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Biomedical subjects

L G Wolfe

Publications and source records attributed to L G Wolfe.

At least 19 recordsLinked to original sources

Cyclooxygenase-2 expression in normal and neoplastic canine mammary cell lines.

Mammary cancer is the most common cancer in female dogs. Induction of cyclooxygenase-2 (COX-2), a key enzyme in prostaglandins (PGs) biosynthesis, has been demonstrated in various cancers in humans and dogs, including mammary cancer. The objective of this study was to investigate the expression and regulation of COX-2 in canine mammary epithelial cells. Cell lines derived from normal and neoplastic canine mammary glands were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and immunoblots, immunocytochemistry, radioimmunoassays, and a cell proliferation assay were used to study COX-2 expression and PGs production. Results showed that the neoplastic cell line CMT12 constitutively overexpressed COX-2 protein whereas other mammary cell lines expressed low to undetectable basal levels of COX-2 protein. Basal PGE(2) production was significantly higher (P < .05) in CMT12 compared to other cell lines. Levels of COX-2 protein in CMT12 decreased in a time-dependent manner with serum starvation, and PMA stimulation induced a strong time-dependent increase in COX-2 protein. Treatment of CMT12 cells with NS-398 (a specific COX-2 inhibitor) significantly blocked PGE(2) synthesis and reduced cell proliferation (P < .05). These results indicate that some neoplastic canine mammary cell lines constitutively overexpress COX-2, and that COX-2 inhibition decreases PGE(2) production and cell proliferation, supporting a role for COX-2 and PGs in canine mammary oncogenesis.

Animals↗

Postoperative complications are not increased in super-super obese patients who undergo laparoscopic Roux-en-Y gastric bypass.

BACKGROUND: It has been suggested that super-super obesity (body mass index [BMI] > or =60 kg/m2) increases the risk of complications after laparoscopic Roux-en-Y gastric bypass (LapRYGB). We hypothesized that a higher BMI does not increase risk the morbidity or mortality rate. METHODS: Complication rates for patients with a BMI > or =60 kg/m2 were compared to those for patients with a BMI <60 kg/m2 who underwent LapRYGB during the same time period. Differences between the groups were analyzed by Fisher's exact test, t-tests, and analysis of variance. RESULTS: Forty-five patients with a BMI > or =60 kg/m2 and 640 patients with a BMI <60 kg/m2 underwent LapRYGB. There were no statistically significant differences between the two groups in the complication or mortality rates. Excess weight loss was less, but actual weight lost was greater in the BMI > or =60 kg/m2 group. CONCLUSIONS: The complication and mortality rates are not increased in super-super obese patients who undergo LapRYGB. Acceptable weight loss can be achieved safely in these patients.

Adult↗

Experience with over 3,000 open and laparoscopic bariatric procedures: multivariate analysis of factors related to leak and resultant mortality.

BACKGROUND: Intestinal leak is a potentially lethal complication of Roux en-Y gastric bypass (GBP). Identification of patients at high risk for leak may reduce complication rates of surgeons early in the procedure learning curve. METHODS: A total of 3073 patients who underwent GBP were analyzed using univariate and multivariate logistic regression analyses of the following preoperative factors: hypertension (HTN), diabetes mellitus (DM), sleep apnea (SA), age, gender, weight, body mass index (BMI), and surgery type. Multivariate logistic regression analysis was performed for each procedure type. RESULTS: There were 48 (1.5%) deaths. Independent risk factors for death included leak, weight, procedure type, and HTN. A total of 102 (3.2%) leaks were found. Independent factors for leak included age, male gender, SA, and procedure type. CONCLUSION: The data suggests that older, heavier male patients with multiple comorbid conditions are at increased risk for leak and mortality. Surgeons early in their learning curve should avoid these high-risk patients to reduce complications.

Adolescent↗

Measurement of hemoglobin synthesis rate in vivo using a stable isotope method.

