Inflammatory effects of autologous, genetically modified autologous, allogeneic, and xenogeneic mesenchymal stem cells after intra-articular injection in horses.

OBJECTIVES: To compare the clinical and inflammatory joint responses to intra-articular injection of bone marrow-derived mesenchymal stem cells (MSC) including autologous, genetically modified autologous, allogeneic, or xenogeneic cells in horses. METHODS: Six five-year-old Thoroughbred mares had one fetlock joint injected with Gey's balanced salt solution as the vehicle control. Each fetlock joint of each horse was subsequently injected with 15 million MSC from the described MSC groups, and were assessed for 28 days for clinical and inflammatory parameters representing synovitis, joint swelling, and pain. RESULTS: There were not any significant differences between autologous and genetically modified autologous MSC for synovial fluid total nucleated cell count, total protein, interleukin (IL)-6, IL-10, fetlock circumference, oedema score, pain-free range-of-motion, and soluble gene products that were detected for at least two days. Allogeneic and xenogeneic MSC produced a greater increase in peak of inflammation at 24 hours than either autologous MSC group. CLINICAL SIGNIFICANCE: Genetically engineered MSC can act as vehicles to deliver gene products to the joint; further investigation into the therapeutic potential of this cell therapy is warranted. Intra-articular MSC injection resulted in a moderate acute inflammatory joint response that was greater for allogeneic and xenogeneic MSC than autologous MSC. Clinical management of this response may minimize this effect.

The value of biomedical research training for veterinary anatomic and clinical pathologists.

Veterinary pathologists traditionally have been actively engaged in research as principal investigators and as collaborators. Pathologists frequently obtain advanced training in research; however, it appears that in the last 10 years there has been a reversal of a previous trend toward increasing numbers of pathologists obtaining PhD degrees. This has arisen despite an established shortage of veterinarians engaged in research. This article evaluates the benefits of research training for individual pathologists, including a wide spectrum of professional opportunities and additional skill development beyond that usually provided by diagnostic pathology training alone. Various training models are discussed, including combined and sequential diagnostic residency and research degree training as well as the nondegree research fellowship programs more commonly pursued in human medicine. Best-practice recommendations for program infrastructure, mentorship, time management, and a team approach to research and research training are advocated to facilitate the development of successful programs and to encourage a continued emphasis on integrated training for pathologists as both clinical diagnosticians and experimentalists. This article is intended to help prospective and active pathology trainees, their mentors, and educational administrators optimize opportunities to ensure the future vitality of veterinary pathologists, and their contributions, in basic and applied research.

Autosomal dominant polycystic kidney disease in Persian and Persian-cross cats.

A form of autosomal dominant polycystic kidney disease (ADPKD) similar in clinical features to human ADPKD occurs in the Persian cat. We characterized the morphologic and immunohistochemical features of this disease in a colony of affected cats. Complete postmortem examinations were performed on 11 normal and 22 affected cats ranging in age from 3 months to 10 years. Kidneys were evaluated by gross and histologic examinations, ultrastructure, lectin staining, bromodeoxyuridine immunochemistry for labeling index and immunochemistry for distribution of Na/K ATPase. Feline ADPKD was characterized by variable numbers of cysts in the renal cortex and medullar. Ultrastructural examination and lectin staining suggested that cysts arose from proximal and distal nephron segments. Bromodeoxyuridine labeling demonstrated increased proliferation of epithelium lining some cysts in young cats. Immunohistochemical staining showed variable translocation of Na/K ATPase from the basolateral membranes of cyst-lining cells to the cytoplasm or luminal membranes. Cystic renal disease commonly was associated with chronic tubulointerstitial nephritis and hepatobiliary hyperplasia and fibrosis. Focal hyperplasia of renal tubular epithelium, hepatic cysts, and cardiac lesions were present in some cats. Feline ADPKD shares many morphologic and pathogenetic features with human ADPKD.

Inheritance of polycystic kidney disease in Persian cats.

