Structural requirements for cocaine-sensitive and -insensitive uptake of phenethylamines into the adrenal chromaffin cell.

The adrenal medullary chromaffin cell is a commonly used model for the adrenergic neuron. Although much work has been done to study the transport system in the adrenal chromaffin vesicles, relatively little is known about cellular transport, especially with regard to structural features of phenethylamines required for intracellular accumulation. We have now investigated the structural requirements of phenethylamine-related compounds for their accumulation into cultured adrenal chromaffin cells. We find that two types of cellular uptake, previously described only for dopamine, norepinephrine, and epinephrine, are also present for [3H]tyramine. Although two types of accumulation occur, tyramine accumulation occurs mainly via a cocaine-insensitive process, whereas dopamine accumulation occurs predominantly via a cocaine-sensitive process. The accumulation of [14C]-phenethylamine and p-methoxyphenethylamine is not affected by cocaine, suggesting that a ring hydroxyl substituent is necessary for cocaine-sensitive accumulation. The compounds p-hydroxyphenylpropylamine and p-hydroxyphenyl-2-aminoethyl sulfide accumulate in the cell only via a cocaine-insensitive process, indicating that lengthening of the aminoalkyl side chain prevents cocaine-sensitive accumulation. We have performed conformational analyses of this series of compounds to determine whether the conformation of these compounds can be related to the kinetic data. For dopamine, tyramine, phenethylamine, and p-methoxyphenethylamine, two groups of energy-minimized conformers were found. We find that there is an approximately linear relationship between the Km values for these phenethylamines and the differences in minimized energies between the low- and highest energy conformer groups of each compound. A similar correlation was found for p-hydroxyphenyl-2-aminoethyl sulfide. These results are consistent with the hypothesis that these compounds undergo a conformational change from the low-energy conformer to the highest energy conformer before their cocaine-insensitive accumulation.

Elimination of malignant clonogenic T cells from human bone marrow using chemoimmunoseparation with 2'-deoxycoformycin, deoxyadenosine and an immunotoxin.

Autologous bone marrow transplantation may contribute to the treatment of several types of lymphoreticular malignancies. Recent studies have suggested that a combination of chemoseparation and immunoseparation may be more effective than either modality alone in eliminating malignant cells from human bone marrow. In this report an immunotoxin has been prepared by conjugating pokeweed antiviral protein (PAP) to the 3A1 murine monoclonal antibody that recognizes a 40 kD (CD7) determinant expressed by most T cell acute lymphoblastic leukemias and a majority of normal mature peripheral T cells. When HSB-2 T lymphoma cells were mixed with normal human bone marrow and incubated with 3A1-PAP and 100 microM chloroquine, approximately 3 logs of clonogenic T cells could be eliminated from a 20-fold excess of bone marrow. Treatment of cell mixtures with 2'deoxycoformycin (2'-dCF) and deoxyadenosine (dAdo) eliminated 2 logs of clonogenic tumor cells. The use of 3A1-PAP and chloroquine with dCF/dAdo was more effective than either single modality, eliminating up to 6 logs of HSB-2 tumor cells in optimal experiments. Anti-tumor activity of the combined treatment extended to T leukemia cells taken directly from patients. Although 3A1-PAP reduced CFU-GM by only 13% and BFU-E by 36%, the addition of 2'-dCF and dAdo was more toxic for normal marrow precursors, further reducing CFU-GM, GEMM and BFU-E as well as preventing recovery of CFU-GM in long-term bone marrow culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisplatin and etoposide: an effective treatment for refractory breast carcinoma.

Thirty-one patients with metastatic breast carcinoma refractory to standard hormonal and chemotherapy were treated with cisplatin 100 mg/m2 per course and etoposide 300 mg/m2 per course divided over 5 days. Courses were repeated at 3-6-week intervals, depending on the speed of recovery from myelosuppression. Of 29 evaluable patients, three had complete responses, eight had partial responses, eight had stable disease, and 10 had progressive disease. Nausea, emesis, anorexia, weakness, and easy fatigability were common but tolerable side effects. Myelosuppression was frequent and occasionally profound but there were no deaths from hemorrhage or infection. No significant renal toxicity was encountered. The combination of cisplatin and etoposide has sufficient antitumor activity with acceptable toxicity in heavily pretreated patients to justify its further study in breast cancer.

High-dose combination alkylating agents with bone marrow support as initial treatment for metastatic breast cancer.

