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Biomedical subjects

D D Morris

Publications and source records attributed to D D Morris.

At least 37 records · Page 2Linked to original sources
PubMed1992

Equine tumor necrosis factor alpha: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and development of a sensitive enzyme-linked immunosorbent assay.

We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNF alpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNF alpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNF alpha was purified by anti-FLAG immunoaffinity chromatography and then used as immunogen for production of murine Mabs and rabbit Pabs. Three Mabs (6H4, 9B10, and 12F6) were obtained from one fusion. All three Mabs recognized rETNF alpha on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTF alpha and neutralized both rETNF alpha and native ETNF alpha (nETNF alpha) in a WEHI cell cytotoxicity assay. A sensitive ELISA was developed using Mab 6H4 and biotin-labeled rabbit Pabs. The ELISA was shown to detect levels of ENTF alpha as low as 100 pg/ml and was used to demonstrate the induction of ETNF alpha in horses with experimental endotoxemia. The rETNF alpha, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.

Animals↗
PubMed1992

Endotoxin-induced production of interleukin 6 by equine peritoneal macrophages in vitro.

A study was performed to determine whether equine peritoneal macrophages produce interleukin 6 (IL-6) in vitro in response to endotoxin. Peritoneal fluid was collected from 14 clinically normal adult horses and was used as the source of peritoneal macrophages. Macrophages from each horse were isolated and cultured separately in vitro in the absence or presence of various concentrations (0.5, 5, 500 ng/ml) of endotoxin (lipopolysaccharide from Escherichia coli 055:B5). Culture medium supernatants were collected after 3, 6, 12, and 24 hours' incubation and were frozen at -70 C until assayed for IL-6 activity. Supernatant IL-6 activity was determined by use of a modified colorimetric assay and the murine hybridoma cell line B 13.29 clone B.9, which is dependent on IL-6 for survival. Results indicated that equine peritoneal macrophages produce IL-6 in vitro and that supernatant medium IL-6 activity was significantly (P less than 0.05) increased by exposure to endotoxin. Significant (P less than 0.05) time and treatment effects on macrophage IL-6 production were apparent. The IL-6 activity peaked at 6 or 12 hours' incubation, then remained high through 24 hours' incubation, regardless of endotoxin exposure. Medium IL-6 activity during 3 and 6 hours' incubation was significantly (P less than 0.05) greater in macrophages exposed to 5 or 500 ng of endotoxin/ml than in those exposed to 0.5 ng of endotoxin/ml; however peak IL-6 activity was similar among all endotoxin concentrations. Endotoxin concentration did not have an effect on medium IL-6 activity from macrophages exposed to endotoxin for 12 or 24 hours.

Animals↗
PubMed1992

Effect of experimentally induced endotoxemia on serum interleukin-6 activity in horses.

A study was conducted to determine whether serum interleukin-6 (IL-6) activity increased in horses during experimentally induced endotoxemia and whether serum IL-6 activity correlated to changes in clinical or laboratory data. Six clinically normal horses were given endotoxin IV (30 ng/kg of body weight) in 0.9% NaCl solution over 1 hour. Five of these and 1 additional horse served as controls and were given only 0.9% NaCl solution. Venous blood, for determination of serum IL-6 activity and WBC count, was collected before and at various times through 8 hours after the start of endotoxin or NaCl infusion. Rectal temperature and heart and respiratory rates were recorded throughout the study period. Serum IL-6 activity was determined by bioassay of proliferation of the B13.29 clone B.9 hybridoma cell line. From 1.5 through 5 hours after start of the infusion, serum IL-6 activity was significantly (P less than 0.05) increased in horses given endotoxin. Mean peak serum IL-6 activity was observed between 3 and 4 hours. In response to endotoxin infusion, horses became lethargic, tachycardic, and febrile. Leukopenia developed by 1 hour, followed by leukocytosis at 8 hours. Significant (P less than 0.05) positive association and linear correlation were apparent between mean serum IL-6 activity and mean rectal temperature in the group of horses that were given endotoxin. Changes from baseline were not evident in any of the clinical or laboratory values in horses given only NaCl solution.

