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Biomedical subjects

A Pihl

Publications and source records attributed to A Pihl.

At least 55 records · Page 3Linked to original sources
PubMed1984

Human tumor xenografts transplanted under the renal capsule of conventional mice. Growth rates and host immune response.

The growth of 29 different human tumor lines under the renal capsule of immunocompetent mice was investigated. The tumors, previously established in athymic mice, included malignant melanomas, colon carcinomas, soft-tissue sarcomas, lung cancers and a mammary carcinoma. The growth rates of 17 tumors, measured repeatedly over a period of several years, were highly reproducible. The different grafts exhibited distinctly different and individual growth rates for up to 6 days. In animals pretreated with an immunosuppressive dose of cyclophosphamide, the growth rate was the same as in non-pretreated animals, but the growth continued for several more days. In the case of 9 different grafts, the subrenal growth rates were compared with those observed when the same tumors were growing subcutaneously in athymic, nude mice. The relative growth rates of the different tumors were practically the same in the two systems. The results indicate that the growth conditions under the renal capsule permit the grafts to express their inherent growth potentials and that the subrenal grafts do not represent a selected sub-population of the tumor cells. The extent of infiltration of the grafts by mouse inflammatory cells was measured by flow cytometry on single-cell suspension as well as by quantitative analysis of serial histological sections. In most cases the mouse cells occupied 15-25% of the total graft volume on day 6. The results indicate that the effect of mouse cell infiltration on the growth of established tumor lines is slight and that it is unnecessary to use athymic mice as host animals when testing new investigational drugs by the SRC assay. The use of established tumor lines in the SRC assay in immunocompetent mice may be useful also in the study of factors influencing the anti-cancer activity of current drugs.

Animals↗
PubMed1984

Estrogen receptors and long-term prognosis in breast cancer.

The long-term prognosis in 233 primary breast cancer patients, most of whom had received ovarian irradiation, has been related to the estrogen-receptor status of the primary tumors. The median observation time was 9 years. The receptor status did influence disease-free interval and survival only in patients having metastases in axillary nodes. Thus, in the postmenopausal patients with axillary metastases, the disease-free interval and survival time were longer in receptor-positive than in receptor-negative patients during the first few years of observation. However, this difference disappeared in the course of the subsequent years. In the premenopausal women with axillary metastases the receptor status did not influence the short-term prognosis, and, unexpectedly, seemed to affect adversely the long-term prognosis. It is concluded that the long-term prognosis is no better in breast cancer patients having estrogen receptors in their primary tumors than in those with receptor-negative tumors.

Aged↗
PubMed1984

Pharmacological studies of ricin in mice and humans.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of ricin in serum is presented. Using this method it was found that IV-injected ricin disappeared from the plasma of mice and cancer patients according to first-order kinetics. DBA mice were found to be more sensitive to ricin than C3H and B6D2 mice. When mice of the different strains were given the same dose of ricin, the concentrations found in liver, spleen, and kidneys were highest in the most sensitive mice. Ricin disappeared most rapidly from serum of the mice with the highest sensitivity. The inverse correlation between the rate of disappearance of ricin from serum and the tissue concentrations reached may be due to the fact that ricin is rapidly and firmly bound to cell surface receptors. Whole-body autoradiography after IV injection of 125I-labeled ricin showed the highest amount of radioactivity in liver, spleen, and adrenal cortex. Considerable amounts of radioactivity were also present in bone marrow, showing that the lack of myelosuppressive activity of ricin previously found in mice and dogs cannot be accounted for by the failure of ricin to reach the bone marrow. Part of the ricin in the tissues was present in the form of free chains, the highest fraction being present in the liver. In this tissue both the free A-chains and those present in whole ricin were found to be modified. However, the modified A-chains had retained their full capacity to inhibit protein synthesis in vitro. In cancer patients, toxicity appeared at about the same initial serum levels as in the mice, supporting the view that mouse data have a good predictive value for man. At each dose level the individual variations were modest, a finding that is important for eventual clinical use of this potent drug.

Animals↗
PubMed1984

Reduced antineoplastic activity in mice of cisplatin administered with high salt concentration in the vehicle.

