Susan A. Halter

Development of mammary hyperplasia and neoplasia in MMTV-TGFα transgenic mice

Abstract To study the role of transforming growth factor α (TGFα) in normal mammary development and mammary neoplasia in vivo, we have generated transgenic mice in which a human TGFα cDNA is expressed under the control of the MMTV enhancer/promoter. Overexpression of TGFα in the mammary epithelium, as confirmed by in situ hybridization and immunohistochemistry, is associated with hyperplasia of alveoli and terminal ducts in virgin female and pregnant transgenic mice. A range of morphologic abnormalities including lobular hyperplasia, cystic hyperplasia, adenoma, and adenocarcinoma is seen in mammary tissue of transgenic females. In contrast, no morphologic abnormalities are seen in transgenic males in spite of TGFα overexpression in salivary glands and reproductive organs. TGFα can therefore act as an oncogene in vivo and appears to predispose mammary epithelium to neoplasia and carcinoma.

Characterization of Hela cell vacuoles induced by Helicobacter pylori broth culture supernatant

Helicobacter pylori broth culture supernatants induce eukaryotic cell vacuolation in vitro, a phenomenon that has been attributed to cytotoxic activity. We sought to characterize further the vacuolation of HeLa cells that occurs in response to H pylori culture supernatant. Nascent vacuoles were detectable by electron microscopy after 90 minutes of incubation with H pylori supernatant and were not associated with any identifiable organelle. After 6 days of incubation with H pylori supernatant, vacuoles were membrane-bound structures filled with electron-dense debris, which resembled secondary lysosomes. Acid phosphatase activity was detected within the vacuoles. The vacuoles induced by H pylori supernatant were then compared with vacuoles induced by trimethylamine, a weak base known to induce lysosomal swelling. Neutral red dye rapidly entered the vacuoles induced by either H pylori supernatant or trimethylamine, and both types of vacuoles were reversible. Compared with trimethylamine-induced vacuoles, the vacuoles induced by H pylori supernatant were larger and typically lacked a limiting membrane. In the early stages of formation, vacuoles induced by trimethylamine were labeled by lucifer yellow, a pinocytotic marker, whereas H pylori cytotoxin-induced vacuoles were not. These data suggest that trimethylamine-induced vacuoles arise directly from endocytic compartments, whereas H pylori cytotoxin induces vacuole formation via an autophagic mechanism.

Methylene Blue Staining and Endoscopic Ultrasound Evaluation of Barrett's Esophagus with Low-Grade Dysplasia

This study was performed to determine if either methylene blue staining or endoscopic ultrasound helped direct biopsies in patients with a history of Barretts esophagus with low-grade dysplasia. Patients underwent radial endoscopic ultrasound scanning to measure esophageal wall thickness, followed by endoscopy with methylene blue staining and biopsies. Mean esophageal wall thickness for squamous mucosa (2.3 ± 0.2 mm), nondysplastic Barretts (2.6 ± 0.2 mm), and Barretts with dysplasia (2.9 ± 0.3 mm) were similar. With staining, Barretts mucosa stained blue more often than gastric epithelium (68% vs 15%, respectively; P < 0.001). The sensitivity and specificity for strong staining detecting Barretts were 68% and 85%, respectively. Barretts with low-grade dysplasia stained blue less frequently (52%) than nondysplastic Barretts (74%; P < 0.05), but the positive predictive value for poor staining indicating dysplasia was 41%. Endoscopic ultrasound was not helpful in directing biopsies in these patients. The utility of methylene blue for detecting dysplasia needs further investigation.

Infarction after embolization of the ileocolic artery

Treatment of colonic hemorrhage by therapeutic embolization of the involved artery may, rarely, result in bowel infarction. In one patient with angiodysplasia, bowel infarction resulted from a therapeutic embolization of the ileocolic artery. Because of the arterial anatomy of the cecum, occlusion of the cecal branches can result in devascularization of the cecum and appendix, even if the colic and ileal branches are not occluded. Thus, the ileocolic artery may not be a good candidate for therapeutic embolization.