Animals↗
PubMed1992

Reduced endotoxin-induced production of tumor necrosis factor activity by equine peritoneal macrophages exposed to the dual inhibitor of arachidonic acid metabolism, SK & F 86002.

The purpose of this study was to determine if a structurally novel dual inhibitor of arachidonic acid metabolism, SK & F 86002, would inhibit the endotoxin-induced production of tumor necrosis factor (TNF) activity by equine peritoneal macrophages. Equine peritoneal macrophages were variously pretreated for 0, 0.5 and 2 h with SK & F 86002 at 10(-9) to 10(-4) molar final concentrations or were left untreated. Then, the macrophages were cultured in vitro in the presence of endotoxin (5 ng/mL). Supernatant media were collected after 4 h and stored at -70 degrees C until assayed for TNF activity and immunoreactive thromboxane B2 (iTxB2). Macrophage supernatant TNF activities were estimated by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. Concentrations of iTxB2 were quantitated by radioimmunoassay. Coincubation of macrophages with SK & F 86002 significantly decreased the subsequent supernatant TNF activity. Concentrations of SK & F 86002 from 10(-7) to 10(-4) molar effectively reduced TNF production when added to macrophages 0 and 0.5 h prior to endotoxin. After 2 h of preincubation, SK & F 86002 significantly reduced supernatant TNF activity at 10(-5) and 10(-4) M concentrations. Supernatant concentrations of iTxB2 were reduced when SK & F 86002 was added at 10(-6) to 10(-4) M concentrations, 0 and 0.5 h prior to endotoxin, and at all concentrations (10(-9) to 10(-4)) when preincubated with macrophages for 2 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Hematuria and weight loss in a mare with pancreatic adenocarcinoma.

This clinical report describes an 11-yr-old Thoroughbred mare that presented with clinical signs of weight loss and hematuria. History and clinical findings suggested the mare had neoplasia of the urogenital system. Although ultrasound-guided biopsy of the perirenal mass did not yield abnormal cells, large irregular cells were isolated from the thoracic and peritoneal fluid. At necropsy, multiple firm nodules were disseminated throughout the peritoneal cavity. Histological examination of the nodules revealed cells that were consistent with a pancreatic exocrine adenocarcinoma. This is the first report of pancreatic adenocarcinoma causing dysfunction of the urogenital system.

Adenocarcinoma↗
PubMed1991

Tumor necrosis factor activity in serum from neonatal foals with presumed septicemia.

A study was performed to determine prevalence of tumor necrosis factor (TNF) activity in serum of equine neonates with presumed sepsis and to determine correlation between serum TNF activity and severity and outcome of disease. Twenty foals less than 21 days old were considered suitable for inclusion in this study by satisfying clinical and laboratory criteria suggestive of septicemia. At admission, blood samples were collected from all foals for determination of serum TNF activity, then clinical course and outcome of disease were recorded. Thirty-one clinically normal foals less than 21 days old served as controls for serum TNF activity. Serum TNF activity was estimated by use of an in vitro cytotoxicity bioassay and WEHI 164 clone-13 murine fibrosarcoma cells. Of the 20 foals with presumed sepsis, 5 had high serum TNF activity. Mean heart rate (P less than 0.005), mucosal petechial hemorrhages (P = 0.06), and death rate (P = 0.06) were greater in the group of foals with high serum TNF activity. These foals also had a lower mean neutrophil count (P less than 0.001), greater band-to-segmented neutrophil ratio (P less than 0.0001), and more prevalent neutrophil toxic changes (P = 0.07) than did foals without serum TNF activity (P = 0.02). Joint swelling was more prevalent in foals without serum TNF activity. Results of the study indicate that serum TNF activity is correlated with clinical criteria of sepsis in equine neonates. An association was apparent between disease severity and serum TNF activity in this group of foals with presumed septicemia.

Animals↗
PubMed1991

Effect of dietary alpha-linolenic acid on endotoxin-induced production of tumor necrosis factor by peritoneal macrophages in horses.

