David V. Habif

Adenylcyclase-phosphodiesterase system in arterial smooth muscle

Abstract Isoproterenol, epinephrine, and norepinephrine increase cyclic AMP formation in rat aorta through their stimulatory effect on adenyl cyclase. Isoproterenol, and epinephrine and norepinephrine in the presence of phentolamine, have a relaxing effect on the rat aortic strip. Propranolol prevents the rise in cyclic AMP with the three catecholamines and modifies the epinephrine-induced contraction. Inhibition of phosphodiesterase is also accompanied by relaxation of arterial smooth muscle.

Immunostaining of estrogen receptor in paraffin sections of breast carcinomas using monoclonal antibody D75P3 gamma: effects of fixation.

Immunostaining of estrogen receptor was carried out on paraffin sections of breast carcinomas using an anti-estrophilin monoclonal antibody (D75P3U03B3) and the avidinbiotin technique. The tumors were fixed in Bouins solution or in formalin for varying periods of time at room temperature or at 4°C. Best results were obtained following fixation in Bouins at room temperature or in formalin at 4°C. The staining was localized in the nuclei of carcinoma cells and was heterogeneous in intensity and extent. Prolonged fixation resulted in decreased immunoreactivity and in the appearance of nonspecific cytoplasmic and background staining. The estrogen receptor immunostaining on parafffin sections was found to be in concordance with that on frozen sections (Abbott ERICA) and with the steroid-binding assay (dextran-coated charcoal) in over 90% of the cases. This method is of easy and rapid execution and yields reliable and reproducible results

An immunohistochemical evaluation of progesterone receptor in frozen sections, paraffin sections, and cytologic imprints of breast carcinomas

Two monoclonal antibodies to progesterone receptor (PR), JZB39 and KD68, were used for the immunocytochemical visualization of PR in different kinds of breast cancer specimens including (1) cryostat sections of tumors frozen at −80°C; (2) paraffin sections of tumors fixed in formalin or in Bouins fixative for varying periods of time at room temperature or at 4°C; and (3) imprints and cryostat sections prepared from the tissue used for frozen section diagnosis and stored at −80°C after fixation in Zambonis solution. Sections of conventionally frozen specimens as well as imprints and cryostat sections stored for varying periods of time were stained with the peroxidase–antiperoxidase technique, whereas the avidin–biotin technique was used for paraffin sections. In all types of specimens the PR immunostaining was localized to the nuclei of carcinoma cells and displayed considerable heterogeneity both in intensity and in distribution of positive cells. Close correspondence was found between the different immunohistochemical techniques as well as between immunostaining and steroid‐binding assays. PR staining was more frequently positive in well‐differentiated than in moderately or poorly differentiated carcinomas, whereas no meaningful correlation was found between PR staining and extent of the disease. Similar results were obtained with the immunostaining of estrogen receptor in the same material using monoclonal antibodies H222 and D75P3γ. Thus, by choosing the technique that best suits the type of specimen available, it is possible to obtain valid information on the receptor status of any breast carcinoma, regardless of its size and clinical presentation.

Antiproliferative effects of natural interferon beta alone and in combination with natural interferon gamma on human breast carcinomas in nude mice

SummaryNude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 4 5-day cycles of intralesional (i.l.) injections of human natural interferon beta (nIFN-β) alone or in combination with human natural interferon gamma (nIFN-γ). The injections were administered to only 1 of the 2 tumors in each animal, thus making it possible to assess at the same time local therapeutic effects in the injected tumors and systemic effects in the contralateral ones. When n-IFN-β was used as a single agent only mild local antitumor effects and virtually no systemic effects were observed. In contrast, the combined administration of nIFN-β/nIFN-γ produced marked antiproliferative effects, presumably as a result of the synergistic action of type I and type II IFNs. These effects ranged from complete regression documented histologically in 2 MCF-7 tumors to varying degrees of growth inhibition with persistence of residual microscopic or grossly detectable tumor. Local effects were more pronounced than systemic effects. The therapeutic efficacy of nIFN-β proved to be greater than that of recombinant interferon beta (rIFN-β). In MCF-7 tumors nIFN-β appeared to be less effective than nIFN-α, whereas the opposite was true for BT 20 tumors.

Immunohistochemical assessment of estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas

A peroxidase-antiperoxidase technique was used to visualize estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas that were prepared at the time of biopsy or frozen section diagnosis. This was done to provide an alternate technique for the assessment of the receptor status of tumors that could not be adequately assayed with other biochemical or immunocytological methods. Fixation in Zambonis fixative followed by passage through cold methanol and acetone before storage at -80 C insured good preservation of the receptor proteins over extended periods of time (up to 56 weeks). Immunostaining of these stored preparations with monoclonal antibodies against estrogen receptor (H222) and progesterone receptor (JZB39 and KD68) showed a high degree of correspondence with immunocytochemical assays (ER-ICA and PR-ICA) and biochemical analysis. This technique is easy to perform and provides reliable information, even in tumors that are too small and/or ill defined to permit separate sampling for receptor assays.

