Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease.

Evidence of in vivo oxidant-induced injury in inflammatory bowel disease (IBD) is largely indirect. Colon epithelial crypt cells (CEC) from paired specimens of histologically normal and inflamed bowel from IBD patients with active disease were examined for altered protein thiol redox status as an indicator of oxidative damage. When CEC preparations from 22 IBD patients were labeled with the reduced-thiol-specific probe [14C]-iodoacetamide (IAM), there was decreased labeling of a number of proteins indicating oxidation of thiol groups in CEC from inflamed mucosa compared to paired normal mucosa, especially the loss of thiol labeling of a 37-kD protein which was almost completely lost. The loss of reduced protein thiol status for the 37-kD band was paralleled by loss of epithelial cell glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) enzyme activity, an enzyme known to contain an essential reduced cysteine (Cys149) at the active site. The identity of the 37-kD protein as GADPH monomer was confirmed by NH2-terminal amino acid sequence analysis. To examine whether this type of in vivo injury could be attributed to biologically relevant oxidants produced by inflammatory cells, CEC prepared from normal mucosa were exposed to H2O2, OCl-, nitric oxide (NO), and a model chloramine molecule chloramine T (ChT) in vitro. Dose-dependent loss of IAM labeling and GAPDH enzyme activity was observed. The efficacy (IC50) against IAM labeling was OCl- >> ChT > H2O2 > NO (52 +/- 3, 250 +/- 17, 420 +/- 12, 779 +/- 120 microM oxidant) and OCl- >> ChT > NO > H2O2 (89 +/- 17, 256 +/- 11, 407 +/- 105, 457 +/- 75 microM oxidant), respectively, for GAPDH enzyme activity. This study provides direct evidence of in vivo oxidant injury in CEC from inflamed mucosa of IBD patients. Oxidation and inhibition of essential protein function by inflammatory cells is a potential mechanism of tissue injury that may contribute to the pathogenesis of the disease and supports the exploration of compounds with antioxidant activity as new therapies for IBD.

Detection of c-erbB-2 related protein in sera from breast cancer patients. Relationship to ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour.

The level of a c-erbB-2 related protein was determined in sera from 168 breast carcinoma patients, 12 females with benign breast disease, and 66 female controls using an ELISA (enzyme linked immunosorbent assay) kit. Elevated c-erbB-2 related protein level was detected in one of 13 preoperative sera (8%), two of 62 postoperative sera from patients without recurrent disease (3%), and 55 of 93 sera collected at recurrent disease (59%). Elevated serum levels were detected significantly more often in patients with distant metastases than in patients with recurrent disease restricted to loco-regional areas (68% versus 19%). Presence of elevated serum level was associated with ERBB2 gene amplification and c-erbB-2 protein overexpression in tumour. None of the patients who had normal ERBB2 gene copy number in tumour had elevated serum levels. Although the usefulness in postoperative prediction of the presence of micrometastases is somewhat questionable, the results suggest c-erbB-2 related protein to represent a novel tumour marker in serum and other body fluids from breast cancer patients at the time of diagnosis and during treatment monitoring.

Effects of mastery criteria and contingent reinforcement for family-based child weight control.

This study tested the effects of mastery criteria and contingent reinforcement in a family-based behavioral weight control program for obese children and their parents over two years. Families with obese children were randomized to one of two groups. The experimental group was targeted and reinforced for mastery of diet, exercise, weight loss, and parenting skills. The control group was taught behavior-change strategies and provided noncontingent reinforcement at a pace yoked to the experimental group. Both groups received the same behavioral family-based educational components over 6 months of weekly meetings and six monthly follow-up meetings. Results showed significantly better relative weight change at 6 months and 1 year for children in the experimental compared to the control group, but these effects were not maintained at 2 years. These results suggest the introduction of mastery criteria and contingent reinforcement for mastery can improve outcome during treatment in behavioral treatments for childhood obesity.

Serum levels of HER-2 neu (C-erbB-2) correlate with overexpression of p185neu in human ovarian cancer.