To evaluate the effect of high-dose chemotherapy in the treatment of metastatic breast cancer, we performed a phase II trial of a single treatment with high-dose cyclophosphamide (5,625 mg/m2), cisplatin (165 mg/m2), and carmustine (600 mg/m2), or melphalan (40 mg/m2) and bone marrow support as the initial chemotherapy for metastatic breast cancer. Twenty-two premenopausal patients with estrogen receptor negative, measurable metastatic disease were treated. Twelve of 22 patients (54%) obtained a complete response at a median 18 days. The overall response rate is 73% (complete and partial response). Median duration of response in the patients achieving complete response was 9.0 months with a median duration of survival for complete responders that is currently undefined. Relapse occurred predominantly at sites of pretreatment bulk disease or within areas of previous radiation therapy. Toxicity was frequent and five patients died of therapy-related complications. The results indicate that a single treatment with intensive combination alkylating agents with bone marrow support can produce more rapid and frequent complete responses than conventional chemotherapy when used as initial chemotherapy for metastatic breast cancer, although median disease-free and overall survival is not improved. Three patients (14%) remain in unmaintained remission beyond 16 months.

Altered immunologic reconstitution after standard-dose chemotherapy or high-dose chemotherapy with autologous bone marrow support.

Altered immunologic reconstitution is observed in patients treated with high-dose chemotherapy consisting of cyclophosphamide, cisplatin, and carmustine, melphalan, or etoposide with autologous bone marrow support, and it is similar to that seen in patients treated with high-dose chemoradiotherapy and allogeneic bone marrow transplantation. A decrease in the absolute number and percentage of B cell and CD4 antigen-positive cells and an increase in the absolute number and percentage of CD8 and Ia antigen-positive cells occur along with a decrease in the CD4/CD8 ratio that persists for 6-12 months after high-dose chemotherapy and autologous bone marrow support. These changes have been associated with four serious infectious episodes usually seen only in immunocompromised patients. The above changes were not seen in patients treated with high-dose busulfan, a drug that has relatively specific effects on granulocytes. It is postulated that these alterations result from effects of chemotherapy on the residual lymphocytes or on the environment of repopulating lymphocytes. Functional studies of lymphocyte populations during immunologic reconstitution after standard-dose combination chemotherapy and high-dose chemotherapy with autologous bone marrow support are needed.

Inflammatory breast carcinoma. Effective multimodal approach.

Twenty-two patients with inflammatory breast carcinoma received preoperative chemotherapy consisting of weekly administration of cyclophosphamide, doxorubicin hydrochloride, fluorouracil, and vincristine sulfate for six weeks. Postoperative therapy consisted of 22 weeks of biweekly administration of these drugs. Regional radiotherapeutic consolidation followed chemotherapy. Nineteen patients completed therapy. Twelve of these patients remain disease free (median, 15 months; range, four to 32 months). Median disease-free survival for all 22 patients is 13 months or more (range, zero to 32 months). Median overall survival is 18 months or more (range, one to 33 months). This regimen compares favorably with prolonged adjuvant and maintenance chemotherapy for inflammatory breast carcinoma.

Combined chemoseparation and immunoseparation of clonogenic T lymphoma cells from human bone marrow using 2'-deoxycoformycin, deoxyadenosine, 3A1 monoclonal antibody, and complement.

Chemoseparation and immunoseparation techniques have been combined to eliminate malignant clonogenic T lymphoma cells from human bone marrow. Incubation with 5 microM 2'-deoxycoformycin and 500 microM deoxyadenosine has eliminated 2 logs of HSB-2 T lymphoma cells from a 20-fold excess of irradiated human bone marrow. Multiple incubations with 3A1 antibody and rabbit complement eliminated approximately 2 logs of HSB-2 cells from similar mixtures. Used in combination, the 2 techniques eliminated up to 4 logs of T lymphoma cells. Incubation of normal human bone marrow under similar conditions failed to affect growth of granulocyte-macrophage colony-forming cell units, burst-forming erythroid units, or multipotential erythroid-granulocyte-megakaryocyte-macrophage colony-forming hematopoietic progenitor cells units.

Fine-needle aspiration biopsy of the mediastinum.

Fine-needle aspiration is a useful technique to identify neoplasms of many sites, such as breast, thyroid, and lung. Thirty-two mediastinum aspirates from 29 patients were reviewed. Five aspirates yielded insufficient material. Five aspirates were of benign lesions. Four aspirates were suggestive of but not diagnostic of malignancy. Eighteen aspirates contained malignant cells; in 13 of these, a definite cell type was identified, which usually was metastatic lung carcinoma; in five instances, the cell type could not be unequivocally identified. Complications were minimal, two instances of pneumothorax (6.3 percent) and two of hemoptysis (6.3 percent). No deaths or hemorrhage occurred. In 16 of the 29 patients (55 percent), thoracotomy was avoided because of fine-needle aspiration biopsy. It is concluded that fine-needle aspiration biopsy of the mediastinum is a safe, useful diagnostic tool. This procedure may obviate the need for thoracotomy in persons with inoperable cancer, thus lowering medical costs and length of hospital stay.