Polycystic kidney disease in Persian cats culminates in chronic renal failure after a variable clinical course. An affected 6-year-old Persian cat was used to establish a colony of cats with polycystic kidney disease. In affected cats, cysts could be detected by ultrasonography as early as 7 weeks of age. Absence of cysts on ultrasound examination at 6 months of age was correlated with absence of polycystic kidney disease at necropsy. Both males and females were affected and, of progeny from affected x unaffected crosses, 42% were affected and 58% were unaffected. In affected x affected crosses, 73% of progeny were affected and 27% were unaffected. These results are compatible with autosomal dominant inheritance of this trait. Polycystic kidney disease in Persian cats resembles autosomal dominant polycystic kidney disease (ADPKD) in human beings, and represents a valuable animal model of the human disease.

Hemostatic disorders in cats: a retrospective study and review of the literature.

Hemostasis profiles from 101 cats presented for medical or surgical evaluation to The Ohio State University Veterinary Teaching Hospital from 1986 through 1991 were reviewed retrospectively; 69% were abnormal. Commonly identified abnormalities included a mixed hemostatic defect compatible with disseminated intravascular coagulation, thrombocytopenia, isolated prolongation of the activated partial thromboplastin time (APTT), and prolongation of both the APTT and one-stage prothrombin time. The most common disorders associated with abnormal hemostasis profiles in this study were liver disease, neoplasia, and feline infectious peritonitis.

Macrophage tropism of feline leukemia virus (FeLV) of subgroup-C and increased production of tumor necrosis factor-alpha by FeLV-infected macrophages.

Erythroid aplasia is induced in cats by feline leukemia virus (FeLV) of subgroup C but not by FeLV of subgroup A. In an investigation of the role of macrophages in FeLV-C-induced diseases, the concentrations of FeLV and tumor necrosis factor-alpha (TNF-alpha) were compared between feline peritoneal macrophages incubated with FeLV of subgroup A or C. FeLV of both subgroups infected macrophages, but expression of FeLV-C was 21-fold higher than FeLV-A in peritoneal macrophages (P = .004). The supernatants of FeLV-C-inoculated macrophage cultures contained significantly higher levels of TNF-alpha (70 +/- 14 U/mL) at 72 hours postincubation compared with FeLV-A-inoculated (38 +/- 8 U/mL) and uninoculated (31 +/- 8 U/mL) cultures. Moreover, a positive correlation was shown between cell-associated FeLV surface glycoprotein gp70 and TNF-alpha expression in FeLV-C-infected macrophages by immunofluorescence (r = .6; P = .001), measured with a computer-assisted, laser-based digital imaging system. The addition of TNF-alpha to a uniform population of FeLV-infected cells (feline embryonic fibroblasts) caused an enhancement of viral expression (P < .05). These results indicate that FeLV-C has tropism for macrophages, FeLV expression is positively correlated with TNF-alpha expression in macrophages, and TNF-alpha enhances FeLV replication in fibroblasts. We suggest that FeLV-C infection of macrophages and secretion of TNF-alpha may be important in hematopoietic suppression in FeLV-C-infected cats.

Lymphoma involving large granular lymphocytes in cats: 11 cases (1982-1991).

Medical records of 11 cats with lymphoma involving large granular lymphocytes were reviewed. All 9 cats tested were FeLV-negative. Ten cats had a history of anorexia, lethargy, vomiting, or diarrhea, and had lymphoma involving abdominal viscera. The most common site of tumor in these cats was the jejunum. One cat had cutaneous masses caused by dermal and epidermal infiltration with neoplastic large granular lymphocytes. The most common hematologic abnormality was leukocytosis, characterized by neutrophilia with a left shift (7 cats); 2 cats had a left shift without neutrophilia. None of the cats had lymphocytosis, but immature large granular lymphocytes were found in the blood of 4 cats. The most common serum biochemical abnormalities were hypoalbuminemia (10 cats), hypocalcemia (10 cats), hypoproteinemia (9 cats), high aspartate transaminase activity (9 cats), and hyperbilirubinemia (8 cats). Large granular lymphocytes were characterized by abundant cytoplasm containing distinct azurophilic granules that varied in size and number. The most common cytochemical staining pattern included detection of alpha-naphthyl butyrate esterase, acid phosphatase, and beta-glucuronidase activities. On examination of histologic sections, granules stained weakly eosinophilic with Giemsa and moderately with periodic acid-Schiff reaction. Ultrastructurally, the granules appeared membrane bound and contained an electron-dense matrix in 4 cats.