Effects of ethanol in vitro on rat intestinal brush-border membranes.

Ethanol, at concentrations found in the intestinal lumen after moderate drinking, has been shown to inhibit carrier-mediated intestinal transport processes. This inhibition could occur by direct interaction with membrane transporters, dissipation of the energy producing Na+ electrochemical gradient and/or nonspecific alteration of membrane integrity. The latter alteration may be reflected by changes in membrane fluidity, chemical composition or vesicular size. These possibilities were examined with studies in purified brush border membrane vesicles of rat intestine. Ethanol inhibited concentrative Na+-dependent D-glucose uptake in a dose-dependent manner. In contrast, ethanol did not inhibit concentrative D-glucose uptake under conditions of D-glucose trans-stimulation in the absence of a Na+ electrochemical gradient. Ethanol also inhibited initial, concentrative Na+-dependent taurocholic acid uptake, as well as equilibrium uptake. That ethanol exerted a dual effect on transport by increasing membrane conductance for Na+ while decreasing intravesicular space was supported by direct studies of Na+ uptake. Morphometric analysis confirmed that ethanol-treated membranes had a decreased intravesicular size when compared to untreated membranes. Finally, membrane fluidity measured by EPR showed that ethanol had a significant fluidizing effect without producing qualitative changes in membrane proteins, as determined by SDS gel electrophoresis. These results suggest that ethanol inhibits carrier-mediated transport by dissipation of the Na+ electrochemical gradient and alteration of membrane integrity rather than by direct interaction with membrane transporter.

Effects of orally administered retinol on natural killer cell activity in wild type BALB/c and congenitally athymic BALB/c mice

SummaryRetinoids have been shown to inhibit the growth and development of neoplastic cells in many systems. One mechanism of action may be through activation of the immune system, specifically natural killer (NK) cell activity. The effect of retinol on NK cell cytotoxicity was examined in three groups of mice: BALB/c (wild-type), BALB/c nu/nu (athymic), and BALB/c nu/nu previously injected with human tumor cells. In untreated mice, NK activity was highest in athymic mice without tumors and lowest in wild-type mice, although serum and liver retinol concentrations were identical in all three groups. In mice fed graded, nontoxic doses of retinol daily for 3 weeks, serum retinol levels in all three groups exhibited a sharp peak and decline following daily bolus retinol administration. Retinol stores in the livers showed a dose-dependent increase in all treated animals. However, NK cell activity, differed for each group. Athymic mice without tumors exhibited no change in NK activity as a result of retinol treatment. Athymic mice with tumors had NK levels that tended to increase with increasing retinol doses, but these changes were not statistically significant. Wild-type mice, on the other hand, demonstrated significantly higher NK levels after treatment with retinol doses of 300 and 600 μg/day. In subsequent time course experiments, there was a peak in NK activity 1 h following bolus retinol administration similar to the peak seen in serum retinol concentrations, suggesting either an acute activation or recruitment of cytotoxic cells. Retinol thus appears to increase NK activity in wild-type BALB/c mice, and this activity may be an important component of its antineoplastic activity.

Superconducting quantum interference device magnetometer for diagnosis of ischemia caused by mesenteric venous thrombosis

