The effect of oxytetracycline (OTC) on bovine blood mononuclear cells and neutrophil functions was examined in vitro. Neutrophil functions tested include respiratory burst, peroxidase, and antibacterial activities. Neutrophils were treated with OTC (10 to 1,500 > microgram/ml) before exposure to either opsonized zymosan or bacteria. A dose-response inhibition of antibacterial activity to high concentrations of OTC (500 to 1,000 microgram/ml) was observed. Beginning at a concentration of 15 microgram/ml, OTC treatment of neutrophil Iysates resulted in decreased peroxidase activity. A dose response was not observed. In contrast, respiratory burst, measured by nitroblue tetrazolium dye reduction, increased after OTC exposure, but only at high concentrations (500 and 1,000 microgram/ml) of OTC. Mitogen-induced proliferation of blood mononuclear cells cocultured with OTC and concanavalin A, phytohemagglutinin-P, or pokeweed mitogen was inhibited at an OTC concentration of 100 microgram/ml at 48 and 72 hours of culture. These results indicate that blood mononuclear cells are more sensitive to the inhibitory effects of OTC than are neutrophils. Furthermore, the OTC-mediated inhibition of neutrophil antimicrobial activity is inversely related to the increase in nitroblue tetrazolium reduction. This suggests that OTC is uncoupling the hexose monophosphate shunt from production of secreted oxygen radicals. These results also suggest that the peroxidase enzyme system has a large biological reserve capacity.

Cartilage resurfacing by chondrocyte transplantation, using porous collagen matrices as a vehicle to secure the cells in cartilage defects, has been used experimentally in animals, This in vitro study evaluated the temporal morphologic features and proteoglycan synthesis of chondrocyte-laden collagen matrices. Forty-two porous collagen disks were implanted with a minimum of 6 X 10(6) viable chondrocytes, covered by a polymerized collagen gel layer, and 6 disks were harvested after 0, 3, 7, 10, 14, 18, or 22 days of incubation in supplemented Ham's F12 medium at 37 degrees C and 5% CO2. Histologic and histochemical evaluation of formalin-fixed segments of the cultured disks indicated that the chondrocytes proliferated in the implant, producing small groups and linear segments of cells by day 14. The collagen framework remained intact over the course of the study with thick areas attributable to depositions of matrix material after day 10. Alcian blue-stained matrix was evident in the pericellular region of chondrocytes in sections of disks harvested on days 14, 18, and 22. Glycosaminoglycan (GAG) assay by dimethylmethylene blue dye binding after papain digestion of the disk segments revealed negligible amounts of GAG at day 0. Significant (P < 0.0001) increase in total GAG content was observed by day 3 (0.329 micrograms/mg of disk) and further increases were observed until a plateau in GAG quantity was seen on day 14. Mean peak GAG content was 0.553 +/- 0.062 micrograms/mg. Secondary treatment of the papain-digested implants with keratanase and chondroitinase ABC yielded similar trends in chondroitin sulfate (CS) and keratan sulfate (KS) concentrations. The CS content significantly (P = 0.0002) increased for the first 14 days of incubation, then a plateau was observed for the remainder of the study. Peak CS content was 0.354 +/- 0.037 micrograms/mg. Concentration of KS reached a plateau earlier than did CS content, with peak amount of 0.193 +/- 0.027 micrograms/mg on day 10. Fluctuations in KS content were not significant until an increase on day 22. Chondrocytes actively populated the collagen implants, increasing in number and synthesizing matrix GAG epitopes over the 22 days of incubation. These results indicate that chondrocyte-laden porous collagen matrices may be suitable cartilage analogue materials and the optimal metabolic time for transfer to cartilage defects is 10 to 14 days.

Hexosamine concentration, DNA concentration, and [35S]sulfate incorporation for articular cartilage obtained from various sites in the metacarpophalangeal and carpal joints of horses were measured. The same measurements were made on the repair tissue filling full-thickness articular defects in the intermediate carpal bone and on cartilage surrounding partial-thickness defects 6 weeks after the defects were created arthroscopically. Cellularity (measured as DNA concentration), proteoglycan content (measured as hexosamine concentration), and proteoglycan synthesis (measured as [35S]sulfate incorporation) varied according to the site sampled. Cartilage from the transverse ridge of the head of the third metacarpal bone and the radial facet of the third carpal bone had the lowest hexosamine concentration, whereas rate of proteoglycan synthesis was lowest in cartilage from the transverse ridge of the head of the third metacarpal bone and the distal articular surface of the radial carpal bone. Repair tissue filling a full-thickness cartilage defect at 6 weeks was highly cellular. It was low in proteoglycan content, but was actively synthesizing these macromolecules. In contrast, the cartilage surrounding a partial-thickness defect was unchanged 6 weeks after the original defect was made.

Phosphonoformate (PFA), a noncompetitive inhibitor of reverse transcriptase (RT), inhibited feline leukemia virus FeLV) infection of 2 feline cell lines and inhibited progeny virus RT activity in a chronically FeLV-infected cell line. Feline leukemia virus infection of 3201 cells, an FeLV-negative lymphoma cell line, was inhibited by > 70% at a concentration of only 1 micromole PFA and by > 90% at concentrations of 64 to 256 micromole PFA, as evidenced by RT activity. However, FeLV antigen expression by 3201 cells remained relatively constant over noncytotoxic concentrations of PFA. Because the persistence of viral antigen expression with concomitant suppression of RT activity appears to be unique and because 3201 cells express small amounts of an endogenous retrovirus (RD and 114) contain endogenous FeLV proviral sequences, a possible role of endogenous retroviruses acting as helper viruses was suggested. Feline leukemia virus infection of 81C cells, a sarcoma-positive, leukemia-negative fibroblast cell line, was inhibited by > 50% at a concentration of 64 micromole PFA and by > 98% at concentrations of 256 to 512 micromole PFA, as indicated by suppression of focus formation. The feline lymphoid cell line FL-74 is a large producer of FeLV. When FL-74 cells were cultured in the presence of 256 micromole PFA, virus production (virus budding and viral antigen) was not affected, but progeny virus lost RT activity and infectivity. Direct addition of PFA (256 micromole to FeLV also reduced RT activity and infectivity. These data indicate that PFA can directly and rapidly inactivate retrovirus independent of cellular processing, presumably by inhibiting RT. Long-term PFA administration may curtail spread of retroviral infections within and between hosts via extracellular inactivation of newly produced virus particles. Results of this study also suggest that PFA might be used prophylactically to treat materials potentially contaminated with retroviruses.

