells as cellular adjuvant have entered phase III trials in humans and have been found to be safe, feasible, and potentially efficacious. Canine patients are generally smaller than adult human patients, which makes production of canine dendritic cell (DC) vaccines problematic, given patient size and the small number of available DC precursors. Here we describe feasibility studies of a novel cell‐based vaccine strategy which uses CD40‐activated B‐cells (CD40‐B) loaded with RNA. This strategy is based on our observations that RNA‐transfected human CD40‐B can drive anti‐tumor T cell responses. One advantage of using CD40‐B cells is the ability to expand this cell population ex vivo, allowing for the numbers of cells required for therapeutic vaccines. Methods: Twenty milliliters of blood were drawn from 6 normal dogs and 5 canine lymphoma patients. Peripheral blood mononuclear cells were separated by Ficoll centrifugation. Culture conditions for B cell activation were optimized using CD40‐ligand, canine IL‐4, and Toll‐like receptor stimulus with CpGoligodinucleotides (ODN). Cyclosporine was added to eliminate peripheral T lymphocytes. Proliferation and activation of CD40‐B cells were demonstrated by CFSE dilution of B cells quantified by flow cytometry. Gene transfer was achieved by mRNA electroporation. Results: Marked in vitro stimulation and proliferation of canine peripheral B cells were achieved with soluble trimeric CD40L, canine IL‐4, and ODN. CD40‐B cells showed dramatic upregulation of MHC class II molecules and CD21 (B‐cell activation marker). After two weeks in culture, cells were negative for CD3 and CD4. Canine CD40‐B cells were efficiently transfected with mRNA, with >60% of CD40‐B expressing green fluorescent protein after GFP mRNA electroporation. Conclusion: RNA‐transfected CD40‐B cells can be efficiently generated from normal and tumor‐bearing dogs. These results provide rationale to test tumor RNA‐transfected CD40‐B as a novel therapeutic approach to treating canine malignancies. Clinical trials in canine lymphoma have been proposed.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

Molecular cloning and expression of bottlenose dolphin CD34

In terrestrial mammals, the surface molecule CD34 is used as a marker to identify hematopoietic progenitor cells. To clarify whether CD34 expression can be used to confirm the undifferentiated state of hematopoietic-like cells isolated from the bone marrow of bottlenose dolphin, Tursiops truncates, we determined in this study the sequence of dolphin CD34 cDNA and analyzed its mRNA expression. Dolphin CD34 cDNA can be expressed as two forms, one that encodes a full-length version and a variant, truncated version of the gene. Both forms were detected in bone marrow mononuclear cells and in various tissues using RT-PCR. The truncated form was not detected in peripheral blood mononuclear cells, and neither form was detected in polymorphonuclear leukocytes. This is the first report on CD34 in marine mammals and our results suggest that dolphin CD34 may be a useful marker to identify hematopoietic progenitor cells.     

Autologous Peripheral Blood Hematopoietic Cell Transplantation in Dogs with T‐Cell Lymphoma

Background

Peripheral blood hematopoietic cell transplantation (PBHCT) is a feasible treatment option for dogs with B‐cell lymphoma.

Objective

To examine apheresis and PBHCT outcomes in dogs diagnosed with T‐cell lymphoma (TCL).

Animals

Fifteen client‐owned dogs diagnosed with high‐grade TCL.

Methods

After high‐dose cyclophosphamide and rhG‐colony‐stimulating (rhG‐CSF) factor treatment, peripheral blood mononuclear cells were collected using cell separators. The harvested cells then were infused after varying doses of total body irradiation (TBI). Postirradiation adverse effects were managed symptomatically and dogs were discharged upon evidence of hematopoietic engraftment.

Results

More than 2 × 10⁶ CD34+ cells/kg were harvested from 15/15 dogs. Thirteen of 15 (87%) dogs engrafted appropriately, whereas 2 (13%) of the dogs died in the hospital. One dog developed cutaneous B‐cell lymphoma 120 days post‐PBHCT. The median disease‐free interval and overall survival (OS) of the 13 dogs transplanted in first remission from the time of PBHCT were 184 and 240 days, respectively. Stage and substage of disease at diagnosis had no effect on OS. Two of 13 (15%) dogs were alive 741 and 772 days post‐PBHCT.

