In vitro identification and verification of inflammatory biomarkers in swine

Currently there are no non-steroidal anti-inflammatory drugs (NSAIDs) approved for the control of inflammation in swine due to a lack of validated animal models and suitable biomarkers to assess drug efficacy. This study investigates the differential expression of genes altered in response to Escherichia coli lipopolysaccharide (LPS) induced inflammation which may serve as indicators of NSAID efficacy. Unstimulated whole blood from swine was mixed with tissue culture media, stimulated with LPS, and RNA extracted at the following time points 0 h, 1 h, 3 h, 24 h and 48 h. Total RNA was extracted and analyzed using a commercial swine DNA microarray. The DNA microarray was utilized as a screen to determine potential biomarkers, focusing on the genes that exhibited the greatest degree of differential expression. A master list of 57 genes was formed based on the differential expression as a result of the stimulation. Following analysis, 12 genes whose expressions were significantly altered (8 up- and 4 down-regulated) were chosen for verification via quantitative RT-PCR (qRT-PCR). The qRT-PCR analysis confirmed the differential expression of 11 of the 12 genes chosen via the microarray analyses. Specifically, traditional genes such as SAA, G-CSF, and IL-10 were up-regulated, while CD4 was down-regulated; all of the genes were altered by 24 h or 48 h post-stimulation. We demonstrate here that expression of these 11 genes is altered as a direct result of LPS stimulation and consequently inflammation.     

Absorption and distribution of intubated insecticides in fasted mice

Radioactive solutions of ¹⁴C-labeled insecticides were administered by stomach needle to fasted female mice (1 mg/kg, Dublin strain). They were killed at different time intervals and the amount of radioactivity determined in the gastrointestinal tract (unabsorbed) and in selected organs. T_0.5 values of penetration (min) ranged from 8 to 17 for the carbamates tested, 24 to 78 for the organophosphorus compounds, and 15 to 62 for the chlorinated hydrocarbons. Among other insecticide groups, nicotine penetrated rapidly whereas permethrin was the least rapid of all compounds under study. The insecticides were rapidly distributed to various organs. No consistent correlations between absorption and physicochemical properties of the compounds were noted.     

Twenty-four hour urinary protein loss in healthy cats and the urinary protein-creatinine ratio as an estimate

Urinary protein loss was determined in 12 healthy cats. Voided urine was collected and protein quantitated by the Coomassie blue method. Mean protein loss for all cats was 12.65 mg/kg/24 h (5.45 SD). Protein loss for male cats (n = 6) was 16.62 mg/kg/24 h (3.3 SD), which was significantly different (P less than 0.01) from 8.69 mg/kg/24 h (4.09 SD) for females (n = 6). A single urine protein-creatinine ratio correlated well with the total urinary protein loss in mg/kg/24 h. The correlation coefficient for the protein-creatinine ratio in voided urine (UPCV) vs 24-hour urinary protein (UP-24) loss was 0.968, and that for the protein-creatinine ratio in urine obtained by cystocentesis (UPCC) vs UP-24 was 0.945. The regression equations were UPCV = 0.02145 + 0.02338 x UP-24 (mg/kg), and UPCC = 0.02667 + 0.02133 x UP-24 (mg/kg). Using the mean value plus 3 SD of urinary protein loss from the healthy cats in this study, a healthy cat would be expected to have a urinary protein loss of less than 29 mg/kg/24 h. A protein-creatinine ratio from a single urine sample provides an accurate estimate of urinary protein loss in healthy cats.     