A study was conducted to determine whether dietary supplements with alpha-linolenic acid altered the ability of equine peritoneal macrophages to produce tumor necrosis factor (TNF) in response to endotoxin. Peritoneal macrophages were harvested from 6 healthy adult horses before and after the horses were fed a nutritionally balanced ration that contained 8% linseed oil as a source of alpha-linolenic acid. The macrophages were cultured in media containing no additives (control), endotoxin (0.5 to 50 ng/ml), or the calcium ionophore, A23187. Macrophage supernatants were collected after 6 and 24 hours' incubation and stored at -70 C. Tumor necrosis factor activity was estimated by a modified in vitro cytotoxicity bioassay, using the murine fibrosarcoma cell line, WEHI 164 clone 13. The TNF activity after 6 and 24 hours' incubation was greater in culture media of macrophages exposed to endotoxin than in media from control macrophages. For macrophages cultured in media that contained endotoxin, neither the concentration of endotoxin nor incubation time had any effect on TNF activity. Endotoxin-induced macrophage production of TNF, as determined by measurement of TNF activity, was significantly less after horses were fed the alpha-linolenic acid-rich ration for 8 weeks.

Animals↗
PubMed1991

Serum tumor necrosis factor activity in horses with colic attributable to gastrointestinal tract disease.

Over a 24-month period, serum tumor necrosis factor (TNF) activity was determined in 289 horses with colic attributable to gastrointestinal tract disease. Serum TNF activity was quantitated by use of a modified in vitro cytotoxicity bioassay, using WEHI 164 clone-13 murine fibrosarcoma cells. Causes for colic, determined by clinical and laboratory evaluation, exploratory celiotomy, or necropsy included: gastrointestinal tract rupture (GTR); ileal impaction; small intestinal strangulating obstruction (SIO); proximal enteritis (PE); transient small intestinal distention; large-colon displacement; large-colon volvulus; large-colon impaction; colitis; small-colon obstruction; peritonitis; and unknown. Each diagnosis was placed into 1 of 3 lesion categories: inflammatory disorders (GTR, PE, colitis, peritonitis); strangulating intestinal obstruction (SIO, large-colon volvulus); and nonstrangulating intestinal obstruction (ileal impaction, transient small intestinal distension, large-colon displacement, large-colon impaction, small-colon obstruction, unknown). The prevalence of high serum TNF activity and/or mortality were evaluated. Differences were tested at significance level of P less than 0.05. Approximately 20% of the 289 horses has serum TNF activity greater than that found in clinically normal horses (greater than 2.5 U/ml). Twenty-three horses (8%) had marked increase in serum TNF activity (greater than or equal to 10 U/ml) which was more prevalent among horses with SIO and PE than in horses of other diagnostic groups, except those with GTR. Mortality and marked increase in serum TNF activity were greater in horses with intestinal inflammatory disorders or strangulating intestinal obstruction than in horses with nonstrangulating intestinal obstruction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Dexamethasone reduces endotoxin-induced tumor necrosis factor activity production in vitro by equine peritoneal macrophages.

This study evaluated the effect of dexamethasone on endotoxin-induced production of tumor necrosis factor (TNF) activity in vitro by equine peritoneal macrophages. Peritoneal macrophages from adult horses were cultured in the presence of dexamethasone (1-100 microM) for various time periods (2 hour, 0.5 hour, 0 hour) prior to the addition of endotoxin (5 ng/ml), then the secretion of TNF activity was evaluated. Macrophage supernatant concentrations of TNF activity were estimated by a modified in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line, WEHI 164 clone 13. An experiment was performed to determine whether dexamethasone interfered with the cytolytic bioassay's ability to detect TNF activity. The endotoxin-induced TNF activity production by equine peritoneal macrophages was significantly reduced by co-incubation with 100 microM dexamethasone, but not by tested concentrations of dexamethasone less than 100 microM. This concentration of dexamethasone greatly exceeds those generally attained by therapeutic use of dexamethasone in horses. Preincubation time did not affect the ability of 100 microM dexamethasone to reduce TNF production by equine macrophages. The quantitation of equine TNF activity by its cytolytic bioassay was not altered by dexamethasone.

Analysis of Variance↗
PubMed1990

Septicemia, atrial fibrillation, cardiomegaly, left atrial mass, and Rhodococcus equi septic osteoarthritis in a foal.