Conclusions and Clinical Importance

PBHCT may be considered as a treatment option for dogs with TCL.     

Early-life longitudinal survey of peripheral blood lymphocyte subsets in Beagle dogs

A longitudinal study of peripheral blood lymphocyte subsets from 7 to 15 months of age was performed in Beagle dogs employing a multiparametric flow cytometry. The data were compared with data obtained from adult Beagle dogs that were housed in the same animal facilities and that were subjected to the same controls during the 34 weeks of the study. Absolute counts of total lymphocytes and CD3+ T, CD3+CD4+ Th and CD21+ B lymphocytes decreased during the entire 34 weeks period of the study in the young dogs group. The same was observed with regard to the percentage of CD3+CD4+ Th lymphocytes and the CD4/CD8 ratio, while the percentage of CD3+CD8+ Tc lymphocytes increased from 7 to 15 months of age. These age-related changes found in lymphocyte subsets distribution of young dogs led to level the absolute and relative values of adult dog lymphocytes. The observations of this longitudinal study illustrate the changes related to maturation of lymphocyte subsets that occur during early life in Beagle dogs.     

Lymphocyte subpopulations and hematologic variables in dogs with congestive heart failure

Alterations in lymphocyte subpopulations and in other hematologic variables have been documented in people with heart failure. The purpose of the current study was to compare flow cytometric and hematologic variables in dogs with congestive heart failure (CHF) to healthy controls. CD4+ peripheral blood mononuclear cells (PBMC) and CD8+ lymphocytes were analyzed by flow cytometry, and white blood cell count, platelet count, hematocrit, and hemoglobin were determined by a complete blood count. Twenty-five dogs with CHF (International Small Animal Cardiac Health Council [ISACHC] class 2 [n = 12] and ISACHC class 3a [n = 13]) and 13 healthy controls were enrolled in the study. Compared with the controls, dogs with CHF had markedly lower percentages of CD4+ PBMC, CD8+ lymphocytes, hematocrit, and hemoglobin, but markedly higher leukocytes, neutrophils, and platelets. There were no differences in these variables between dogs with dilated cardiomyopathy (n = 6) and those with chronic valvular disease (n = 19). Dogs in ISACHC class 3a had a markedly lower total lymphocyte number, CD4+ and CD8+ cells, and hematocrit, but markedly higher leukocyte and neutrophil numbers relative to the control group. CD4+ and CD8+ subpopulations and other blood cell variables are altered in dogs with CHF. Future studies to determine possible clinical implications of these changes are warranted.     

Class II major histocompatibility complex expression and cell size independently predict survival in canine B-cell lymphoma

Background: Class II major histocompatibility complex (MHC) is an independent predictor of outcome in human B‐cell lymphoma. We assessed class II expression together with other markers for their impact on prognosis in canine B‐cell lymphoma. Hypothesis: Low class II MHC expression, large cell size, and expression of CD34 will predict a poorer outcome in canine B‐cell lymphoma. Expression of CD5 and CD21 on tumor cells also may be associated with outcome. Animals: One hundred and sixty dogs with cytologically confirmed lymphoma. Methods: Patient signalment, treatment type, and flow cytometry characteristics were analyzed for their influence on outcome. A multivariable predictive model of survival was generated using 2/3 of the patients and validated on the remaining 1/3 of the dataset. Results: Class II MHC expression had a negative association with mortality and relapse. Treatment type also influenced relapse and mortality, whereas cell size and patient age was only associated with mortality. CD34, CD21, and CD5 expression was not associated with disease outcome. The constructed model performed variably in predicting the validation group's outcome at the 6‐month time point. Conclusions and Clinical Importance: Low levels of class II MHC expression on B‐cell lymphoma predict a poor outcome, as in human B‐cell lymphoma. This finding has implications for the use of dogs to model human lymphomas. Class II expression, cell size, treatment, and age can be combined to predict mortality with a high level of specificity.