Structure and immunogenic behaviour of methylated tobacco mosaic virus.

Tobacco mosaic virus was methylated, using various concentrations of dimethylsulfate. The methylated virus sample with still intact particles was subjected to sequential analysis. The sites and the degree of methylation were determined in the tryptic peptides. Tyrosine 139 and cysteine 27 are more accessible to methylation than tyrosine 72, lysine 68 and tyrosine 2. A limited number of carboxyl groups was also methylated. The ability of methylated and original tobacco mosaic virus to initiate the formation of humoral antibodies and the capacity to induce a delayed-type hypersensitivity reaction were investigated in STU mice. Original tobacco mosaic virus could not induce a delayed-type hypersensitivity reaction but methylated tobacco mosaic virus induced a delayed-type reaction, not depending on whether the virus particles were intact or disintegrated. This phenomenon was strictly linked with the presence of methylester groups.

Randomized phase II study comparing topotecan/cisplatin administration for 5 days versus 3 days in the treatment of extensive stage small cell lung cancer (SCLC).

BACKGROUND: Topotecan (T) is an active drug in SCLC. A combination of topotecan with cisplatin (DDP) was suggested to be highly synergistic. This phase II trial was initiated to assess the activity of T/DDP in chemotherapy-naive patients suffering from extensive disease small cell lung cancer (SCLC) and to compare the conventional 5-day regime with an experimental 3-day schedule. PATIENTS AND METHODS: A total of 86 patients were included. Patients were randomized to receive either T 1.0 mg/m2 d 1-5 and DDP 75 mg/m2 d 5 (arm A) or T 1.5 mg/m2 d 1-3 and DDP 75 mg/m2 d 3 (arm B). Six cycles were given at a 3-week interval. RESULTS: Data of 84 evaluable patients (67 males and 17 females) were analysed. All patients had metastatic disease. The best response rate was 61.9% in arm A and 59.5% in arm B. Median overall survival was 8.7 months in arm A and 7.6 months in arm B (p=0.6809). CONCLUSIONS: Combination of T and DDP is active in ED SCLC. Toxicity and median survival were comparable in both arms. Three days treatment seems to be similar to the 5 days regime.

Colonic spreading of a non-chlorofluorocarbon mesalazine rectal foam enema in patients with quiescent ulcerative colitis.

BACKGROUND: Rectal foam enemas provide for drug delivery to the distal colon for treatment of left sided ulcerative colitis. However, currently available formulations contain chlorofluorocarbons which are due to be phased out in the near future. The objective of this study was therefore to determine the degree of dispersion of a newly developed non-chlorofluorocarbon rectal foam preparation in ulcerative colitis patients. METHODS: This was an open label non-controlled study of a single administration of a mesalazine foam enema (two actuations containing 2 g of mesalazine in approximately 120 mL foam) in 10 patients with quiescent ulcerative colitis. Spreading of the 99mTc-labelled foam enema was assessed over a 4-h period by the non-invasive technique of gamma scintigraphy. RESULTS: All patients retained the enema for the full 4-h imaging period. In nine out of the 10 patients, the enema was observed to spread as far as the descending colon and on average 23% of the dose was present in the descending colon at 4 h post-dose. CONCLUSIONS: The extent of spreading observed in the study supports the use of the formulation in the treatment of left sided ulcerative colitis.

Subnuclear distribution of DNA topoisomerase I and Bax protein in normal and xeroderma pigmentosum fibroblasts after irradiation with UV light and gamma rays or treatment with topotecan.

Immunohistochemical methods were used to determine abundance and subnuclear distribution of DNA topoisomerase I and the Bax protein in normal and excision-repair-deficient xeroderma pigmentosum (XP) fibroblasts after irradiation of cells with gamma rays or UV light, or exposure to the topoisomerase I inhibitor topotecan. DNA topoisomerase I and Bax were monitored using antisera raised against the human proteins. In addition, topoisomerases IIalpha and IIbeta were made visible with specific antibodies. In untreated cells, DNA topoisomerase I was found to occur in the cytoplasm and in nucleoli. Irradiation with gamma rays (2-12 Gy) or UV light (0.3-1.2 mW/cm2) changed the staining pattern in nuclei such that a multitude of small topoisomerase-I-rich centers occurred, which were evenly distributed over the karyoplasm. Simultaneously nucleoli disintegrated. Treatment of fibroblasts with topotecan (6-100 microM concentrations) resulted in similar alterations although the changes were much more pronounced. Combinations of topotecan and gamma irradiation caused additive effects. We conclude that the increase in the number of topoisomerase-I-positive spots and the high fluorescence intensity of the latter may reflect three biological processes: (i) enhanced transcriptional activity (e.g. of DNA damage response genes), (ii) tagging of damaged DNA sites for repair, or (iii) initiation of apoptosis. In separate assays using normal and XP cells, a dose-dependent increase in protein reacting with Bax antibody was observed in nuclei, following treatment with gamma rays or topotecan. In addition, topotecan induced a netlike arrangement of this Bax protein in nuclei. The meshes of the net structure resembled vesicles. DNA staining with 4',6-diamidino-2-phenylindole dihydrochloride revealed that the vesicle-type structures contained DNA. Upon further incubation with topotecan, cells showing the netlike Bax arrangement eventually died. We conclude that topotecan-induced changes made visible by nuclear Bax protein are associated with apoptosis. XP cells, when treated with topotecan, responded more readily than normal cells with both an increase in nuclear Bax protein and rearrangement of Bax, indicating that UV repair functions may be required to process DNA damage inflicted by topotecan. Monitoring of DNA topoisomerases IIalpha and IIbeta in gamma-irradiated cells with antibodies revealed a dramatic increase in the IIalpha form and a redistribution of the IIbeta form representing fragmentation of nucleoli.