We developed a method to measure hemoglobin synthesis rate (SynHb) in humans, assuming that free glycine in the red blood cell (RBC) represents free glycine in bone marrow for hemoglobin synthesis. The present rat study examines this assumption of the method and quantifies SynHb in rats. Sprague-Dawley rats (n = 9) were studied, [2-(13)C]glycine was intravenously infused over 24 h (2.5 mg kg(-1) h(-1)), blood was drawn for glycine and heme isolation, and bone marrow was harvested for glycine isolation. Isotopic enrichments of glycine and heme were measured, fractional hemoglobin synthesis rate (fSynHb% day(-1)) was calculated, and from this a value for SynHb (mg g(-1) day(-1)) was derived. Mean body weight was 446 +/- 10 g (mean +/- SE) and hemoglobin concentration was 14 +/- 0.5 g dl(-1). At 24 h, the mean isotopic enrichment, atom percentage excess (APE), of the RBC free glycine (1.56 +/- 0.18 APE) was similar to the bone marrow (1.68 +/- 0.15 APE). The rate of incorporation of (13)C into heme increased over time from 0.0004 APE/h between 6 and 12 h, to 0.0014 APE/h between 12 and 18 h, and 0.0024 APE/h between 18 and 24 h. Consequently, fSynHb (1.19 +/- 0.32, 2.92 +/- 0.66, and 4.22 +/- 0.56% day(-1), respectively) and SynHb (0.11 +/- 0.03, 0.28 +/- 0.05, and 0.42 +/- 0.05 mg g(-1) day(-1), respectively) showed similar patterns over the 24-h study period. We conclude that (1) enrichment of free glycine in the circulating RBC approximates enrichment of bone marrow free glycine for heme formation and (2) this pattern of hemoglobin synthesis rate is reflecting the characteristic release and gradual maturation of reticulocytes in the circulation.

Animals↗

Nitric oxide is a neurotransmitter in the chloride secretory response to serotonin in rat colon.

BACKGROUND: Serotonin (5-hydroxytryptamine [5-HT]) has been shown to induce chloride secretion through a nonadrenergic/noncholinergic neural pathway, mediated by a 5-HT(3) receptor. We hypothesized that 5-HT(3)-induced Cl(-) secretion is ultimately mediated by nitric oxide (NO). METHODS: Unstripped sheets of rat distal colon were mounted in Ussing chambers and short-circuited. The 5-HT(3) receptor agonist, 2-methyl-5-HT, was added in the absence and presence of the NO synthase inhibitor, L-NAME. Companion studies involved the addition of sodium nitroprusside to tissue that was incubated with or without tetrodotoxin. RESULTS: L-NAME caused a significant reduction in the 2-methyl-5-HT-induced change in circuit current, in a concentration-dependent manner. Sodium nitroprusside caused a change in circuit current over baseline in 5 minutes. The addition of tetrodotoxin did not significantly alter the change in circuit current; however, the apical Cl(-) channel blocker, anthracene-9-carboxylic acid, abolished this response. CONCLUSIONS: Neurally mediated Cl(-) secretion in response to 2-methyl-5-HT is inhibited by an NO synthase inhibitor. Exogenous NO mimics this response, which is unaffected by tetrodotoxin. These data suggest that neurally mediated serotoninergic Cl(-) secretion is, in part, mediated by NO. The ability of exogenous NO to induce a change in circuit current in the presence of tetrodotoxin suggests that NO is a final neurotransmitter in this neural-mucosal reflex and therefore acts directly on the enterocyte to induce secretion.

Animals↗

Isolation and characterization of the canine melanoma antigen recognized by the murine monoclonal antibody IBF9 and its distribution in cultured canine melanoma cell lines.

OBJECTIVE: To characterize the canine melanoma antigen recognized by the murine monoclonal antibody IBF9 as to its cellular location, molecular size, protein and glycogen contents, and distribution in cell lines. SAMPLE POPULATION: 7 cultured canine melanoma cell lines. PROCEDURE: Molecular characteristics of the antigen were determined by western blotting, enzymatic digestion studies, and tunicamycin inhibition studies. Distribution of the antigen in the cultured melanoma cell lines was determined by flow cytometry. RESULTS: The antigen consists of 2 proteins with molecular mass of 89 and 85 kd. Tunicamycin and enzymatic digestion studies indicated that these proteins contained little glycosylation. Immunogold and immunofluorescence studies localized the antigen to the cell surface. Antigen expression was consistent within each cell line, with > 90% of the cells positive for all cell lines except 1 (80%). Percentage of positive cells and relative intensity of immunostaining were constant throughout all phases of the cell cycle. CONCLUSIONS: The antigen identified by MAB IBF9 is a well-conserved and highly expressed cell surface protein present during all phases of the cell cycle in all malignant canine melanoma cell lines examined. CLINICAL RELEVANCE: Because of consistency in expression, the antigen may have potential for use in dogs for melanoma immunodiagnostics and immunotherapy.

