Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system.

A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB.

A proposed design for conducting target animal safety studies for developing new veterinary pharmaceuticals.

Target animal safety studies are required for the regulatory approval of any new veterinary pharmaceutical registered almost anywhere in the world. Both the United States and Europe have extensive guidelines for conducting these studies. Due to new veterinary drug labeling requirements in the United States, changes in the overall design of target animal safety studies will be required for the United States. The guideline changes proposed in this paper are more evolutionary than revolutionary. While many new ideas are suggested, a number of existing practices that seem to work well were retained. Several new abbreviated drug development pathways are also proposed, an idea missing from current guidelines. This proposal, and others like it, should serve as a basis for representatives from the U.S. Food and Drug Administration, the European Commission on Veterinary Medical Products, the veterinary pharmaceutical industry, and practicing veterinarians to develop internationally harmonized target animal safety study guidelines that are clear in their intent, scientifically sound, and practical.

Safety of albendazole in developing bovine fetuses.

Albendazole, administered orally at a dose rate of 25 mg/kg of body weight to presumed pregnant cows or heifers on days 21, 31, 41, 51, and 61 of gestation, did not induce toxicosis in embryos or fetuses, and all calves born were structurally normal. Albendazole administration at a rate of 25 mg/kg to cows at 7 and/or 14 days of gestation decreased the apparent conception rate (ie, embryolethality), but did not have a teratogenic effect. Apparent embryolethality was greater in cows administered 25 mg/kg only on day 14, compared with those administered the drug only on day 7. Single dosage of 25 mg/kg given in the final 3 months of gestation did not induce abortion. There were no adverse effects of albendazole at a dosage of 10 or 15 mg/kg on developing embryos or fetuses when administered to presumed pregnant cows at various times in early gestation.

Comparison of Microbiological Methods for Detecting Penicillin and Penicillin-Like Antibiotic Residues in Milk.

Five microbiological methods for detecting residues of penicillin and penicillin-like antibiotics were compared, and the relative incidence of residues in whole milk samples was determined. Five groups of 10 lactating cows each and five commercial antibiotic products (penicillin G in oil, penicillin G in a water-dispersible oil base, cloxacillin, cephapirin and ampicillin) used for the treatment of mastitis were studied. Milk samples collected from the cows were tested for antibiotic content by the Sarcina lutea or Bacillus subtilis cylinder plate, International Dairy Federation-Association of Official Analytical Chemists (IDF-AOAC) Bacillus stearothermophilus ATCC 10149 disc plate, Delvotest-P, Antibiotic Residue Branch (ARB) B. stearothermophilus C 953 quantitative disc plate and ARB B. stearothermophilus C 953 quantitative cylinder plate methods. All samples tested by the original S. lutea or B. subtilis cylinder plate methods, with the exception of the penicillin G in oil product (Uni-Biotic), were free of antibiotic residues and showed no inhibitors beyond the labeled milk withdrawal time. The newer, more sensitive official IDF-AOAC method, Delvotest-P and both ARB B. stearothermophilus quantitative disc and cylinder plate methods detected measurable amounts of cephapirin and penicillin G in the post-treatment milk for one to three additional milkings beyond the labeled milk discard time. Cloxacillin, as determined by the ARB B. stearothermophilus cylinder plate method, was generally depleted by 48 h. The excretion rate of ampicillin product coincided with the labeled milk discard time. The level of detection of both disc plate methods and Delvotest-P for cephapirin and ampicillin was comparable. Variations in the concentration in milk and the persistence of two penicillin G products were probably due to differences in the formulation and the dosing regimen. Inhibitory substances and non-specific reactions were observed with the IDF-AOAC disc method, the Delvotest-P and both ARB B. stearothermophilus methods in some control and post-treatment milk samples. These results are consistent with reports of other investigators and suggest that the use of more sensitive procedures for monitoring antibiotic residues in milk may result, for some infusion preparations, in an increased incidence of violative residues of penicillin G and cephapirin.

Comparative pharmacokinetics of chlortetracycline in milk-fed versus conventionally fed calves.

Plasma and tissue concentration and pharmacokinetics of chlortetracycline (CTC) was determined in milk-fed and conventionally fed Holstein calves. A two-compartment open model was used after a single intravenous dose (11 mg CTC/kg body weight). There were no significant differences between dietary treatments. The drug was rapidly distributed from plasma into the peripheral compartment but was slowly eliminated, with detectable concentration of CTC continuing for 72 h after dosing. A single-compartment model was used after a single oral dose (22 mg CTC/kg body weight). All but four of the kinetic parameters were significantly different for the two dietary treatments. Milk-fed calves had a larger area under the plasma level curve, a larger fraction of the dose absorbed, a smaller volume of distribution and a smaller overall body clearance rate. Estimated recovery of CTC in the urine of the milk-fed calves was greater, regardless of route of administration. The concentration of CTC in tissues following an oral dose was greatest in kidney, followed by liver, heart, skeletal muscle, spleen and brain. Tissue depletion of CTC closely paralleled the decline in plasma concentration.

Selected blood chemistry and hematologic characteristics in milk-fed and conventionally fed calves.

Effects of diet and age on selected blood chemistry and hematologic characteristics were studied in 20 Holstein bull calves. Milk-fed calves (treatment 1) were maintained exclusively on a milk-replacer diet. Conventionally fed calves (treatment 2) were provided a calf-starter diet and alfalfa hay beginning at 2 wk of age and were weaned from milk replacer at 7 wk of age. Blood samples were collected by jugular puncture from all animals just before the morning feeding at 6, 7, 9, and 11 wk of age. Milk-fed calves had greater glucose concentration in blood serum. Urea nitrogen of blood increased in conventionally fed calves from 6 through 11 wk and decreased in milk-fed calves. Hemoglobin concentration and packed cell volume decreased in calves receiving milk-replacer diet. Total protein and albumin concentrations were greater in the conventionally fed group. Treatment differences also existed for calcium and cholesterol concentrations and in alkaline phosphatase activity.

Experimental hexachlorophene intoxication in young swine.

Hexachlorophene (HCP) intoxication in swine was studied as a possible model of HCP poisoning in human beings. Swine were topically or orally given HCP each day, and observations were made on central nervous system function, hematologic measurements, serum chemical analyses, necropsy, microscopic examination of tissues, and concentrations of HCP residues in blood and tissue. Intoxication was observed in orally exposed swine, but the syndrome was not consistently produced in topically exposed swine. Signs and microscopic lesions were all related to dysfunction of the central nervous system, e.g., incoordination, paresis, and status spongiosus. Occurrence of signs and lesions, mortality rates, and concentrations of HCP residue were positively correlated with the dosage of HCP given.

The relative influence of sex of progeny on the lethal expression of the sonless gene in Drosophila melanogaster.

The sex-linked maternally influenced lethal gene, sonless (snl), in D. melanogaster was shown to affect the survival of daughters as well as sons of snl/snl mothers but to a lesser degree. Interaction studies of sonless with the sex altering mutants transformer (tra) and doublesex (dsx) revealed that any alteration toward increased masculinity of progeny from snl/snl females reduced their zygotic viability, independent of snl dosage or Y chromosome presence or absence. Possible implications of these results are discussed and the original gene action hypothesis for snl is extended to incorporate these new findings.-Sonless is non-allelic but closely linked to rudimentary (r) and fused (fu), two mutants with similar sex-ratio patterns.