Ovine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein. First, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography. The protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays. Purified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

The role of interleukin 1 (IL-1) as an inflammatory mediator during mastitis and the therapeutic effect of recombinant human IL-1 receptor antagonist (IL-1ra) for bovine mastitis was studied. Cows were intramammarily infused with lipopolysaccharide (25 g) in 1 mammary gland. Half the cows also received infusions of 5 mg of IL-1ra into the same mammary gland just prior to endotoxin infusion and 4, 8, and 12 hours later. After endotoxin infusion, tumor necrosis factor and high IL-1 bioactivity were detected in whey from infused glands. Vascular permeability changes and neutrophil accumulation in milk paralleled the appearance of cytokines. A systemic reaction, characterized by pyrexia and an increase in blood cortisol concentration, also were observed. Milk yield was inhibited and milk composition was altered in infused and noninfused glands. The increase in IL-1 bioactivity in milk after endotoxin infusion was almost completely prevented in glands receiving IL-1ra. However, IL-1ra had no effect on local inflammation, systemic reaction, or impairment in productive performance. These results indicate that IL-1 does not mediate its effect within the milk compartment, and suggest either that IL-1 is not critical to the mastitic response or that intramammary infusion of IL-1ra does not place the antagonist where IL-1 interacts with its receptor.

Acyloxyacyl hydrolase (AOAH) is a lysosomal enzyme found in neutrophils and macrophages that acts to partially deacylate the lipid-A component of the endotoxin of gram-negative bacteria rendering it less toxic, yet maintaining much of its immunostimulatory potential. We have found that the activity of neutrophil AOAH per cell increased during localized inflammation. The purpose of this study was to determine the mechanism(s) responsible for these increases in neutrophil AOAH activity. Because changes in neutrophil maturity commonly are associated with inflammation, intravascular infusion of purified gram-negative bacterial lipopolysaccharide and SC injection of bovine recombinant granulocyte colony-stimulating factor was used to induce large numbers of circulating immature neutrophils. Immature neutrophils were found to have AOAH activity equal to that of mature cells; however, when neutrophils were stimulated in vitro with known activators, AOAH activity of activated cells was more than that of unstimulated cells. The increase in AOAH activity was inversely related to prestimulation activity. Increases in AOAH activity after neutrophil activation were not a result of de novo synthesis of the enzyme, because cycloheximide did not prevent activation-induced increases in activity.

Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.

The absorption kinetics and glycemic effects of 3 long-acting insulin preparations (protamine zinc beef-pork insulin, ultralente beef-pork insulin, and ultralente human insulin) were evaluated in 9 healthy, adult, domestic shorthair cats (6 males, 3 females). A triple crossover study was performed, in which the serial serum concentrations of insulin and glucose were determined over a 24-hour period after sc administration of the 3 insulin preparations (dosage, 1.0 U/kg of body weight) at 3-week intervals. A control study was also performed in 4 of the cats by serially collecting samples for insulin and glucose determinations after administration of insulin diluent. After administration of protamine zinc insulin (PZI), mean (+/- SEM) serum insulin concentration increased significantly (P < 0.05) above baseline, reached a peak value (484 +/- 287 pmol/L) at 1 hour, and remained significantly (P < 0.05) higher than baseline at 24 hours. After administration of ultralente human insulin, the serum insulin curve was similar to that obtained after PZI administration, but mean serum insulin concentration took longer to peak (538 +/- 177 pmol/L at 4 hours). After administration of ultralente beef-pork insulin, mean peak serum insulin concentration was lower (220 +/- 54 pmol/L, not statistically significant) than that obtained after administration of PZI and ultralente human insulins; it then decreased to values statistically indistinguishable from baseline by 16 hours. The area under the serum insulin concentration curve for PZI (5,063 +/- 681 pmol.h/L) and ultralente human insulin (4,138 +/- 439 pmol.h/L) was significantly (P < 0.05) larger than that for ultralente beef-pork insulin (2,378 +/- 561 pmol.h/L). Serum glucose concentration decreased after administration of all 3 insulins, but the decrease was not different from that observed after diluent (control) administration. Results of this study indicate that differences may exist between absorption of PZI, ultralente human, and ultralente beef-pork insulins. Of the 2 ultralente insulin preparations, human insulin appears better absorbed than beef-pork insulin, but these findings need to be confirmed in cats with naturally acquired diabetes mellitus.