Animals↗

Expression of the oncogene c-erbB-2 in canine mammary cancers and tumor-derived cell lines.

OBJECTIVE: To determine, for canine mammary tumors, whether malignancy, with or without local invasion or regional metastasis, was associated with overexpression of the oncogene c-erbB-2. DESIGN: c-erbB-2 expression was measured in canine mammary tumor-derived cell lines and in mammary tumor tissues from clinical cases. Clinical samples were examined histologically to determine whether they were benign or malignant and, if malignant, whether they had evidence of local invasion or regional metastasis. Canine fibroblast cultures and normal canine mammary epithelial tissues were used as reference standards for cell lines and mammary tumors, respectively. SAMPLE POPULATIONS: 28 canine mammary tumor tissue samples obtained surgically from clinical cases and samples from 7 canine mammary tumor cell lines derived from primary canine mammary tumors. PROCEDURE: c-erbB-2 mRNA levels were determined by means of hybridization of total polysomal RNA with a 32P-labeled human c-erbB-2 probe on dot blots, and results were quantified by means of scanning densitometry. Overexpression of c-erbB-2 was defined as an autoradiographic density > or = 2 times the density of reference samples on the same blot. RESULTS: Overexpression of c-erbB-2 was detected in 17 of 23 malignant tumors, 0 of 5 benign tumors, and 2 of 7 mammary tumor cell lines. c-erbB-2 overexpression was correlated with a histopathologic diagnosis of malignancy (P = 0.005) but not with the presence of local invasion or regional metastatic disease (P = 0.621). CONCLUSIONS: Results suggest that overexpression of c-erbB-2 occurs prior to the development of metastatic disease in canine mammary tumors and plays a role in the development of malignancy.

Animals↗

Detection of tumor-associated antigens in sera of canine cancer patients by monoclonal antibodies generated against canine mammary carcinoma cells.

Two murine monoclonal antibodies (MAbs), 1A10 and SB2, generated against a canine mammary carcinoma cell line, were used in a competitive enzyme-linked immunosorbent assay (ELISA) to measure tumor-associated antigens (TAAs) in canine serum samples. Sera were tested from disease-free dogs and from dogs diagnosed with mammary carcinoma, non-mammary carcinoma, sarcoma, benign mammary tumor, benign non-mammary tumor, or non-neoplastic disease. Serum antigen concentrations measured by ELISA were expressed as inhibitory units (IU). The upper limit of normal, defined as the mean plus 2 SD of the TAA concentration in disease-free dogs, was 20 IU with antibody 1A10 and 22 IU with antibody SB2. Compared with disease-free dogs, the frequency of TAA-positive sera was significantly greater (P < 0.05) among dogs with mammary or non-mammary carcinoma when tested with MAbs 1A10 or SB2, and also with sarcoma when tested with MAb SB2. Testing a serum sample with both antibodies rather than just one increased the sensitivity of the competitive ELISA for TAA detection. The presence of TAA in serum might serve as a useful marker for certain types of carcinomas or sarcomas in canine cancer patients.

Animals↗

Effect of triiodothyronine on postischemic myocardial function in the isolated heart.