BACKGROUND: The HER-2 neu (c-erbB-2) oncogene product p185neu is expressed by most ovarian cancers and overexpressed in approximately 30%. METHODS: Sera from patients with ovarian cancer were evaluated for neu antigen using an enzyme-linked immunoassay and for CA 125 antigen by radioimmunoassay. Tissue levels of neu from the same patients were determined by immunohistochemical staining with anti-neu monoclonal antibody. RESULTS: Elevated levels (> 2050 human neu unit [HNU]/ml) of circulating neu determinants have been detected in sera from 15% of 48 patients. Of 45 patients for whom tumor tissue had been cryopreserved, overexpression of neu was found in 17 by immunohistochemical analysis; of these 17, serum neu levels were elevated in 5 (29%). Among the 28 patients with normal to moderate tissue expression of neu, only 2 (7%) had elevated serum neu levels. Thus, elevated serum neu levels predicted tissue overexpression with a specificity of 93%. Serum neu levels were not related to serum levels of CA 125. CONCLUSION: Serum and tissue levels of neu correlate in patients with epithelial ovarian cancer.

Immunoconjugates containing novel maytansinoids: promising anticancer drugs.

The potential of immunoconjugates of cytotoxic drugs for the treatment of cancer has not yet been realized owing to the difficulty of delivering therapeutic concentrations of these drugs to the target cells. In an effort to overcome this problem we have synthesized maytansinoids that have 100- to 1000-fold higher cytotoxic potency than clinically used anticancer drugs. These maytansinoids are linked to antibodies via disulfide bonds, which ensures the release of fully active drug inside the cells. The conjugates show high antigen-specific cytotoxicity for cultured human cancer cells (50% inhibiting concentration, 10 to 40 pM), low systemic toxicity in mice, and good pharmacokinetic behavior.

Diagnostic utility of oncogenes and their products in human cancer.

The first clear cut association of an oncogene with a specific cancer is the c-abl translocation in chronic myelogenous leukemia and acute lymphocytic leukemia; it has been observed in 90% of CML cases examined. This is the major contributing factor to its being the target of the first oncogene-based FDA-approved diagnostic test. Although the role of the abl translocation in the tumorigenic process is not yet understood, it is clear that somehow it must be causally related to the disease, and thus is an ideal target for a diagnostic test. The association of this oncogene with a specific cancer is the model on which all others may be based in the future. Second generation tests could easily include PCR on mRNA, and/or in situ hybridization, both of which could be performed using blood samples. Both methods would provide a faster means of testing a large number of cells, however, the methodologies must be improved through automation and computer-aided image analysis, respectively, in order to become useful routine tests. Both neu and epidermal growth factor receptor (EGFR) appear to have a close correlation between overexpression of the gene product and outcome of disease in breast cancer; valuable information for prognosis of the disease. And again, although the actual mechanism of action of these molecules and how this relates to the tumorigenic process is not yet known, it is believed from the very nature of the molecules that they must in some way contribute to the progression of the disease. In both cases, the protein products are overexpressed in tissue, and in the case of Neu, it appears as through at least some of the patients have a Neu-related protein in their serum. These molecules present relatively easy targets for the development of diagnostic/prognostic assays, as antibodies are easily made and can be incorporated into a variety of assay formats. Current assays available, an ELISA for Neu and a radio-ligand binding assay for EGFR, are highly sensitive, reproducible and relatively easy to perform. Only the ELISA is commercially available, however, and hence allows for easy comparison between laboratories. An abvious step towards the routine measurement of EGFR then is the development of a comparable commercially available test. An improvement for both types of assay would be the incorporation of an internal control to gauge the cellular component of the tissue samples that are tested. The outcome of the applications of myc and ras to cancer diagnostics is not so easily predictable, with a couple of exceptions.(ABSTRACT TRUNCATED AT 400 WORDS)

Conditioned tolerance to the heart rate effects of smoking.

This study extended our findings that behavioral tolerance to nicotine in animals can be influenced by conditioning to cardiovascular tolerance in humans. Subjects smoked one-half a cigarette during each of five trials. In the ten-minute intersmoking interval the contexts that preceded smoking were varied. Smokers in the Changing group attended to a different five-minute segment of a Sherlock Holmes radio mystery before each trial, while those in the Repeated group listened to the same segment of the tape. Presmoking heart rates were stable across the groups from trials 1 to 5. As predicted, heart rate for subjects who smoked in the same context showed tolerance to smoking from trials 1 to 5 (84.5 to 78 bpm), while subjects who smoked in in the same context showed tolerance to 83.9 bpm). COa levels increased equally for both groups over the five trials. The results of this study suggest tolerance to smoking can be influenced by learning.

The extracellular domain of p185/neu is released from the surface of human breast carcinoma cells, SK-BR-3.