The effect of high NaCl concentration in the vehicle on the toxic and antineoplastic activities of cisplatin [cis-dichloro-diammineplatinum(II)] (CDDP) was reinvestigated in mice. The toxicity, as measured by the survival of mice given CDDP iv, was reduced by 50-60% when the NaCl concentration in the vehicle was raised from 0.9 to 4%. In ascitic P388 leukemia the antineoplastic activity of CDDP given ip was not reduced significantly. However, in all other systems studied the antitumor activity was reduced when the CDDP was dissolved in high NaCl solution. The tumor models studied included systemic P388 and L1210 leukemias, Lewis lung carcinoma, and 5 human tumor xenografts. The human tumors were studied by the subrenal capsule assay. In the case of a malignant melanoma and a malignant fibrous histiocytoma, the effect also was demonstrated in the subcutaneous nude mouse model. In one of the malignant melanomas a 50% increase in the CDDP dose did not compensate for the reduced antitumor activity caused by the high NaCl concentration in the vehicle. These results, which stand in contrast to current views, question the experimental basis for the use of high-NaCl vehicles in the "high-dose" CDDP regimens.

Animals↗
PubMed1984

Phase I study of the plant protein ricin.

A Phase I study was carried out with ricin, a plant toxin acting by inhibiting protein synthesis, on 54 cancer patients with advanced disease. Ricin was given as i.v. bolus injections every two weeks at dose levels ranging from 4.5 to 23 micrograms/sq m of estimated body surface area. Ricin was well tolerated at doses up to 18 to 20 micrograms/sq m. At these levels and at higher levels, flu-like symptoms with fatigue and muscular pain appeared and, in some patients, nausea and vomiting occurred also. No myelo-suppression was seen. Antibodies to ricin were detected in serum after two to three ricin injections. Ricin was eliminated from blood according to first order kinetics. At each dose level, the plasma concentrations, as well as the side effects, showed only minor differences between patients. The highest dose given, 23 micrograms/sq m, gave plasma concentrations twice those found previously to be therapeutically effective in tumor-bearing mice. Of 38 evaluable patients, one patient with lymphoma had a partial response. Stable disease was observed in four patients with renal cancers, in two with soft tissue sarcomas, and in one patient each with mesothelioma, thyroid, and rectal cancer. A dose of 23 micrograms/sq m is recommended for Phase II trials of ricin.

Abrin↗
PubMed1983

Effect of protein synthesis inhibitors on the fidelity of translation in eukaryotic systems.

Factors influencing the accuracy of poly(U)-directed poly(Phe) synthesis in a wheat germ and in a reticulocyte system were studied. Addition of preformed phenylalanyl-tRNA, as well as increasing the ratio of poly(U) to ribosomes, significantly enhanced the poly(Phe) synthesis and concurrently reduced the misincorporation of leucine. The protein synthesis inhibitors cycloheximide, abrin and ricin had little or no effect on the misreading when the system was supplemented with 100 microM phenylalanyl-tRNA, but they reduced the relatively high error rate observed when the poly(U) system was not supplemented with the cognate substrate. Raising the incubation temperature enhanced the accuracy to the same extent whether or not ricin was present i.e., at widely different rates of elongation. The results show that the translational accuracy is not linked to the elongation rate as such. Translational inhibitors affect the fidelity by influencing the kinetics of the system. In systems containing limiting concentrations of cognate substrate, translational inhibitors will cause an increase in the limiting aminoacyl-tRNA species and thereby increase fidelity.

Abrin↗
PubMed1983

Antibody formation against the cytotoxic proteins abrin and ricin in humans and mice.

Antibody formation may limit the therapeutic use of cancerostatic proteins. To study the significance of antibody formation against abrin and ricin, highly sensitive ELISA procedures for determination of anti-abrin and anti-ricin were developed. In mice treated weekly with therapeutic doses of ricin, antibodies appeared after 2-3 weeks and then rose rapidly, whereas after abrin treatment the antibody formation was slower. Ricin A-chain was found to be more immunogenic than either intact ricin or human serum albumin (HSA). Cyclophosphamide inhibited the antibody response to both abrin and ricin and a combination of cyclophosphamide and prednisolone totally inhibited both anti-abrin and anti-ricin formation during the 6-week observation period. In mice treated weekly with HSA, abrin treatment strongly reduced the anti-HSA formation, showing that abrin has an immunosuppressive effect which appeared to be stronger than that of cyclophosphamide. The existence of circulating antigen-antibody complexes could be demonstrated in the sera of toxin-treated mice by precipitation with polyethyleneglycol, whenever antibodies were detectable with ELISA. The life-span of animals given lethal ricin doses was appreciably enhanced in animals having antibody levels in excess of 10-20 ng/ml. In cancer patients treated i.v. every second week with therapeutic toxin doses, the 10-20 ng/ml levels of anti-ricin and anti-abrin were reached 6-8 weeks and 7-10 weeks after the first injection of ricin and abrin, respectively. The data indicate that the effective therapeutic use of abrin and ricin as single agents may be limited to these time frames, but that the period of effective use may be substantially prolonged if the toxins are given together with conventional cytostatic agents having immuno-suppressive activity.

