Effects of growth factors (EGF,PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing

Objective The physiologic mechanisms involving growth factors, including PDGF‐BB, EGF, and TGF‐β1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types. Procedures Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF‐BB, EGF, TGF‐β1, keratocytes (1 × 10⁵/well) and epithelial cells (2 × 10⁵/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF‐BB between 0 and 75 ng/mL; and TGF‐β1 between 0 and 10 ng/mL. Cell proliferation was measured using ³H‐thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05). Results Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF‐BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF‐β1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL. Conclusions Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF‐BB and EGF stimulate, while TGF‐β1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.     

Effects of nonesterified fatty acids and beta-hydroxybutyrate on functions of mononuclear cells obtained from ewes

OBJECTIVE: To assess the effects of nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) on functions of mononuclear cells obtained from ewes. ANIMALS: 6 Sardinian ewes. PROCEDURE: Mononuclear cells were cultured with concentrations of NEFA (0, 15.6, 31.2, 62.5, 125, 250, 500, 1,000, or 2,000 micromol/L) and BHBA (0, 0.45, 0.9, 1.8, or 3.6 mmol/L). Concentrations of NEFA and BHBA were intended to mimic those of ketotic or healthy ewes, and NEFA and BHBA were tested alone and in combination. Synthesis of DNA was stimulated by use of concanavalin A (Con A) or pokeweed-mitogen (PWM). Secretion of IgM was stimulated by use of PWM. RESULTS: Synthesis of DNA stimulated by Con A and PWM was significantly inhibited by high concentrations of NEFA (> or = 250 micromol/L) or by a combination of high concentrations of NEFA (> or = 250 micromol/L) and all concentrations of BHBA (> or = 0.45 mmol/L). In contrast, DNA synthesis was not inhibited by low concentrations of NEFA (< or = 125 micromol/L) or by a combination of low concentrations of NEFA (< or = 125 micromol/L) and the lowest concentration of BHBA (0.45 mmol/L). Secretion of IgM was significantly inhibited by all concentrations of NEFA and by all combinations of NEFA and BHBA concentrations. When used alone, none of the concentrations of BHBA inhibited DNA synthesis or IgM secretion. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced immunoresponsiveness during ketosis is likely to be associated with an increase in plasma concentration of NEFA and not with an increase in plasma concentration of BH BA.     

Effects on functions of ovine blood mononuclear cells for each of several fatty acids at concentrations found in plasma of healthy and ketotic ewes

OBJECTIVE: To assess effects on functions of peripheral blood mononuclear cells (PBMC) obtained from ewes for each of several fatty acids represented in ovine plasma at concentrations mimicking those of ketotic or healthy ewes. SAMPLE POPULATION: Blood samples obtained from 6 Sardinian ewes. PROCEDURE: The PBMC were cultured in media that contained oleic (OA), palmitic (PA), stearic (SA), linoleic (LA), or palmitoleic (POA) acid at concentrations similar to those of ketotic or healthy ewes. Synthesis of DNA was stimulated by use of concanavalin A or pokeweed mitogen (PWM). Secretion of IgM was stimulated by use of PWM. RESULTS: High concentrations (900, 450, and 225 micromol/L) of OA significantly inhibited DNA synthesis and IgM secretion of PBMC. Conversely, low concentrations (56 or 28 micromol/L) of OA significantly enhanced DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, or 75 micromol/L) significantly inhibited DNA synthesis of PBMC. High concentrations of PA (600, 300, 150, 75, 375, or 18.7 micromol/L) and SA (300, 150, 75, or 38 micromol/L) also significantly inhibited IgM secretion of PBMC. None of the concentrations of LA and POA affected PBMC functions. CONCLUSION AND CLINICAL RELEVANCE: Impaired immunoresponsiveness of ketotic ewes is likely associated with an increase of plasma concentrations of OA, PA, or SA and not with that of LA or POA. At physiologic concentrations, single fatty acids are likely to participate in modulation of immunoresponsiveness by exerting suppressive or stimulatory effects on immune cells.     

Association between endometritis and urocystitis in culled sows

Slaughterhouse sampling and examination of urogenital tracts of 499 sows and gilts culled for reproductive reasons from 21 Hungarian herds were performed over a 6-year period. The aim was to estimate the prevalence of different urogenital tract lesions, and to provide sensitivity and specificity estimates for macroscopic and bacteriological examinations in the diagnosis of urocystitis and endometritis. Furthermore, the association between endometritis and urocystitis was assessed. The prevalence of main lesions of the urogenital tract was similar to that reported in other studies. The 'sensitivity' of macroscopic and bacteriological methods was determined statistically by taking histopathology as the 'Gold Standard'. As a result, the 'sensitivity' of macroscopic methods for the diagnosis of endometritis and urocystitis was < or = 18.1% and 47.9%, respectively, while the 'sensitivity' of bacteriology for the diagnosis of the same conditions was < or = 31.8% and 63.0%, respectively. The presumed positive association between urocystitis and endometritis was confirmed; it was not confounded by parity. Animals affected by urocystitis had a 3.5 times higher odds to simultaneously have endometritis than animals without urocystitis.     

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.     

RADIOGRAPHIC ANATOMY OF THE CRIBRIFORM PLATE (LAMINA CRIBROSA)

The radiographic appearance of the cribriform plate was investigated in 16 canine cadaver heads. The cribriform plate appeared as a "V"-shaped multilinear bone-opaque stripe in the caudal nasal region in projections perpendicular to the hard palate in 6 dogs with a skull index between 50.00 and 54.00. In 9 dogs with a skull index between 55.40 and 74.40, the cribriform plate had a more "C"-shaped and sharp appearance. In vertically oblique projections with an obliquity greater than 20 degrees, the cribriform plate lost its sharp outline and finally (40 degrees) disappeared. In lateral projections the cribriform plate appeared as a "C"-shaped interrupted bone-opaque stripe in all 16 dogs. In more brachycephalic dogs frontal bone structures superimposed on the cribriform plate on ventrodorsal and dorsoventral views and accentuated the radiographic appearance of the plate. Vertically oblique views separated both structures to produce two lines.     

Baclofen intoxication in a dog successfully treated with hemodialysis and hemoperfusion coupled with intensive supportive care

Objective: To describe a case of confirmed baclofen intoxication in a dog that was successfully treated with hemodialysis and hemoperfusion (HD/HP) and to report the serum baclofen kinetics. Case summary: A 2.5‐year‐old, 23 kg, spayed female Brittany Spaniel‐mix was treated after ingesting 21‐52 mg/kg of baclofen. The dog was comatose and was receiving manual ventilation at the time of presentation. Extracorporeal HD/HP was started 10 hours after admission. Within 3 hours of starting HD/HP the dog began initiating breaths and was extubated 18 hours after admission. Serial serum samples that were obtained during the first 24 hours of hospitalization were later analyzed for baclofen concentrations. The dog had elevated creatine phosphokinase and liver enzymes that correlated with an agitated recovery period. The dog had thrombocytopenia that resolved by 10 days after presentation. New or unique information provided: HD/HP shortened the baclofen serum elimination half‐life from 5 to 1.5 hours in the initial 2 hours of treatment. The intrinsic elimination rate constant (K_intr) for this dog was 0.138/hour and the total elimination rate constant (K_tot) during the first 2 hours of HD/HP treatment was 0.458/hour. In this dog, HD/HP was an effective method for rapidly decreasing serum baclofen concentration after an acute overdose.