Thyroid dysfunction has been shown to have a significant impact on hemodynamic status and cardiac function. The purpose of this study was to determine the influence of triiodothyronine (T3) on cardiac functional recovery after ischemia in a dose-dependent manner. Postischemic functional recovery was assessed in isolated rabbit hearts mounted in a modified Langendorff preparation. Left ventricular systolic, diastolic, and peak developed pressures were measured before and after ischemia, and calculated as a percentage of preischemic function. Two cohorts of hearts were studied: the first was exposed to warm ischemia until a myocardial contracture of 4 mmHg was produced; the second cohort was exposed to warm ischemia until a contracture of 15 mm Hg was observed. In each cohort, T3 was added to the perfusion solution after ischemia in a physiologic concentration (2.5 x 10(-9) g/mL; 1 x T3), as well as ten times (2.5 x 10(-8) g/mL; 10 x T3) and a hundred times (2.5 x 10(-7) g/mL; 100 x T3) the physiologic concentration. One group, given the carrier only but without T3, served as the control. Rabbit hearts exposed to a short period of ischemia (4-mmHg diastolic contracture) showed increased recovery with 1 x T3 and 10 x T3. 100 x T3 did not bring about improved left ventricular recovery versus that in the control group. Rabbit hearts in the 15 mm Hg-diastolic contracture cohort showed increased recovery with 10 x T3 but not with 1 x T3. 100 x T3 led to decreased recovery in this cohort versus that in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Antigen expression in normal and neoplastic canine tissues defined by a monoclonal antibody generated against canine mesothelioma cells.

Monoclonal antibody (MAb) 3B5 generated against canine mesothelioma cells was applied to canine tumors and normal tissues via immunohistochemical and immunoblotting techniques to evaluate antigen binding. By use of an avidin-biotin immunoperoxidase complex (ABC) method, immunoreactivity was noted in reactive mesothelial cells and in normal tissues was observed primarily in mesothelial cell linings, endothelial cells, and smooth muscle of blood vessels and soft tissues; the reactivity was nearly equivalent in frozen or formalin-fixed, paraffin-embedded tissue sections. Use of the ABC method on formalin-fixed, paraffin-embedded tumors yielded moderate to strong cytoplasmic immunostaining of neoplastic cells in 10/11 (91%) mesotheliomas, 18/23 (78%) hemangiosarcomas, 4/10 (40%) intestinal and lung carcinomas, and < or = 20% of hemangiomas, leiomyosarcomas, leiomyomas, mammary carcinomas, and squamous cell carcinomas. No immunostaining of tumor cells was observed in fibrosarcomas, hemangiopericytomas, perianal gland carcinomas, and melanomas. Immunoblotting was performed on samples that demonstrated strong immunoreactivity with MAb 3B5 by the ABC method: mesothelioma, hemangiosarcoma, urinary bladder (smooth muscle), and lung (alveolar capillaries). These analyses showed that MAb 3B5 bound a major antigen of 78 kilodaltons (kd) and minor antigens at 56 and 54 kd in normal and neoplastic tissues. The preliminary immunohistochemical results suggest that MAb 3B5 may possess utility in diagnosis of mesotheliomas and hemangiosarcomas, discrimination of cell types in proliferative serosal lesions, and demonstration of vascularity or angiogenesis in neoplastic and inflammatory lesions.

Animals↗

Estrogen and progesterone receptor status of mammary carcinomas and correlation with clinical outcome in dogs.

Estrogen and progesterone receptors (ER, PR) were measured in cytosol fractions from 18 primary canine mammary carcinomas by use of biochemical assays. One or both receptors were detected (> 10 fmol/mg of cytosol protein) in 11 tumors: 5 ER and PR; 2 ER only; 4 PR only. Mean cytoplasmic receptor concentrations (fmol/mg of cytosol protein) were 22.8 +/- 2.9 (SEM) for ER and 51.0 +/- 10.3 for PR in tumors containing ER and PR, 28.8 +/- 12.1 for ER in tumors containing only ER and 13.2 +/- 1.5 for PR in tumors containing only PR. Estrogen or progesterone receptors or both were identified in 6 of 9 tubular adenocarcinomas, 4 of 5 papillary adenocarcinomas, and 1 of 1 squamous cell carcinoma. These receptors were not identified in solid carcinomas (n = 2) or a single spindle cell carcinoma. Although the number of cases was limited, survival times of dogs tended to be longest in those with tumors containing ER alone or in combination with PR, intermediate in those with tumors containing only PR, and shortest in those with tumors without ER or PR. A correlation was not apparent between receptor status and age, presence of ovaries, tumor size, or histologic classification of the tumor. In the analysis of this series, the extent of surgery (mastectomy of the involved gland vs unilateral or bilateral mastectomy) did not appear to influence the outcome of the disease, and metastasis to regional lymph nodes did not appear to be a reliable prognostic indicator.