Effects of tumor necrosis factor-alpha on normal feline hematopoietic progenitor cells.

The effects of tumor necrosis factor-alpha (TNF-alpha) on feline bone marrow hematopoietic progenitors were evaluated by exposing bone marrow mononuclear cells from specific pathogen-free cats to different concentrations of TNF-alpha (ranging from 50 to 800 pg/ml) for 2 h before plating for clonal assays of colony-forming units. TNF-alpha caused a dose-dependent suppression of feline erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E), whereas granulocyte-macrophage colony-forming units (CFU-GM) were minimally affected. TNF-alpha concentrations as low as 200 pg/ml significantly inhibited growth of erythroid progenitors. Addition of polyclonal rabbit anti-TNF-alpha antibodies completely neutralized the suppressive effect of TNF-alpha on erythroid progenitors. At higher concentrations of TNF-alpha (800 pg/ml), 35% of CFU-E and 21% of BFU-E still survived, indicating that some erythroid progenitors are not sensitive to a single exposure of TNF-alpha in vitro. These results suggest that TNF-alpha may play a role in regulating hematopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukemia virus.

Cytotoxicity in feline leukemia virus subgroup-C infected fibroblasts is mediated by adherent bone marrow mononuclear cells.

The pathogenesis of retrovirus-induced erythroid aplasia in cats is unknown. In studies to define mechanisms of cytotoxicity associated with retroviral infections, bone marrow mononuclear cells (BMMC) from healthy specific pathogen-free cats were co-cultured with uninfected feline embryonic fibroblasts (FEA cells) and FEA cells infected with feline leukemia virus (FeLV) of subgroup A (FEA-A) or subgroup C (FEA-C). Moderate to marked cytotoxicity (CPE) developed in co-cultures of BMMC and FEA-C cells on Days 5 to 7 of incubation but not in co-cultures of BMMC and FEA-A or BMMC and uninfected cells (FEA-CT). Cytotoxicity was associated with adherent cells of light density (1.056) from bone marrow and peripheral blood, which were positive for alpha naphthyl butyrate esterase activity. Stimulation of adherent cells with phorbol ester or addition of recombinant human tumor necrosis factor-alpha (rhTNF-alpha) caused similar CPE in FEA-CT cells. The TNF-alpha concentrations in the culture supernatants of BMMC+FEA-C were higher than those of BMMC+FEA-A or BMMC+FEA-CT, and addition of anti-TNF antibodies to the cultures blocked the CPE. These data support the hypothesis that macrophages exposed to FeLV-C cause CPE in co-cultures of BMMC and FEA cells by a mechanism involving TNF-alpha. It is suggested that TNF-alpha may be involved in the suppression of hematopoiesis in cats which develop FeLV-C induced erythroid aplasia.

Variable suppression of feline bone marrow fibroblast colony-forming units by two isolates of feline leukemia virus.

Bone marrow fibroblast colony-forming units (CFU-F) were evaluated in cats experimentally infected with different, isolates of FeLV. Cats infected with the Kawakami-Theilen isolate of FeLV (FeLV-KT) had progressive decrease in the number of CFU-F at 2, 4, and 6 weeks after infection. The number of CFU-F in FeLV-KT-infected cats ranged from 38 to 70% of the preinoculation CFU-F value. Of 3 cats with FeLV-KT-induced suppression of CFU-F, 2 developed fatal nonregenerative anemia. Cats infected with the Rickard isolate of FeLV (FeLV-R) had more moderate decrease in the number of CFU-F at 2, 4, and 6 weeks after infection. The number of CFU-F in FeLV-R-infected cats ranged from 62 to 82% of the preinoculation CFU-F value. The FeLV-R-infected cats did not become anemic.