Polymorphonuclear neutrophils (PMN) from 4 cows were preincubated (30 minutes, 37 C) in either actinomycin D (100 micrograms/ml) or puromycin (10 micrograms/ml), inhibitors of mRNA transcription and protein translation, or in medium 199. The PMN were incubated for a further 4.5 hours in medium containing 100 U of recombinant bovine interferon-gamma (rboIfn-gamma). The PMN were then incubated with bovine IgG1, IgG2, IgM, or aggregated IgG (aIgG; 4 C, 12 hours) for flow cytometric analysis, using fluoresceinated isotype-specific antibody. The percentage of PMN binding the ligand and the logarithmic mean fluorescent channel (LMFC), an indicator of the amount of receptor (R) expression, were recorded. Competitive inhibition of ligand binding was measured by incubating PMN with fluoresceinated IgG2 in the presence or absence of 100-fold excess of IgG1, IgG2, and aIgG. Activation with rboIfn-gamma induced a 4.5-fold increase in binding of IgG1 and a fivefold increase in LMFC for IgG2. These increases were inhibited by actinomycin D and puromycin. Percentage of PMN binding aIgG decreased after activation by rboIfn-gamma. Interferon-gamma treatment did not affect binding or LMFC of IgM. However, binding of IgM was reduced by treatment with actinomycin D. Binding of fluoresceinated IgG2 was inhibited by unlabeled IgG1, IgG2, and aIgG. Results indicate that bovine PMN Fc receptors (FcR) for IgG1 and IgG2 were rboIfn-gamma inducible, that induction required de novo transcription and translation, that a heterogeneous population of FcR exist on bovine PMN, and that IgG1 and IgG2 share a common FcR. Further, bovine PMN are capable of gene activation and are responsive to changes in their environment, thus being amenable to modulation for effective pathogen destruction.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of IL-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the IL-1-induced model of osteoarthritis. Osteoarthritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Recombinant equine interferon-beta 1 (reqIFN-beta 1) induces an antiviral state in blood mononuclear cells (BMC) of horses. Maximal protection against replication of vesicular stomatitis virus is achieved 6 hours after treatment with IFN in vitro and in vivo. Duration of the protective effect depends on the dose of IFN in vitro and in vivo. Availability of reqIFN-beta 1 in cultures of BMC for up to 48 hours does not prolong the antiviral state. The protective effect on BMC after treatment with IFN has similar duration in vivo and in vitro. Monitoring of the effect of IFN in vivo is, thus, simplified because the antiviral state may be recorded by testing cells twice (ie, before and 6 hours after application of interferon). All further tests may be performed in vitro. Multiple administrations of reqIFN-beta 1 do not prolong duration of the protective phases after each administration. Duration of the antiviral state depends only on the dose of reqIFN-beta 1.

Three kinds of recombinant vaccinia virus (RVV)--mO-HA/ATI, LO1-HA/ATI and mO-HA/7.5kD--expressing bovine leukosis virus (BLV) envelope glycoprotein (gp60) were constructed. The BLV envelope gene of RVV mO-HA/ATI and LO1-HA/ATI or of RVV mO-HA/7.5kD was expressed under control of the promoter of A-type inclusion body (ATI) protein gene of cow-pox virus or vaccinia virus 7.5-kD protein gene, respectively. The vaccinia virus strain, LC16mO, was used as vector for RVV mO-HA/ATI and mO-HA/7.5kD, and strain LO-1 was used for RVV LO1-HA/ATI. Strains LC16mO and LO-1 are attenuated vaccine virus strains originating from the Lister original vaccinia virus. All 3 kinds of constructed RVV expressed gp60 in cultured rabbit kidney cells after infection; mO-HA/ATI expressed more antigen than did mO-HA/7.5kD. Rabbits vaccinated with RVV produced considerable antibody capable of inhibiting syncytium formation, as well as antibody with virion-binding ability. The RVV that used ATI promoter induced higher antibody titer than did the RVV that used 7.5-kD promoter. Results indicate that BLV gp60 is responsible for induction of neutralizing antibodies that suppress in vitro formation of syncytia among BLV-infected cells. Applicability of RVV, especially those using ATI promoter, was evaluated in a vaccine against bovine leukosis.

Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbGM-CSF)/ ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxocity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbGM-CSF significantly increased (P < 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbGM-CSF-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.