A foal with vegetative bacterial endocarditis affecting the wall of the left atrium was treated successfully with cefotaxime, erythromycin, and rifampin. Bacterial isolates included Escherichia coli from blood and Rhodococcus equi from a P-type osteomyelitic lesion of the left third metatarsal bone and from synovial fluid from the left metatarsophalangeal joint. Cardiac complications included cardiomegaly and atrial fibrillation, which responded to treatment with digoxin and quinidine sulfate. Cardiac function was considered normal 18 months after treatment. Bacteriologic cure of osteoarthritis was achieved by use of surgical debridement, lavage, and local and systemic antimicrobial treatment; however, lameness developed 18 months after treatment when training for flat racing was begun. Radiography revealed chronic degenerative joint disease.

Actinomycetales Infections↗
PubMed1990

Correlation of clinical and laboratory data with serum tumor necrosis factor activity in horses with experimentally induced endotoxemia.

Serum tumor necrosis factor (TNF) activity was quantitated in 8 horses given an IV infusion of endotoxin (0.03 micrograms of lipopolysaccharide/kg of body weight, from Escherichia coli 055:B5) in 0.9% NaCl solution over 1 hour. Serum TNF activity was likewise measured in 6 horses given only 0.9% sterile NaCl solution at the same rate. The duration of serum TNF activity was determined, and serum TNF activity was correlated with clinical and laboratory changes during the induced endotoxemia. Horses had no serum TNF activity prior to endotoxin administration, but geometric mean serum TNF activity was significantly higher from 1 to 4 hours after the start of the infusion. In response to endotoxin, horses seemed depressed, had signs of mild to moderate abdominal pain, developed tachycardia and fever, and had leukopenia followed by leukocytosis. Association between serum TNF activity and temperature, heart rate, attitude abnormality score, and WBC count of horses given endotoxin was significant. Serum TNF activity had a significant positive linear correlation with attitude abnormality and heart rate and a negative linear correlation with the WBC count during endotoxemia. Geometric mean serum TNF activity peaked approximately 1.5 hours prior to mean peak fever, and these data were significantly correlated. Results of this study suggest that TNF is an important mediator of endotoxemia in horses.

Animals↗
PubMed1990

Endotoxin-induced procoagulant activity, eicosanoid synthesis, and tumor necrosis factor production by rat peritoneal macrophages: effect of endotoxin tolerance and glucan.

Macrophages release pro-inflammatory substances that may augment intravascular coagulopathy associated with endotoxemia. In the present study, the effect of Salmonella enteritidis endotoxin on expression of procoagulant activity (PCA), eicosanoid metabolism, and production of tumor necrosis factor (TNF) by rat peritoneal macrophages was determined. Endotoxin induced significant dose-dependent increases in the concentrations of immunoreactive (i) thromboxane B2 (TxB2), 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha), and TNF in culture media. Calcium ionophore (A23187; 0.5 microM) induced an approximate two-fold greater increase (P less than 0.05) in iTxB2 and i6-keto-PGF1 alpha than that stimulated by the maximal endotoxin dose. Endotoxin (0.5, 5, and 50 micrograms/ml) induced similar increases in PCA in macrophage lysates which paralleled the production of iTxB2 and i6-keto-PGF1 alpha. In contrast to its marked effect on eicosanoid metabolism, A23187 elicited little increase in PCA. After the responses of peritoneal macrophages from normal animals were characterized, we hypothesized that procedures which alter the in vivo response of rats to endotoxin would similarly alter the in vitro responses of their peritoneal macrophages. In subsequent studies, the effect of altered endotoxin sensitivity on expression of PCA, eicosanoid synthesis, and TNF activity were assessed. Endotoxin tolerance, induced by repeated injection of sublethal doses of endotoxin in vivo, rendered rat peritoneal macrophages refractory to in vitro endotoxin-induced production of iTxB2, i6-keto-PGF1 alpha, PCA, and TNF activity. In contrast, pretreatment of rats with the macrophage stimulant glucan, which enhances endotoxin lethality, augmented the in vitro production of iTxB2, PCA, and TNF by endotoxin-stimulated peritoneal macrophages. These studies demonstrate that endotoxin-induced macrophage arachidonic acid metabolism is associated with expression of PCA and secretion of TNF. Additionally, macrophage synthesis of these pathogenic mediators is reduced under conditions associated with endotoxin resistance (endotoxin tolerance) and is augmented during endotoxin hypersensitivity (glucan stimulation).