The cytologic diagnosis of neoplasia.

With minimal skill, most veterinarians can use cytology to differentiate inflammation from neoplasia and thus provide useful information for the direction of further diagnostic testing. The experienced cytologist can definitively diagnose several specific neoplasms and make a tentative diagnosis of neoplasia for many other types of tumors. This information is useful in establishing a prognosis and in directing appropriate therapy. Cytologic findings should be correlated with other clinical and laboratory information. When the cytologic diagnosis of neoplasia is uncertain, the presence of tumor and tumor cell type should be confirmed histopathologically.

A feline large granular lymphoma and its derived cell line.

A lymphoma cell line (MCC) was derived from an abdominal mass from a 13-yr-old castrated male cat. The cells resemble natural killer precursor cells, have membrane-bound granules, and are positive for chloroacetate esterase, alpha-naphthyl butyrate esterase, and tartrate-resistant acid phosphatase activities. The MCC cells are negative for rearranged feline T-cell receptor genes, negative for feline T-cytotoxic antigen, Ia, and surface mu, tau, and lambda chains and do not form E-rosettes. The MCC cell line is negative for the feline leukemia virus (FeLV); e.g., negative for exogenous FeLV (exU3) sequences, negative for cytoplasmic and surface FeLV major core protein of 27,000 daltons (p27) by indirect immunofluorescence assay, negative for helper FeLV by clone 81 assay, and negative for release of soluble FeLV p27 by enzyme-linked immunosorbent assay. Electron microscopy reveals budding type C retrovirus particles and MCC cells react with anti-RD-114 (anti-endogenous feline retrovirus) reference serum. After in vitro infection, MCC replicate FeLV readily, but replication is noncytopathic.

Lymphocytosis of large granular lymphocytes in three dogs.

The clinical course and hematologic changes of three dogs with lymphocytosis of cells morphologically resembling large granular lymphocytes are presented. Hemograms from all dogs showed leukocytosis with marked lymphocytosis. Lymphocytes were characterized by abundant basophilic cytoplasm containing distinct granules which varied in size and number. Electron microscopically the granules were membrane-bound with an electron-dense core. Lymphocytes from one dog were positive for alkaline phosphatase activity, and lymphocytes from another dog were positive for alpha naphthyl butyrate esterase activity. Lymphocytes from one dog were positive for surface receptors for the crystalline fraction portion of gamma immunoglobulins.

A macrophage-monocyte cell line from a dog with malignant histiocytosis.

The DH82 cell line was established from the neoplastic progenitor cells of canine MH and was characterized as histiocytic in origin based on light microscopic and ultrastructural morphology, positive staining reactions for alpha naphthyl acetate esterase and acid phosphatase, presence of Fc receptors, phagocytosis of latex beads, and plastic adherence in culture.

Suppression of feline bone marrow fibroblast colony-forming units by feline leukemia virus.

Bone marrow fibroblast colony-forming units (CFU-F) were evaluated in cats experimentally infected with feline leukemia virus (FeLV). Cats that developed persistent viral infection and anemia (progressor cats) had a progressive decrease in the number of CFU-F at 2, 4, 6, 8, and 10 weeks after inoculation with FeLV. This suppression of CFU-F number in progressor cats ranged from 16 to 44% of the preinoculation CFU-F value. Cats that did not develop persistent viral infection or anemia (regressor cats) had decreased numbers of CFU-F (24% of the preinoculation CFU-F value) at 2 weeks after inoculation, but normal CFU-F numbers at 4, 6, 8, and 10 weeks after inoculation. In vitro incubation of bone marrow mononuclear cells from healthy cats with the 15,000-dalton envelope protein of FeLV resulted in decreased number of CFU-F (21% of that of untreated cultures). The number of CFU-F from bone marrow mononuclear cells incubated with the 27,000-dalton core protein of FeLV was similar to that from untreated cultures.