The human breast carcinoma cell line SK-BR-3, expresses the neu oncogene product, p185, which is a receptor tyrosine kinase. Using a double monoclonal antibody capture enzyme-linked immunosorbent assay for p185, activity was detected in conditioned media from cultures of SK-BR-3 cells. Two monoclonal antibodies specific for the extracellular domain of p185/neu immunoprecipitated a protein with a molecular mass of approximately 105 kDa. p105 was further shown to compete with p185 for binding to monoclonal antibodies and pulse-chase experiments indicate that it was generated by post-translational processing. Peptide maps showed that p105 and p185 are related polypeptides. Since p105 is close to the predicted size for the extracellular domain of p185/neu, we propose that SK-BR-3 cells specifically process and release this portion of the receptor into the medium. The release of the extracellular domain may have implications in oncogenesis and its detection could prove useful as a cancer diagnostic.

Strategies for the analysis of oncogene overexpression. Studies of the neu oncogene in breast carcinoma.

The development of a consistent strategy for the analysis of oncogene expression at the cellular level is essential for understanding the roles of these genes in the development and progression of human neoplasia. Detection of the neu oncogene products in breast carcinoma was selected as a model for analysis of oncogene expression. Fifty-two primary human breast carcinomas were evaluated by quantitation of neu DNA amplification and mRNA expression and by localization of neu mRNA and protein (p 185) at the cellular level by in situ hybridization (ISH) and immunohistochemistry (IHC). The specificity and sensitivity of the molecular and immunologic probes for neu were established with the use of genetically engineered cell lines that overexpressed either neu or epidermal growth factor receptor (EGFR). Twenty-nine percent of breast carcinomas demonstrated neu DNA amplification and mRNA overexpression, and there was close correlation between the level of neu mRNA expression and detection of neu gene products by ISH and IHC. Thirty-two percent of carcinomas demonstrated neu mRNA overexpression by ISH. The immunohistochemical method using TA1 monoclonal antibody for p185 was exquisitely sensitive in acetone-fixed frozen sections and provided an excellent approach for judging overexpression as confirmed by the various molecular analyses. All areas of nonmalignant breast epithelium stained weakly, and a wide range of staining intensity was observed in malignant breast epithelium, with 31% of carcinomas judged to be p185 overexpressors. Heterogeneous expression of p185 was seen in some carcinomas. This study provides a strategic approach for the evaluation of oncogene expression in human tumors.

neu oncogene protein and epidermal growth factor receptor are independently expressed in benign and malignant breast tissues.

The neu oncogene protein, p185, and epidermal growth factor receptor (EGFR) were localized immunohistochemically in benign and malignant human breast tissues using monoclonal antibodies. Both benign and malignant epithelial cells were positive for these oncogene proteins in acetone-postfixed frozen sections. Stromal cells were negative for p185, but occasionally positive for EGFR. Myoepithelial cells were consistently positive for EGFR, and p185 was localized predominantly in duct-lining cells, where the basolateral plasma membrane was the normal expression site of both substances. Paraformaldehyde-prefixed frozen sections were less sensitive for antigen demonstration. Based on the intensity of immunoreactivity, 11 of 37 acetone-postfixed breast carcinomas (30%) were judged neu overexpressors, while none of 24 benign tissues overexpressed neu. Epidermal growth factor receptor was demonstrated in 18 of 36 acetone-postfixed cancer tissues (50%) and was overexpressed in three (8%). At the cellular level, heterogenous expression of p185 and EGFR was occasionally observed in both benign and malignant tissues, and a single case of cancer overexpressing both neu and EGFR showed reciprocal patterns of staining, indicating their independent expression. In some carcinomas, EGFR was localized only in stromal cells. Our findings confirmed mutually independent expression of the two closely related protooncogenes in benign and malignant breast tissues.

Analysis of c-erbB-2 expression in breast carcinomas with clinical follow-up.