Abrin↗
PubMed1982

Isolation and characterization of viscumin, a toxic lectin from Viscum album L. (mistletoe).

A toxic protein, viscumin, was isolated from extracts of mistletoe by affinity chromatography on acid-treated Sepharose 4B. Viscumin was selectively bound to the column and could be eluted with lactose. It migrated in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate corresponding to Mr = 60,000. In addition, two bands migrating corresponding to Mr = 29,000 and 32,000 were found. After treatment with 2-mercaptoethanol, only 2 bands (Mr = 29,000 and 34,000) were found. Apparently, viscumin consists of two chains which, in some of the molecules, are disulfide-linked. Protection experiments with antiserum against viscumin indicated that the major part of the cytotoxic activity in mistletoe extracts is due to viscumin. Gel filtration experiments on Sephacryl 200 indicated that, at low concentrations, viscumin occurs as a monomer and at higher concentrations as a dimer. Viscumin was found to inhibit protein synthesis in cell-free systems. When the two constituent peptide chains of viscumin were eluted from polyacrylamide gels and tested for ability to inhibit cell-free protein synthesis, this property was found to be associated with the fastest migrating chain, here denoted the A chain. The heavier chain was denoted the B chain. The A chain was found to inhibit protein synthesis by inactivating the ribosomes catalytically. Reconstitution experiments with isolated ribosomal subunits from untreated and A chain-treated ribosomes showed that the 60 S ribosomal subunit was selectively inactivated.

Animals↗
PubMed1982

Action of viscumin, a toxic lectin from mistletoe, on cells in culture.

A toxin from mistletoe, viscumin, inhibited the incorporation of leucine in cells more rapidly than the incorporation of uridine and thymidine, indicating that the toxins act by inhibiting cellular protein synthesis. The presence of galactose, lactose, and melibiose in the medium protected cells against viscumin. The sensitivity to viscumin of different cell lines differed considerably. The cytotoxic properties of viscumin were compared to those of the related toxins abrin, ricin, and modeccin. The profile of the sensitivity of a panel of cell lines to viscumin was dissimilar from those obtained with abrin and modeccin. Cell lines selected for resistance to modeccin and ricin were fully sensitive to viscumin. Ca2+ was required for viscumin to express its toxic effect. In contrast, the Ca2+ ionophore A23187 protected the cells against viscumin. Whereas the sensitivity of cells to viscumin did not vary much between pH 7 and 9, the cells were much less sensitive at pH 6. Also, cells treated with the two metabolic inhibitors, 2-deoxyglucose and NaN3, were insensitive to the toxins. Viscumin taken up by cells in the absence of Ca2+ or at pH 6 was able to intoxicate the cells when Ca2+ was added or when the pH was adjusted to neutrality. In contrast, cells exposed to viscumin in the presence of 2-deoxyglucose and NaN3, which inhibit endocytosis, were not intoxicated when the cells were treated with antiviscumin and then transferred to normal medium. The results indicate that endocytosis is involved in the entry of viscumin.

Animals↗
PubMed1982

Preparation and properties of chimeric toxins prepared from the constituent polypeptides of diphtheria toxin and ricin. Evidence for entry of ricin A-chain via the diphtheria toxin pathway.