Characterization of fibroblast colony-forming units in bone marrow from healthy cats.

The adherent cell layer of bone marrow from healthy cats was characterized in vitro, and the mean fibroblast colony-forming unit (CFU-F) was determined. The majority (82%) of the cells in the adherent cell layer were spindle-shaped fibroblastic cells. These cells were weakly positive for acid phosphatase activity and negative for alpha-naphthyl butyrate esterase and alkaline phosphatase activities. They did not phagocytose latex beads. The remaining cells (18%) in the adherent cell layer resembled macrophages. They were strongly positive for acid phosphatase and alpha-naphthyl butyrate esterase activities, and they phagocytosed latex beads. The mean CFU-F per 10(6) mononuclear cells in bone marrow from healthy kittens and adult cats was 62 and 65, respectively. The CFU-F assay was linear over a range of 0.25 to 1.25 x 10(6) bone marrow mononuclear cells cultured. Variation in the feline CFU-F assay was similar to that reported for the human CFU-F assay. Bone marrow collections repeated at 1-month intervals (from the same bone) did not affect CFU-F concentration. A difference was not observed between CFU-F cultured from the feline humerus or femur. Bone marrow adherent cells in cats resembled those described for other species. Results of the feline CFU-F assay were consistent and repeatable and were similar to those reported for other species.

Guinea pig erythrocyte rosette formation as a nonspecific cell surface receptor assay in the cat.

The specificity of guinea pig erythrocyte (GPE) rosettes for feline peripheral blood lymphocytes was studied. Of the GPE rosette-positive cells from peripheral blood, 54% were monocytes, 29% were granulocytes, and only 17% were lymphocytes. Results were similar for rosettes incubated at 4 C and those incubated at 37 C. Mononuclear cells separated with polyvinylpyrrolidone-coated silica formed fewer monocyte rosettes (49%) and more granulocyte rosettes (34%) than did cells separated with sodium diatrizoate-Ficoll (60% monocyte rosettes and 18% granulocyte rosettes), whereas the percentage of lymphocyte rosettes was similar for both media. Mononuclear cells suspended in Eagle's minimum essential medium had a higher percentage of monocyte rosettes (75%) and a lower percentage of granulocyte rosettes (12%) than did cells suspended in RPMI 1640 medium (59% monocyte rosettes and 27% granulocyte rosettes). The percentage of lymphocyte rosettes was similar in the 2 media. Two sequential 45-minute plastic adherent cell depletions decreased monocyte rosettes to 51% and increased lymphocyte rosettes to 23% compared with 63% monocyte rosettes and 12% lymphocyte rosettes before adherent cell depletion. The granulocyte rosettes were unchanged by plastic adherent cell depletion. The percentage of rosette-positive cells (9%) was not significantly affected by incubation at 4 C or 37 C, cell separation with polyvinylpyrrolidone-coated silica or lymphocyte separation medium, or suspension in Eagle's minimum essential medium or RPMI 1640 medium. Plastic adherent cell depletion decreased the percentage of rosette-positive cells. Feline thymocytes were 38% to 80% GPE rosette-positive and a feline leukemia virus-infected lymphoblastic cell line (F422) was 88% GPE rosette-positive.(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant histiocytosis in four dogs.

The clinical and pathologic features of 4 dogs with malignant histiocytosis were evaluated. The most common clinical signs were weight loss, lethargy, lymphadenopathy, hepatosplenomegaly, and anemia. Neoplastic histiocytic infiltrates most commonly were found in the spleen, bone marrow, liver, or lymph nodes. Malignant histiocytosis was considered as a differential diagnosis for anemic dogs with lymphadenopathy and/or hepatosplenomegaly.