Various monoclonal antibodies reactive with protooncogene products or tumor-associated antigens have been utilized to investigate breast carcinoma biology or antigen expression with potential prognostic relevance. Murine monoclonal antibody TA1, generated by immunization of BALB/c mice with whole c-erbB-2 (neu) transformed NIH/3T3 cells, recognizes the extracellular domain of the c-erbB-2 protein and binds a Mr 185,000 protein by immunoprecipitation. Using avidin-biotin-peroxidase techniques and monoclonal antibody TA1, 313 archival primary adenocarcinomas of the breast were evaluated for c-erbB-2 overexpression; 290 of these were used for multiparametric statistical analysis. Historical, clinical (age, laterality), histological (nuclear grade, tumor size, lymph node status, lymphatic or blood invasion), and hormone receptor data as well as clinical outcome (minimal follow-up, 6 years; median follow-up, 8.5 years) were compared to TA1 staining. For these 290 patients Cox regression multivariate analysis showed the strongest correlation between lymph node status or estrogen receptor status and overall survival (P = 0.0001 and 0.049, respectively). TA1 staining did not significantly correlate with survival (P = 0.395). However, univariate analysis of certain patient subpopulations showed a significant correlation if the examined tumors were subdivided into negative or focally reactive and those with greater than or equal to 40% cellular reactivity. For T3, T4 patients, strong TA1 immunoreactivity correlated with a shortened disease-free survival (log rank P = 0.0018; Wilcoxon p = 0.0078) and overall survival (log rank P = 0.0002; Wilcoxon P = 0.0013). For these patients the overall survival at 6 years was markedly different between the strongly reactive tumors (0%) and the negative to weakly reactive tumors (55%). In lymph node-positive patients a trend between high TA1 reactivity and a worse overall survival was also noted (log rank P = 0.128; Wilcoxon P = 0.054), with a 6-year survival of 42% in the strongly reactive tumors (n = 16) and 65% in the negative to weakly reactive carcinomas (n = 105). No correlation between TA1 immunoreactivity and other historical, clinical, and histological features were noted. c-erbB-2 overexpression as measured by immunohistochemical techniques, therefore, may have clinical significance in certain patient subpopulations.

Generation and characterization of monoclonal antibodies specific for the human neu oncogene product, p185.

A series of monoclonal antibodies specific for the extracellular domain of the human neu gene product (p185) have been produced. The generation of these monoclonal antibodies, and their biochemical and immunological characterization is described. The immunization protocol utilized a series of injections of NIH3T3 cells, cyclophosphamide, and a neu transfected NIH3T3 cell line (designated 18-3-7) which expressed the full length human neu-encoded protein. This immunization regimen induced an immune response to the extracellular portion of p185 on the 18-3-7 cells. A panel of ten hybridomas were identified which secreted monoclonal antibodies with a variety of epitope specificities, and reacted with p185 in a number of different experimental formats. As the neu gene product has been associated with human breast cancers, a series of monoclonal antibodies such as these could prove useful in the diagnosis, prognosis and/or treatment of these human malignancies.

Temporal stability of psychophysiological responding: a comparative analysis of mental and physical stressors.

Although extensive research has been conducted on psychophysiological reactivity, there is a paucity of data concerning the temporal stability of such procedures. Test-retest reliability of experimental stressors from both mental and physical modalities were assessed using a wide range of psychophysiological measures. Absolute baseline and test values demonstrated adequate test-retest reliability for skin temperature, skin resistance, vasomotor response, heart rate, systolic and diastolic blood pressure, while forearm EMG had low reliability. Difference scores, which represent change from baseline to test conditions, did not have adequate reliability. These data represent a necessary step towards standardization of psychophysiological assessment techniques and thus may guide further use of more reliable methods.

Cholera toxin B subunit as a carrier protein to stimulate a mucosal immune response.

Horseradish peroxidase (HRP) was covalently coupled to the binding subunit of cholera toxin (CTB) via a two-step glutaraldehyde procedure. The HRP-CTB conjugate was characterized by physiochemical as well as immunochemical methods. Mice were immunized intraduodenally with the HRP-CTB conjugate, with HRP alone, or with a mixture of uncoupled CTB and HRP. The functionally active dose of CTB was 50 micrograms and the HRP dose was in the 30- to 90-micrograms range. Both IgA and IgG antibody responses were measured in serum, intestinal washes, and bile by using a solid phase immunoradiometric assay. Mice immunized with the HRP-CTB conjugate showed a significantly higher level of IgA anti-HRP in intestinal washes and bile, as well as increased levels of serum IgG anti-HRP. Animals that received only HRP or the mixture of CTB and HRP had reduced levels of HRP-specific antibody of either class in both gut washes and bile. The IgA anti-HRP responses in the gut washes were 33- to 120-fold higher when the conjugate was used as the immunogen in comparison with immunization with the CTB + HRP or the HRP alone. Vaccines to stimulate mucosal immunity to any antigenic determinant might thus be prepared by covalent conjugation to effective mucosal immunogens such as CTB.