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Endotoxin-induced tumor necrosis factor activity production by equine peritoneal macrophages.

A study was performed to determine whether equine macrophages produce tumor necrosis factor (TNF) activity in vitro in response to endotoxin and to study the effects of endotoxin concentration and incubation time on the amount of TNF produced. Equine peritoneal macrophages were isolated and cultured in vitro for 2, 6, 12, or 24 hr in tissue culture media containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 ng/ml, 5 ng/ml, or 5 micrograms/ml), or 3) the calcium ionophore A23187 (0.95 microM). The supernatant media concentrations of TNF activity were determined by an in vitro cytotoxicity bioassay using the murine fibrosarcoma cell line WEHI 164 clone 13. At all incubation times, macrophages exposed to endotoxin produced significantly more TNF than did nonstimulated control cells (P less than 0.05). Among the three doses of endotoxin tested, there were no differences in concentrations of TNF activity in the supernatant media. The mean concentration of TNF activity in media from nonstimulated macrophages was significantly less after 24 hr incubation than after 2, 6 or 12 hr. Incubation time had no effect on TNF production by macrophages exposed to 0.5 ng/ml and 5 micrograms/ml endotoxin, but macrophages exposed to 5 ng/ml endotoxin produced significantly more TNF after 6 and 12 hr than after 2 hr. To address the reason for the decrease in TNF activity in media from nonstimulated macrophages over time, a second study was designed to determine the effect of incubation on the activity of TNF under tissue culture conditions. Human recombinant TNF was added to media that was incubated 2-24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Plasma plasminogen concentrations in clinically normal horses: the effect of age, sex and pregnancy.

Plasma concentrations of plasminogen were determined in 28 clinically normal horses, including 13 adult geldings, five non-pregnant mares, five pregnant mares and five yearlings (two fillies, three geldings). Plasminogen was quantitated by a chromogenic assay based on activation of plasmin by excess urokinase. The overall mean plasma plasminogen for these horses was 2.94 +/- 0.54 CTA units (casein units, as defined by the Committee on Thrombolytic Agents) per ml. There were no significant differences in mean plasma plasminogen values among adult geldings, non-pregnant mares, pregnant mares or yearling horses (P greater than 0.05).

Age Factors↗
PubMed1989

Antibody titres to core lipopolysaccharides in horses with gastrointestinal disorders which cause colic.

Serum immunoglobulin (Ig) titres to core lipopolysaccharide (LPS) were determined in 102 horses admitted to a university referral hospital during a 12-month period for evaluation of colic. Serum samples were collected again 10-14 days later from 84 of the horses. Titres to core LPS were quantitated by an indirect enzyme-linked immunosorbent assay (ELISA), utilising the J-5 mutant of Escherichia coli 0111:B4 as the solid-phase antigen. All horses had natural antibodies to core LPS at the time of admission and the titre was not affected significantly by age, sex or type of gastrointestinal disorder. The geometric mean titres to core LPS increased significantly within 14 days of admission in those horses with large colon displacement (25), ileal impaction (13), small intestinal strangulating obstruction (11) and small colon obstruction (4). Twenty four (28.6 per cent) of the horses had at least a 4-fold rise in titre (seroconversion) to core LPS within 14 days of admission. The incidence of seroconversion to core LPS was significantly greater (P < 0.05) in horses with disorders requiring surgical intervention (35.8 per cent) than in those with disorders (proximal enteritis, colitis, large colon impaction and unknown) which only required medical treatment (16.1 per cent). Seroconversion rate was not statistically different between groups of horses with diseases of the small intestine which required surgical or medical treatments. The results of this study indicate that gastrointestinal disorders that cause colic in horses result in IgG production to core LPS, and the latter is more prevalent in disorders requiring surgery.

Aging↗