Although mesenteric venous thrombosis carries a better prognosis than arterial thrombosis, mortality and morbidity are still high. Previous studies have shown that the basic electrical rhythm (BER) of the bowel decreases early after induction of arterial ischemia. Furthermore, our studies have shown that these changes occur prior to pathologic changes and that they can be recorded noninvasively using a superconducting quantum interference device (SQUID). SQUIDs measure magnetic fields that are created by the electrical activity of the gastrointestinal smooth muscle and have been used to measure the BER of the small intestine in human volunteers. This study was conducted to determine if a SQUID could be used for early noninvasive detection of mesenteric venous ischemia in an animal model. Simultaneous recordings from serosai electrodes and a SQUID outside the abdomen were taken from anesthetized New Zealand rabbits. Recordings were made for 15 minutes before and 90 minutes after injection of thrombin into the superior mesenteric vein. The basic electrical rhythm of the small bowel dropped from 16.42 ± 0.69 to 8.80 ± 0.74 cycles per minute at 30 minutes and to 6.82 ± 0.722 after 90 minutes (p < 0.0001, paired t-test). The correlation coefficient between the SQUID and electrical recordings was 0.954 (p < 0.0001). These data suggest that the ischemia caused by mesenteric venous thrombosis results in changes in the bioelectrical activity, which can be noninvasively detected using a SQUID.RésuméLe pronostic de la thrombose veineuse mésentérique est meilleur que celui de la thrombose artérielle mais la morbidité et la mortalité restent élevées. Des études antérieures ont montré que le Basic Electrical Rhythm (BER) de l’intestin diminue lorsque l’on provoque une ischémie artérielle. Nos études indiquent que ces modifications précèdent les changements anatomopathologiques et qu’elles peuvent être enregistrécs de façon non-invasive en utilisant l’appareil SQUID (Superconduction QUantum Interference Device). Le SQUID mesure le champs magnétique créé par l’activité électrique du muscle lisse de l’intestin grêle et a déjà été utilisé pour mesurer le BER de l’intestin grêle chez des volontaires humains. Cette étude a eu pour but de déterminer si le SQUID peut être utilisé pour la détection non-invasive de l’ischémie veineuse mésentérique dans un modèle animal. Des enregistrements simultanés à partir d’électrodes sur la séreuse et d’un SQUID placé en dehors de l’abdomen ont été réalisés chez le lapin New Zealand. On a enregistré l’activité 15 minutes avant et 90 minutes après l’injection de thrombine dans la veine mésentérique supérieure. L’activité électrique de base a chuté de 16.42 ± 0.69 à 8.80 ± 0.74 cycles/minute après 30 minutes et à 6.82 ± 0.722 après 90 minutes (p < 0.0001, test de Student apparié). Le coefficient de corrélation entre le SQUID et les enregistrements électriques étaient de 0.954 (p < 0.0001). Ces données indiquent que l’ischémie en rapport avec la thrombose mésentérique veineuse est responsable d’une modification d’activité bioélectrique détectable de façon non-invasive par le SQUID.ResumenAunque la trombosis venosa mesentérica conlleva un mejor pronóstico que la trombosis arterial, su mortalidad y morbilidad todavía son altas. Estudios previos han demostrado que el Ritmo Eléctrico Básico (REB) del intestino se disminuye luego de la inducción de isquemia arterial. Además, nuestros propios estudios han señalado que tales cambios se presentan con anterioridad a las alteraciones patológicas y que pueden ser registrados mediante una técnica no invasora que utiliza un SQUID (Superconducting Quantum Interference Device). Los SQUIDs miden los campos magnéticos que se crean por la actividad eléctrica de la musculatura lisa gastrointestinal y se han utilizado para medir la REB del intestino delgado en voluntarios humanos. El presente estudio fue realizado para determinar si se podría utilizar un SQUID para la detección temprana y no invasora de isquemia venosa mesentérica en un modelo animal. Registros simultáneos a partir de electrodos serosos y de un SQUID por fuera del abdomen fueron tomados en conejos anestesiados. Se tomaron registros a los 15 minutos antes y a los 90 minutos después de la inyección de trombina en la vena mesentérica superior. El REB de intestino delgado disminuyó de 16.42 B1 0.69 a 8.8 B1 0.74 ciclos por minuto a los 30 minutos y a 6.82 B1 0.722 a los 90 minutos (p< 0.0001, prueba apareada). El coeficiente de correlación entre los registros del SQUID y los eléctricos fue 0.954 (p< 0.0001). Estos datos sugieren que la isquemia causada por trombosis venosa mesentérica resulta en cambios en la actividad bioeléctrica que puedan ser detectados de manera no invasora utilizando un SQUID.