Animals↗

Antigen expression in canine tissues, recognized by a monoclonal antibody generated against canine melanoma cells.

A murine hybridoma monoclonal antibody (MAB), IBF9, was generated by fusing myeloma cells (P3X63Ag8.653) with spleen cells from a BALB/c mouse immunized with the canine melanoma cell line CML-10c7. Initial screening of hybridoma antibodies was performed by use of an indirect immunoperoxidase assay on formalin-fixed CML-10c7 cells. The isotype of MAB IBF9 was IgG1 as determined by radial gel immunodiffusion. The antibody was tested for reactivity against a panel of formalin-fixed, paraffin-embedded normal and neoplastic canine tissues, using immunoperoxidase staining. Immunostaining was observed in melanomas (24 of 38), a few carcinomas, basal cell tumors, and cutaneous lymphosarcomas. Immunostaining was not observed in fibrosarcomas, hemangiosarcomas, hemangiopericytomas, or histiocytomas. Staining of normal adult canine tissues was limited to a few epithelial tissues and a small percentage of lymphocytes. Fetal tissues were not reactive with MAB IBF9. There were statistically significant differences in frequency of reactivity among melanomas with regard to oral vs non-oral, malignant vs benign, and mitotic indices greater than or equal to 1 vs mitotic indices less than 1. Differences were not significant when tumors were compared for degree of pigmentation or histologic type. On the basis of these findings, we suggest that MAB IBF9 may be of assistance in diagnosis of nonpigmented melanomas and in assessing the malignant potential of melanomas.

Animals↗

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Conserved antigen expression in epithelial tumors recognized by monoclonal antibody 4A9 generated against canine mammary carcinoma cells.

Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.

Animals↗

Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri.

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.

Animals↗

Biologic characterization of canine melanoma cell lines.

Eight canine melanoma cell lines were established from tissues from 6 dogs with spontaneous primary or metastatic melanomas. Cell lines were characterized for morphologic features and growth patterns on plastic, pigmentation, ultrastructure, cloning efficiency in soft agar, and tumorigenicity in nude mice. Biologic properties of cell lines were distinct and preserved during 40 to 120 passages in vitro. All cell lines were clonogenic and tumorigenic.

Animals↗

Biologic properties of cell lines derived from canine mammary carcinomas.

Six canine mammary tumor cell lines (5 epithelial, 1 myoepithelial) were established from dogs with spontaneous mammary carcinomas. The cells were characterized for features of morphology and growth patterns on plastic, ultrastructure, cloning efficiency in soft agar, tumorigenicity in athymic nude mice, and hormonal receptor status. Biologic properties of the cell lines were distinct and preserved during long-term culture in vitro. All cell lines were clonogenic, 5 of 6 were tumorigenic, and 3 of 5 were receptor positive for estrogen and/or progesterone.

Animals↗

Antibodies directed against human liver specific membrane lipoprotein (LSP) in marmosets experimentally infected with the hepatitis A virus.

Autoantibodies directed against liver plasma membrane antigens have recently been described in patients with acute viral hepatitis, type A (AVH-A). To further investigate this phenomenon, the antibody against one such liver membrane antigen, liver specific membrane lipoprotein (LSP), was assayed in six marmosets orally inoculated with hepatitis A virus (HAV). Using a sensitive radioimmunoassay technique, anti-human LSP antibodies were detected in five of six animals. Two peaks of 125I-HLSP binding were observed: a minor peak at 20 days post-inoculation (dpi) in two animals, and a major peak at 38-45 dpi in five animals. There was no correlation between 125I-HLSP binding and liver histology score, ALT level, IgG concentration, anti-HAV P/N ratio, or E rosette lymphocyte count. A statistically significant correlation was observed, however, between 125I-HLSP binding and IgM anti-HAV antibody P/N ratios. 125I-HLSP binding was blocked by both marmoset and human LSP, but not by a marmoset kidney protein prepared in an identical manner. In summary, marmosets infected with HAV are a suitable animal model for the further investigation of anti-LSP autoantibody formation in AVH-A.

Acute Disease↗