Immunostaining of Estrogen Receptors in Paraffin Sections of Breast Cancers Using Anti-Estrophilin Monoclonal Antibodies

A monoclonal antibody against estrophilin was used to visualize estrogen receptors in paraffin sections of 59 specimens of primary and metastatic or recurrent breast carcinomas from 28 patients. Immunostaining was carried out with the avidin-biotin technique. Positive staining in the form of brown granules, was seen predominantly in the nucleus of carcinoma cells and less frequently in the cytoplasm. Variations in intensity and extent of the immuno-reactivity was observed in all the sections, among different samples from the same specimens, and among different specimens from the same patients. These staining variations are probably related to heterogeneity of the tumor cell population and suggest the need of examining multiple sections from every specimen and as many specimens as possible from any patient with metastases or recurrences. A good correlation was obtained between immunostaining and receptor content of the corresponding specimens examined by dextran-coated charcoal analysis. About one half of the patients with positively stained tumors responded favorably to anti-estrogen therapy, whereas most of those with negatively stained tumors failed to respond. This study indicates that immunostaining of estrogen receptors in paraffin sections of breast carcinomas is a reliable technique and correlates well with biochemical assays for estrophilin. In this group of patients, positive immunostaining could not be used as a predictor for response to anti-estrogen therapy. On the contrary, negative immunostaining appeared to indicate that the patient was unlikely to benefit from anti-estrogens.

Applicability of Monoclonal Antibodies Against Estrogen and Progesterone Receptors in the Immunocytochemical Evaluation of Breast Carcinomas

The development of monoclonal antibodies against receptor proteins for estrogen (ER) and progesterone (PR) has opened a new perspective in the endocrinological evaluation of breast cancer patients. Indeed, a great deal of information can be obtained by applying these antibodies to tissue sections, including information that cannot be provided by steroid-binding assays. Of particular interest is the assessment of the percentage of receptor-positive cells in any given tumor, which can be easily evaluated in stained sections, but cannot be estimated by biochemical assays on tissue homogenates. Basic research in this field has been done on frozen sections which have proved to be quite reliable as documented by several reports presented at a recent symposium on Estrogen Receptor Determination with Monoclonal Antibodies (Cancer Res. Suppl. 46: 4231–4314, 1986). However, for the purpose of using immunocytochemistry for the practical assessment of the ER and PR status of breast cancer patients it should be realized that tissue for frozen sections is not always available and for this reason it is important to be able to apply these techniques to paraffin embedded tissues or to cytological preparations.

Immunoglobulins, Complement and Foreign Antigens in Human Tumor Cells

Fluorescein-labeled antibodies (FA) to human immunoglobulins, complement, fetal proteins and other antigens were used to search for these immunologic reactants in malignant and benign neoplasia as well as in control tissues. The FA reacted with five or more target antigens in 42 of 59 (70%) malignant tumors and 21 of 23 (40%) benign tumors. The remaining neoplasia and normal tissues showed positive staining with fewer than five specific antibodies. IgM, IgA, B1C, fetal proteins and HSV were the antigens found most frequently. This indicates that neoplasia exhibits immunologic activity more frequently than normal tissue.

Experimental Immunotherapy of Breast Cancer Using Alpha Interferon Conjugated to Monoclonal Antibody Mc5

In recent years, rapidly advancing knowledge on biological response modifiers and the advent of monoclonal antibodies (MoAbs) have provided renewed incentives for immunotherapy of malignant neoplastic diseases. Indeed, important progress has been made in the management of several solid tumors and leukemias through a variety of immunological manipulations, 1,2 while other tumors, including carcinomas of the breast, have thus far been regarded as poor candidates for immunotherapy. Nevertheless, some encouraging local results have been obtained in breast cancer recurrences and metastases treated with intralesional (i.l.) injections of interferons (IFNs). 3-5 A recent study has shown that natural interferon-α combined to natural interferon-γ (nIFN-α/nIFN-γ) delivered i.l. to recurrences and metastases of carcinomas of the breast can effectively eradicate the tumor cells apparently through direct antineoplastic effects and enhancement of cell-mediated immunological responses. 5

Effect of trypsin on prothrombin.

Summary 1. Trypsin converts prothrombin into thrombin. Calcium and accessory clotting factors are not essential. Calcium does, however, accelerate the rate of thrombin formation but without affecting the thrombin yield. 2. Conversion of prothrombin by trypsin is incomplete. 3. Heparin, in low concentrations, can inhibit the clotting effect of trypsin by increasing plasma antithrombic activity.