Explants were prepared from skeletal muscle tissue from 5 nondystrophic pups and from 5 pups with X-linked muscular dystrophy; pups were 2 to 17 weeks old. A serial reexplant method resulted in optimal cell density with minimal fibroblast growth. Cultures were examined daily by use of phase-contrast microscopy; differentiated (postfusion) cultures were examined by electron microscopy. Moderate nuclear pleomorphism and cell clustering were observed in cultures of normal and dystrophic muscle cells. Cultures were maintained to 27 days after plating. Minimal myofilament synthesis was observed in multinucleate cells from nondystrophic and dystrophic pups, but spontaneous contraction of myotubes was not observed during this period. Differences in growth, fusion, or differentiation of myogenic cells into multinucleate cells and myotubes were not found between dystrophic and normal muscle.

Cell proliferation kinetic values were established for the hair root matrix of primary anagen follicles of 14 Beagles and 4 Cocker Spaniels with healthy skin and 9 Cocker Spaniels with primary idiopathic seborrhea. Indices were established by intradermal pulse labeling with tritiated thymidine, followed by cutaneous biopsy and autoradiography. The hair root matrix cell labeling index was 23.4 +/- 3.5% for Beagles, 24.4 +/- 4.0% for healthy Cocker Spaniels, and 24.9 +/- 4.3% for seborrheic Cocker Spaniels. These values indicate a rapidly proliferating cell population. Differences among these cell kinetic data for the 3 groups of dogs were not statistically significant. Although significant cell kinetic differences have been reported for other epidermal structures (interfollicular epithelium, upper hair follicle external root sheath, sebaceous glands) in seborrheic Cocker Spaniels, proliferation of hair root matrix cells apparently remains unaffected.

The use of cultured tissue has not yet become wide-spread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consisted of Dulbecco modified Eagle medium with 10% whole fetal bovine serum (wFBS) and 20 micrograms of endothelial cell growth supplement/ml. The EMVE grew readily in culture, had the cobble-stone morphologic feature at confluence, stained positively for factor VIII-related antigen, and metabolized acetylated low-density lipoprotein. Fibroblast and smooth muscle cell contamination was minimal in primary cell cultures, which were successfully passed and maintained in culture for 3 to 5 serial passages, using various media and substrates. Preliminary studies were undertaken to determine optimal growth conditions with a range of variables: serum concentration, extracellular matrix components, and growth factors. Optimal conditions were achieved with a minimum of 10% wFBS, and with either fibronectin or laminin as extracellular matrix substrates. The EMVE grew adequately in Dulbecco modified Eagle medium plus 10% wFBS, and the added growth factors or serum supplements did not appear necessary for growth of EMVE.

Cell proliferation kinetic values were established for the epidermis, hair follicle epithelium, and sebaceous glands of 8 Cocker Spaniels with primary idiopathic seborrhea. Values were established by intradermal pulse labeling injections of tritiated thymidine followed by cutaneous biopsy and autoradiography.The epidermal basal cell-labeling index was 4.96 +/- 0.97%, and the epidermal nucleated cell-labeling index was 3.33 +/- 0.71%. Calculated epidermal cell renewal time for the viable layers of the epidermis was 7.85 +/- 1.80 days. The hair follicle infundibulum basal cell-labeling index was 5.48 +/- 2.01%, and the sebaceous gland basal cell-labeling index was 5.94 +/- 4.15%. When compared with previously reported cell kinetic values for Cocker Spaniels and Beagles with healthy skin, these data indicate accelerated cellular proliferation in all 3 cutaneous structures in seborrheic Cocker Spaniels.

The early interaction of Lawsonia intracellularis with host cells was examined with the use of porcine ileum models. Two conventional swine were anesthetized, and ligated ileum loops were prepared during abdominal surgery. The loops were inoculated with 10⁸ L. intracellularis or saline. After 60 min, samples of each loop were processed for routine histologic and electron microscopic study. Histologic and ultrathin sections of all the loops appeared normal, with no apposition of bacteria and host cells or bacterial entry events in any loop. Portions of ileum from a single gnotobiotic piglet were introduced as xenografts into the subcutis of each flank of 5 weaned mice with severe combined immunodeficiency disease. After 4 wk, 10⁸ L. intracellularis were inoculated into each of 4 viable xenografts with a sterile needle; the other 3 viable xenografts received saline. Histologic and ultrathin sections of all the xenografts 3 wk after inoculation showed relatively normal porcine intestinal architecture, with normal crypts, crypt cell differentiation, and low villous structures; the xenografts treated with the bacteria also showed intracytoplasmic L. intracellularis within crypt and villous epithelial cells. Thus, entry of L. intracellularis into target epithelial cells and multiplication may not be sufficient alone to directly cause cell proliferation. A proliferative response may require active division of crypt cells and differentiation in conjunction with L. intracellularis growth.