Mitoxantrone in the treatment of relapsed and refractory acute leukemia.

Eleven academic institutions were selected to study mitoxantrone administered on a schedule of 10 mg/m2/d for five days initially and later at 12 mg/m2/d for five days, each given as a 30 minute intravenous (IV) infusion each day. Patients with acute or chronic leukemia were stratified by leukemic type and clinical status and included one group of patients considered to be in relapse after complete remission from previous chemotherapy and another group of patients considered refractory to standard induction and/or salvage chemotherapy. During the initial treatment schedule, complete remissions were obtained in two of seven patients with acute nonlymphoblastic leukemia, in one of three patients with acute lymphoblastic leukemia, but in none of the patients with chronic granulocytic leukemia in blast crisis. The durations of remission for these three patients were 22, 57, and 78 days, respectively. An increase in mitoxantrone dose to 12 mg/m2/d produced complete remissions in 8 of 19 evaluable patients with acute nonlymphoblastic leukemia, in one of ten patients with refractory acute nonlymphoblastic leukemia, and in one of four patients with chronic granulocytic leukemia in blast crisis. Each of these patients required only a single course of mitoxantrone to achieve remission; the median time to remission was 37 days (range 18 to 64 days). Remission duration ranged from 35 days (chronic granulocytic leukemia) to 186 days, with the median duration for those patients with acute nonlymphoblastic leukemia achieving remission being 135 days. Of the six patients with acute lymphoblastic leukemia, none achieved remission at the higher dose level. Drug-related gastrointestinal toxicity included mucositis (25%), diarrhea (21%), and nausea and vomiting (61%). Systemic infection (nonfatal) was experienced by 21% of patients and alopecia by 17%. Other side effects that occurred occasionally were hepatic dysfunction, decreased renal function, confusion, lethargy, anxiety, and fever. Possible drug-related phlebitis developed in one patient, and a single episode of minor epistaxis was reported in another. Cardiovascular toxicity was low. At a mitoxantrone dose of 10 mg/m2/d for five days, one patient developed hypotension, and one episode of congestive heart failure was reported in another. At the higher dose of 12 mg/m2/d, no drug-related hypotension, congestive heart failure, tachycardia, or chest pain were reported. These data indicate that mitoxantrone is a promising single drug for the treatment of acute nonlymphoblastic leukemia and possibly for acute lymphoblastic leukemia.

Alteration of murine mammary tumor metastasis and growth by cytomegalovirus infection.

Host resistance to the development of metastatic lesions is complex and involves both lymphocyte and macrophage functions. Studies in both humans and animals have suggested that cytomegalovirus infection may alter these components of the defense mechanism of the host. In the present study, an experimental model was developed to determine whether cytomegalovirus infection would affect host resistance to the establishment of metastatic tumor nodules in the lungs of C3H mice after i.v. inoculation of a single-cell suspension of mammary tumor cells. The number of tumor nodules in the lungs, the lungs-heart/body weight ratio, and the mean day of death were determined in control animals inoculated i.v. with 10(6) mammary tumor cells and compared with groups of animals also receiving a sublethal i.p. inoculum of murine cytomegalovirus (MCMV) (10(5) plaque-forming units) either 3 days before, on the day of, or 10 or 13 days after tumor cell inoculation. The results suggest a biphasic effect of virus infection on tumor development in the lung. A preexisting or concurrent MCMV infection suppressed tumor growth and prolonged life, while a MCMV infection later in tumorigenesis enhanced tumor growth and shortened survival. These data suggest that MCMV modulates host resistance to the development of metastatic tumor nodules and that this experimental model may be utilized to investigate further the relationship between virus-induced alterations of host defense mechanisms and tumor growth.

A new animal model with hyperbilirubinemia: the indigo snake.

An unconjugated hyperbilirubinemia has been observed in all species of normal indigo snakes. The plasma clearance of large organic anions such as sulfobromophthalein and unconjugated bilirubin was markedly delayed when compared to other snake species. Endogenous bile flow and biliverdin and bilirubin excretory rates and the excretion of bile pigments after a bilirubin load were measured in various snakes. The indigo snake represents a new animal model in which to study mechanisms important to hepatic anion uptake and biliary transport.

Effect of treatment with exogenous interferon, polyriboinosinic-polyribocytidylic acid, or polyriboinosinic-polyribocytidylic acid-poly-L-lysine complex on Herpesvirus hominis infections in mice.