A model of cytochrome P-450-centered hepatic dysfunction in drug metabolism induced by cobalt-protoporphyrin administration

Cobalt-protoporphyrin treatment disrupts cytochrome P-450-centered drug metabolism and is known to decrease significantly the cytochrome P-450 content of the liver. This study assesses further the correlations between biochemical and functional changes induced by Co-protoporphyrin. Specifically, it confirmed the fall in cytochrome P-450 levels in liver and demonstrated that both NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities decreased in a dose-dependent manner, albeit to a lesser degree, upon Co-protoporphyrin administration. Furthermore, plasma clearance of the marker drug aminopyrine fell off abruptly with a minimal decrease in cytochrome P-450 content, and then monotonically with its further depletion. Both aminopyrine and caffeine demethylation, as measured by the amount of radiolabeled CO2 exhaled, also decreased with diminishing cytochrome P-450 content. With aminopyrine the decrease was abrupt but with caffeine biphasic, consistent with preferential isozyme depletion. The drop in oxidative drug metabolism measured by these two in vivo techniques occurred in the absence of organellar damage to hepatocytes, as observed by electron microscopy. In vitro studies of aminopyrine metabolism in microsomes prepared from rats with and without Co-protoporphyrin injection proved to be consistent with the in vivo studies. Moreover aminopyrine Vmax decreased and Km increased with decreasing cytochrome P-450 content, suggesting preferential isozyme depletion. Furthermore, the changes in aminopyrine intrinsic clearance predicted by the in vitro Vmax and Km values agreed with those measured by in vivo plasma clearance. Taken together, these data suggest that Co-protoporphyrin treatment can be used to produce a model of altered cytochrome P-450-centered drug metabolism, as measured consistently by several techniques. However, this model appears to be more complex than one involving nonspecific depletion of cytochrome P-450 alone, and may be influenced also by concomitant changes in the electron transport chain or other aspects of hepatic metabolism.

Carcinoembryonic Antigen Expression and Patient Survival in Carcinoma of the Breast

Immunoperoxidase staining was used to examine carcinoembryonic antigen (CEA) expression in 167 breast cancer cases. Patients with histological evidence of regional or localized breast cancer who lived less than 3 years or greater than 10 years were assessed. Overall expression of CEA was 65%. There was no significant correlation in CEA expression and survival in either regional or localized breast cancer cases. There was no association between CEA expression and number of lymph nodes involved, size of tumor, parity, gravidity, blood type, or menopausal status of the patients in either group. When the lymph nodes of cases with regional breast cancer were examined, there was a statistically significant number of short survivors whose primary tumor was negative for CEA, but whose metastatic tumor expressed the marker when compared to long survivors with regional lymph node involvement.

Selective isolation of human breast carcinoma cells resistant to the growth‐inhibitory effects of retinol

The anchorage-dependent and anchorage-independent growth of the human mammary carcinoma cell line MDA-MB-231 is inhibited by vitamin A (retinol). Clones resistant to growth inhibition by retinol were isolated from this cell line in soft agar without the use of mutagens. This paper describes the isolation and characterization of the resistant lines. The clones were selectively resistant to retinol. There was significant growth inhibition after treatment with retinoic acid and 13-cis-retinoic acid. The resistant clones maintain their resistance to retinol through multiple passages. Resistance is specific for inhibition of growth, because treatment of the resistant clones results in stimulation of plasminogen activator activity without alteration of proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows no significant qualitative or quantitative difference in the clones when compared with the MDA-MB-231 parent line. Although the clones do not regrow in soft agar, they are tumorigenic in athymic mice. Tumors are produced at a rate similar to the parent line. The advantage of this isolation method is that sensitive and resistant malignant cells derived from the same parent cell line are now available to study the molecular events involved in the inhibition of cellular proliferation after treatment with retinol.