Complications associated with 355 flexible colonoscopic procedures in dogs

Flexible colonoscopy is commonly performed in dogs with signs of large-bowel diseases. Although considered to be a safe procedure, no reports of complications associated with colonoscopy have appeared in the veterinary literature. The purpose of this study was to describe the frequency and types of adverse events that developed during flexible colonoscopy in dogs. Medical records were reviewed from 355 scheduled colonoscopic procedures. Major complications were defined as adverse events in which the dog's life was potentially jeopardized and the complication required intensive treatment or monitoring. Major complications consisting of fatal aspiration of GoLYTELY, colonic perforation, and excessive hemorrhage after biopsy of an adenocarcinoma with rigid forceps occurred in 3 (0.85%) dogs. Minor complications associated with anesthesia or colonoscopy occurred during 3.4% of procedures. Complications were classified as minor if the adverse event required minimal treatment or monitoring, and the complication was not considered a threat to the dog's life. Vomiting of GoLYTELY occurred with the administration of 4.6% of doses in 6.5% of dogs. When administering GoLYTELY, clinicians should be prepared to rapidly remove the orogastric tube and mouth speculum if vomiting occurs to reduce the potential for aspiration. In this group of dogs undergoing flexible colonoscopy, major complications occurred infrequently and minor complications developed uncommonly. Overall, minor or major complications developed during 30 (8.5%) of 355 procedures. Mortality was rare (0.28%). Flexible colonoscopy appears to be a safe procedure in dogs with signs of large-bowel diseases.     

Clinical signs associated with pseudorabies in dogs

Clinical signs in dogs with pseudorabies (Aujeszky's disease) were tabulated from 25 confirmed cases. The duration of disease was short, ranging from 6 to 96 hours. Eight dogs were euthanatized. Of those not euthanatized, 12 (71%) died within 24 hours of onset, 16 (94%) died within 48 hours, and only 1 (6%) lived longer than 48 hours (96 hours) after the onset of clinical signs. All of the dogs had ptyalism, 84% were restless, 84% were anorectic, 76% were atactic, and 64% wandered aimlessly. Sixty-four percent of the dogs had tachypnea, 60% had dyspnea, 56% vocalized, 52% were pruritic, 48% held their necks rigidly, 36% vomited, 36% had muscle spasms, 36% were aggressive, 28% had trismus, and 24% had dysphagia. Five of 25 dogs (20%) had abnormal pupillary light responses. Two of the 25 dogs circled and 2 walked backwards. Each of the following were detected once: blindness, ptosis, facial paresis, excessive lacrimation, head-tilt, head-pressing, signs of abdominal pain, and photophobia. All dogs had been exposed to swine, although in some instances the farmer was unaware pseudorabies existed in the herd or believed it was not in the herd on the basis of negative results on serologic testing.     

Analysis of herbal teas made from the leaves of comfrey (Symphytum officinale): reduction of N-oxides results in order of magnitude increases in the measurable concentration of pyrrolizidine alkaloids

OBJECTIVES: To determine the relative quantities of two hepatotoxic pyrrolizidine alkaloids, symphytine and echimidine, in teas prepared from comfrey leaves (Symphytum officinale), and to determine the potential contribution of the N-oxide forms of these alkaloids to levels of the parent alkaloids. DESIGN: Comfrey leaves were purchased from three commercial sources and used to prepare tea in a manner consistent with the methods used by consumers. An extraction scheme was devised for extraction of the alkaloids, and a gas chromatographic method was developed to quantify the two major alkaloids, symphytine and echimidine. Recognising that the N-oxide derivatives of these alkaloids have also been identified in comfrey preparations, chemical reduction was applied to determine the total quantities of the alkaloids as free bases and as N-oxide derivatives. RESULTS: The concentration of symphytine and echimidine varied considerably between teas prepared from leaves purchased from the different vendors of plant material. Moreover, a much higher concentration of symphytine was found in the tea when steps were included to reduce N-oxides prior to analysis. The treatment of pure symphytine with hot water did not generate the N-oxide derivative de novo. CONCLUSIONS: Since the pyrrolizidine alkaloids are known to be hepatotoxic, consumption of herbal teas made from comfrey leaves may be ill-advised. The concentration of pyrrolizidine alkaloids in such teas may be underestimated substantially unless the concentration of N-oxides is taken into consideration.     

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.