Glenn A. Herbst

Oncogenic transformation of rat embryo fibroblasts with photoinactivated herpes simplex virus: rapid in vitro cloning of transformed cells.

Summary Rat embryo fibroblasts (REF) were transformed in vitro with photoinactivated herpes simplex virus. Low passage (7 to 10) HSV-transformed rat cells (t-REF-line G) produced multiple tumours in 49% of newborn rats with a latent period of 20 to 24 weeks. An in vitro cloning procedure for transformants in the uncloned t-REF-line G cells produced clonal lines which varied from non-oncogenic to clonal lines producing tumours with shorter latent periods (10 to 14 weeks) compared to uncloned cells. At passage 30, t-REF-line G-clone 1 cells produced rapidly growing tumours in 100% of the newborn rats with a latent period of only 2 to 3 weeks. Tumour cells (RFS 12-22-75) established in culture produced tumours within 2 weeks after subcutaneous (s.c.) inoculation of weanling rats (100% with tumours) and they were transplantable to 100% of inoculated adult rats. Histopathological examination of all tumours produced in newborn, weanling or adult rats revealed large, poorly differentiated malignant fibrosarcomas; metastatic tumours were observed in the lungs of 10 to 20% of newborn rats inoculated s.c. with RSF cells. Approx. 25 to 50% of the clonal transformed or tumour cells synthesized HSV-specific-antigens detected by immunofluorescence. HSV-transformed and tumour cells are resistent to superinfection by the homologous transforming virus. Since the in vitro cloning procedure for transformant cells can readily segregate cells producing clonal lines varying in oncogenic potential, the procedure might have useful application in elucidating HSV oncogenesis.

Studies of lymphocyte populations in pre-eclampsia-eclampsia

The possibility that the etiology of toxemia might be immunologic has been held for over 70 years. In the past decade, numerous studies have been instituted in attempts to verify the possible role of the immune system in this disease. The present study was undertaken as a probe to determine if a gross difference in the numbers of T and B cell lymphocyte populations might exist between pre-eclamptic and normally pregnant women. Twenty-five normally pregnant women in the third trimester were compared to 25 pre-eclamptic women, and significant differences were noted. If, indeed, there is an immunologic basis for pre-eclampsia, it is more subtle than the methodology used in this study is capable of detecting.

Phagocytosis and erythroblastosis: I. Modification of the neonatal response by promethazine hydrochloride

Phagocytic cells were obtained from children at ages comparable to those at which the disease is most commonly seen during pregnancy. The effect of P-HCl on the phagocytic action of these cells on opsonized red blood cells was studied in vitro.

Chemotherapy of advanced ovarian epithelial carcinoma with melphalan and levamisole: A pilot study of the Gynecologic Oncology Group

Twenty-three patients with Stage III, Stage IV, or recurrent epithelial ovarian cancer were treated with a combination of melphalan and levamisole to determine a tolerable dosage schedule, possible adverse effects, and a general estimate of response rate and duration. In seven patients with measurable disease there were four complete responses (57%) with a median duration of 75 weeks. Two of the complete responders have had negative second-look laparotomies while the other two patients have had subsequent progression. Of 16 patients with nonmeasurable disease two have had negative second-look laparotomies and two remain progression free. Thus 8 of 23 patients (35%) had complete responses or remain progression free whereas 4 of 23 patients (17%) have had negative second-look laparotomies. No serious toxicity was encountered. Immunologic monitoring did not indicate significant immunologic reconstitution in these immunosuppressed patients.

The effect of promethazine hydrochloride on fetal and maternal lymphocytes.

THIS LABORATORY has been involved in the study of promethazine hydrochloride and its use in erythroblastosis for many years. Our interest in this drug was originally aroused by the report by Bierme-Alie-Enjalbert’ of a possible amelioration of the effects of erythroblastosis in 1 I patients treated with that drug during pregnancy. The possibility that its ability to ameliorate this immunologically mediated disease by an immunosuppressive capability was studied by us in patients and in the laboratory. Our most recent publication reviews our prior work and documents the in vitro inhibition, by promethazine hydrochloride, of the ability of macrophages of the newborn infant to form rosettes with anti-D opsonized red blood cells.’ We have previously shown that fetal red blood cell destruction in erythroblastosis caused by the Rh antigen was not accomplished directly by the antibody and that the anti-D was not complement-fixing. In addition, we have also documented the ability of promethazine hydrochloride to inhibit lymphocyte-mediated functions in vivo in animals. The first paper we published concerning the effectiveness of promethazine hydrochloride in the treatment of erythroblastosis involved only 15 patients. A subsequent paper involving more than 40 patients is in preparation. However, we have maintained a concern regarding the effects which promethazine hydro-

Purification of naturally occurring antibody to human placental lactogen by affinity chromatography

Antibodies against human placental lactogen (HPL) were isolated from maternal postpartum sera and nonpregnant female sera by the technique of immunoadsorption (or affinity chromatography). HPL was linked to Sepharose gel by cyanogen bromide activation and the resultant chromatographic resin was used in a repetitive column procedure to absorb and fractionate naturally occurring antibodies to HPL. The antibody to HPL was concentrated after elution from the column with a chaotropic ion, and tested for immunologic acitivity. It was determined to be an IgG molecule, and only radioimmunoelectrophoresis was sensitive enough, among the several tests performed, to show specific anti-HPL activity after treatment.