A highly toxic conjugate of ricin A-chain and diphtheria toxin fragment B was prepared by disulfide exchange reaction. A similar conjugate between diphtheria toxin fragment A and ricin B-chain was nontoxic. Like native diphtheria toxin, the conjugate ricin A/diphtheria toxin B was much more toxic to Vero than to HeLa cells. Ricin was equally toxic to these cell lines. Lactose, which inhibits ricin binding, did not protect against the conjugate. Cells resistant to ricin, partly due to a reduced number of ricin-binding sites, were fully sensitive to the conjugate, indicating that the conjugate binds to diphtheria toxin receptors. The conjugate was fully toxic to two Vero cell mutants, resistant to diphtheria toxin because the elongation factor 2 could not be ADP-ribosylated by the diphtheria toxin A-fragment. Therefore, the inhibition of protein synthesis by the conjugate must be caused by the ricin A-chain. Ammonium chloride which prevents entry of diphtheria toxin, but not of ricin, also protected against the conjugate. Like diphtheria toxin, the conjugate was most toxic at low pH, whereas ricin is most active at pH above neutrality and inactive at low pH. The results indicate that the conjugate ricin A/diphtheria toxin B binds to diphtheria toxin receptors and inhibits cellular protein synthesis due to the action of ricin A-chain which appears to enter the cell by the diphtheria toxin pathway.

Ammonium Chloride↗
PubMed1982

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts.

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.

Animals↗
PubMed1982

Colony growth and chemosensitivity in vitro of human melanoma biopsies. Relationship to clinical parameters.

To study the usefulness of an in vitro colony-forming assay in predicting individual clinical responses to chemotherapy, tumor cells obtained from 150 melanoma metastases (119 patients) were grown in soft agar according to the method of Courtenay and Mills (1978), and tested for sensitivity to DTIC, CCNU, vinblastine, procarbazine, abrin and ricin. In 83% of the cases colony formation was observed (plating efficiency, PE, greater than 0.01%). Twenty-seven per cent of the tumors gave PEs greater than 1%, 45% gave PEs in the range 0.1-0.9%, whereas 11% of the tumors gave 0.01-0.09%. The PEs were not correlated with the degree of pigmentation or with the clinical course. Evaluable chemosensitivity data were obtained on 104 metastases from 83 patients. Large differences in sensitivity were seen. In cases which were evaluable both in vivo and in vitro a clear correlation was found between the in vitro chemosensitivity, expressed as the expected growth delay, and the clinical response to chemotherapy. Tumors from patients with partial response, mixed response or stable disease after prior progression, all had rather high in vitro sensitivity to the drug used (expected growth delay greater than 2.0), whereas patients with progression had lower sensitivity. The results confirm that the soft agar method used here provides good culture conditions for human melanoma cells and show that chemosensitivity data can be obtained in a high percentage of melanoma patients. The approach used seems promising in aiding clinicians to adjust chemotherapy to individual patients.

Antineoplastic Agents↗
PubMed1982

Inhibition of mitochondrial protein synthesis and energy coupling by fragment A of diphtheria toxin.

The effect of intact diphtheria toxin and its fragment A on energy-dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case. Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 micrograms fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I-labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A. At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP-ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria. The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.

Animals↗
PubMed1981

The usefulness of human tumor cell lines in the study of chemosensitivity. A study of malignant melanomas.

The chemosensitivity of human melanoma cells has been studied before and after continuous in vitro culture. Altogether, nine cell lines were studied, two derived from patients' biopsies, and seven from xenografts in athymic mice. The sensitivity to the agents DTIC (Dacarbazine), CCNU (Lomustine), procarbazine, vinblastine, abrin and ricin was assayed. Furthermore, in five cases the chemosensitivity of the cell lines was compared to that of tumors obtained by injecting the cell lines into athymic mice. In all cases the sensitivity was measured in an in vitro soft agar assay. Upon cultivation in vitro, two of the cell lines, one derived from a patient's metastasis and one from a xenograft in athymic mice, showed marked increases in sensitivity to some of the drugs, whereas sensitivity to other drugs showed little or no change. For the other cell lines small, but definite increases or decreases in chemosensitivity were observed. Permanent cultures showed the same chemosensitivity as early subcultures. The tumors formed by injecting the cell lines into athymic mice showed moderate changes in chemosensitivity, as compared to the cell lines in vitro. The data indicate that considerable changes in chemosensitivity may occur when cells are brought from in vitro to in vitro conditions and vice versa and that such changes may be highly specific. Therefore, although cell lines may be useful in some respects, they should be used with caution in attempts to evaluate quantitatively the sensitivity of human tumors to cancerostatic drugs.

Animals↗