The effect of treatment with exogenous interferon was compared with those of two interferon inducers, polyriboinosinic-polyribocytidylic acid [poly(I) . poly(C)] and poly(I) . poly(C)-poly-L-lysine complex [poly(ICLC)], in three model Herpesvirus hominis type 2 infections of mice. After intraperitoneal inoculation of H. hominis type 2, all drugs significantly protected animals against death and increased the mean day of death when administered as late as 48 hr after viral inoculation. With intranasal inoculation of H. hominis type 2, pretreatment with poly (I) . poly (C) and poly (ICLC) increased the mean day of death; however, no drug prevented death. In mice inoculated intravaginally, local treatment with exogenous interferon or poly(I) . poly(C) appeared to reduce the mean titers of virus in genital secretions and resulted in earlier clearance of infection in some animals. Systemic treatment of genital H. hominis type 2 infections with all drugs resulted in significant numbers of infected animals surviving the infection, although the mean titers of virus in genital secretions were unchanged. Therapeutic efficacy varied depending on the route of viral inoculation.

Treatment of a murine cytomegalovirus infection with exogenous interferon, polyinosinic-polycytidylic acid, and polyinosinic-polycytidylic acid-poly-L-lysine complex.

Treatment of a cytomegalovirus infection of mice with exogenous murine interferon did not alter final mortality or mean day of death. Pretreatment with two interferon inducers significantly reduced mortality, but treatment initiated after infection was not effective.

Alteration of host defense mechanisms by murine cytomegalovirus infection.

An animal model of a sublethal infection, utilizing murine cytomegalovirus (MCMV), was developed to determine whether immunological factors could contribute to the establishment of a persistent viral infection. Adult female C3H mice inoculated intraperitoneally with 10(5) plaque-forming units of MCMV developed splenomegaly 5 to 12 days after infection. Virus replicated to peak titers (10(3) to 10(6) plaque-forming units per g of tissue) in liver, spleen, lung, kidney, and salivary gland tissue during the acute phase of the infection (3 to 12 days); it then decreased to undetectable levels in all tissues except salivary gland. Serum interferon was detected as early as 12 h after infection, peaked at 36 h (1,093 U/ml), and was undetectable by 4 days after infection. MCMV-infected animals were hyporeactive to interferon induction with New castle disease virus on days 5 to 9 of the infection. Splenic lymphocyte reactivity to phytohemagglutinin and lipopolysaccharide was normal early during the course of the infection, was suppressed during the acute phase of the infection, and had returned to normal by day 18. These data indicate that several parameters of host defense are transiently suppressed during the course of a MCMV infection. The capacity of cytomegaloviruses to alter host resistance may be one factor that contributes to the establishment of a persistent infection.

Effect of treatment with exogenous interferon, polyinosinic acid-polyctyidylic acid or polyinosinic acid-polycytidylic acid-poly-L-lysine complex on encephalomyocarditis virus infections in mice.

The effect of treatment with exogenous interferon was compared with two interferon inducers, polyinosinic acid-polycytidylic acid [poly(I:C)] and [poly(I:C)]-poly-l-lysine complex (P-L-L complex), in two model encephalomyocarditis virus infections of mice. Although both inducers stimulated the production of interferon, the peak serum levels induced by P-L-L complex were five- to eightfold greater than those induced with poly(I:C). When encephalomyocarditis virus was inoculated by either the intraperitoneal or the intranasal route, interferon and both of the inducers protected mice against mortality and prolonged the mean day of death when the compounds were given prior to or immediately after viral challenge. In general, treatment with interferon was not as successful as treatment with poly(I:C) or P-L-L complex. In these infections, P-L-L complex appeared to be the most effective agent in that successful treatment resulted when drug therapy was initiated as late as 48 h after virus inoculation. An examination of the effect of treatment on the pathogenesis of the infection indicated that protection was associated with the prevention of viremia and subsequent seeding of target organs, particularly the central nervous system.

The use of muscle stimulation for inducing scoliotic curves. A preliminary report.

Based on a clarification of the anatomical mechanisms involved in the lateral stability of the spine, electrical stimulation was applied unilaterally to paraspinal muscles of 20 growing dogs in an attempt to produce thoracic scoliotic curves. These experiments were considered critical in the development of a new procedure for correcting human scoliosis. Various alternating voltages and wave forms were used. The curvature varied with the voltage applied, frequency being maintained at a constant value. Curves can be developed in the spines of normal dogs by unilateral electronic muscle stimulation without clinical discomfort or significant muscle atrophy. Curves always develop in the predicted direction (concave to stimulation). Stimulation of more than 4-6 weeks produced curves with significant wedging and rotational deformation. In one experiment curves created in one direction were reversed by stimulating opposite side musculature. Because of the similarity of human and canine anatomy, the results obtained gave impetus to further experimentation with human application. Electromyographic tests, biopsy studies, direct deformation measurements of post mortem dissections and weekly X-rays substantiate the above observations.