Lymphocyte Cyclic AMP Levels Antepartum and Postpartum as an Indirect Assessment of the Immune System During Pregnancy

ABSTRACT: The literature dealing with the immunological state of the pregnant woman has been conflicting. The concentrations and activity of a number of hormones and proteins which modify lymphocytic activity have been measured both in vivo and in vitro during pregnancy. Most of the differences between reported studies can be reconciled to technical or experimental variations. In some instances, the purported suppressive effects of embryonic proteins such as HCG have actually been caused by the impurity of the preparation studied. We have attempted to approach the question of whether or not the pregnant woman is immunosuppressed by studying a regulatory material in the lymphocytes. It is known that cAMP is a mirror of lymphocytic activity and that low levels of cAMP may indicate a high degree of reactivity, while high levels are present when lymphocyte reactivity is low. In an initial study, ten women volunteered to have blood drawn in the last trimester and two months postpartum. Cyclic AMP was extracted from lymphocyte‐enriched leukocytes and stored until all samples were available from all patients so that the analysis could be made simultaneously. Five samples were obtained from healthy nonpregnant women in the same age range. Postpartum and nonpregnant women were found to have significantly elevated levels of cAMP as compared to the lymphocytes obtained in the third trimester of pregnancy. The experiments were then repeated using seven more patients. The same significant increase in postpartum lymphocyte cAMP concentrations were found. The precise reason(s) for this is not known, but may be due to increased suppressor cell activity.

Localization of anti-HPL in fetal, placental, and maternal renal tissues.

Anti-human placental lactogen (HPL) was given to pregnant rats. An analysis of various tissues including lung, muscle, placenta, kidney and fetal localizes the heterologous antibody at the placenta. It is presumed that whatever destructive effect it has takes place there.

Evaluation of the Shake Test to Predict Respiratory Distress Syndrome

An analysis of the relationship of amniotic fluid shake test titers and the subsequent fetal lung maturity as evinced by the development of the respiratory distress syndrome (RDS) has been conducted. Over a four-year period, 131 amniotic fluid samples were tested within 48 hours of delivery. RDS was diagnosed in 16 infants. When the shake test was positive at a 1:2 dilution, one child developed a mild case of RDS (2.3%). If the test was positive at a 1:4 dilution, none developed RDS. RDS occurred in 15% of the cases with a positive test at a 1:1 dilution and in 30% of the cases with a negative test. This test has proved to be an excellent screening method for predicting fetal lung maturity if it is positive at 1:2 or greater. If the result is positive at a 1:1 dilution, or negative, other methods must be used for assessment. Its advantages are its rapidity, simplicity, and low cost.

The inhibition of polymorphonuclear and lymphocyte-mediated fetal red blood cell lysis by promethazine in vitro.

Abstract An in vitro method for the measurement of fetal red blood cell destruction by Fc receptor-bearing polymorphonuclear leukocytes and lymphocytes has been developed. Cord blood was collected at the time of delivery from infants who were Rh-D positive and compatible with their mother, as well as infants who were Rh-D positive and incompatible with their Rh-D-sensitized mothers. Red blood cells, lymphocytes, and polymorphonuclear preparations were individually separated from each cord blood sample. Rh-D-positive cord red blood cells from compatible and incompatible pregnancies complicated by erythroblastosis were coated with anti-Rh-D. All target red blood cells, opsonized and not opsonized, were labeled with chromium-51 and cultured with autologous lymphocytes or polymorphonuclear leukocytes at an effector:red blood cell ratio of 10:1. Cultures containing either no promethazine hydrochloride or varying amounts of the drug were studied. Antibody alone did not cause red cell lysis. Both lymphocytes and polymorphonuclear leukocytes were effective in causing the lysis of red blood cells bearing antibody. Lysis of red blood cells by polymorphonuclear leukocytes or lymphocytes was inhibited by promethazine hydrochloride. Excessive amounts of promethazine hydrochloride alone cause lysis of the red cells. At the low doses utilized for inhibition in this experiment, the action of promethazine hydrochloride on the lymphocytes was found to be reversible. Promethazine is believed to have exhibited two different types of activity in this study: (1) It inhibited antibody-dependent, cell-mediated lysis of autologous red blood cells coated with antibody; (2) it stabilized the red blood cell membrane so that even spontaneous lysis of red blood cells was decreased 50 per cent.