In vivo induction of neoplastic growth in nude mouse connective tissue adjacent to xenografted human tumors.

Induction of neoplastic growth of murine stroma cells within the human tumor xenograft was observed after serial passage of CEA and beta 2-microglobulin producing human colonic SLu tumor xenografts in nu/nu BALB/c mice. Mouse tumors within the human tumor xenografts were identified using specific immunohistologic staining techniques for mouse histocompatibility marker or human CEA. These mixed tumors could be distinguished from normal human tumor xenografts by a different relationship between development of the tumor marker in the serum and tumor size. We were able to establish transformed murine cells from human xenografts, either induced by SC injection of 1 X 10(6) tumor cells of the SLu cell line or by human SLu or mammary carcinoma tissue serially passaged in athymic animals. The established human and murine cell lines were characterized by cytogenetic methods. Transformed murine cells were then continuously passaged in tissue culture. The transformed mouse fibroblasts proved to possess tumorigenicity in nude mice. In the case of SLu-derived mouse tumor cells, tumors also developed in the immunocompetent BALB/c mice using 1 X 10(6) to 5 X 10(6) tumor cells for SC transplantation.

Investigations of droloxifene and other hormone manipulations on N-nitrosomethylurea-induced rat mammary tumours. 1. Influence on tumour growth.

The effect of droloxifene, a new anti-oestrogenic drug, on N-nitrosomethylurea-induced mammary tumours of Sprague-Dawley rats was investigated and compared with that of tamoxifen. The response of tumour growth to ovariectomy or to treatment with aminoglutethimide or high doses of oestradiol was also studied. Ovariectomy was by far the most effective treatment for mammary-tumour-bearing animals. More than 75% of the tumours in ovariectomized rats did not grow progressively but remained in remission for up to 12 weeks after castration when the experiment was terminated. The inhibitory effects of droloxifene and tamoxifen on mammary tumour growth were similar, but body weight loss of animals treated with tamoxifen was more marked than that of animals treated with droloxifene at the same dose and schedule.

Investigations of droloxifene and other hormonal manipulations on N-nitrosomethylurea-induced rat mammary tumours. 2. Influence on oestrogen receptor.

In N-nitrosomethylurea-induced rat mammary tumours, tamoxifen is found to compete at the binding sites of the oestradiol receptor if a receptor determination is performed 1 day following the last drug application to animals. Despite a higher binding affinity of droloxifene (3-OH-tamoxifen) to oestradiol receptor, compared to tamoxifen, its influence on the measurable receptor quantity is only very weak or not demonstrable. Therefore, binding affinity is not a valid explanation for the different influences of the two anti-oestrogens on the receptor. These only can be attributed to different behaviour patterns of both substances in relation to their half-lives and metabolism and accumulation in the organism. Owing to the short half-life of droloxifene, even 1 day after the last application too little drug is available to compete for oestradiol binding sites. In the case of both anti-oestrogenic substances, cessation of drug application for 8 weeks abolished any influence on the oestradiol receptor. Furthermore, failure of aminoglutethimide to influence the oestradiol receptor could be observed because this substance does not act via this receptor. The experiments performed confirm literature data regarding the effect of aminoglutethimide therapy on oestradiol receptors in breast tumour tissue of human beings. In summary: receptor investigations of N-nitrosomethylurea-induced rat mammary tumours, used as a model to test therapy regimens with droloxifene or other drugs with a short half-life, may be of limited value only.

Is serum beta 2-microglobulin a tumor marker in gastrointestinal cancer?

Serial determinations of serum beta 2-microglobulin (beta 2m) and carcinoembryogenic antigen (CEA) were performed in 314 patients with histologically confirmed gastrointestinal cancer. The data were correlated with a set of clinical parameters. Pre-operative serum beta 2m levels did not discriminate different classes of tumor extension nor different stages of resectability of tumors in contrast to CEA. During post-operative surveillance the correlation of the time courses of serum beta 2m and CEA with the clinical course of malignant disease was studied in a selected group of 165 patients with resected primary carcinoma of the gastrointestinal tract. During the follow-up 74/165 patients showed disease progression or recurrence. In the beta 2m follow-up 66% false negative indications (49/74) of malignant disease were observed, whereas in the CEA follow-up it was 5% (4/74). The ratio of correct positive/false positive indications was 25/10 in the beta 2m follow-up and 70/10 in the CEA follow-up. The data indicate that the formation of serum beta 2m is not directly tumor associated in gastrointestinal cancer.

Eighty-four potential second-look operations based on sequential carcinoembryonic antigen determinations and clinical investigations in patients with recurrent gastrointestinal cancer.

In our study of patients with resected primary gastrointestinal cancer, slope analysis of the post-operatively increasing carcinoembryonic antigen time courses signaled relapse in about 80 percent of the patients up to 12 months before positive clinical diagnosis. In 29 patients, clinical confirmation of the relapse could be obtained only after second-look surgery. Slope analysis generally differentiated localized from metastatic disease and therefore also predicted the site of relapse. A first evaluation of 84 patients with potential cases of second-look operations provided evidence for a significant increase in survival. Recently, the evaluation of individual carcinoembryonic antigen doubling times was used to derive an individual prognosis since doubling times strongly correlated with the survival of untreated patients. On this basis, it was clearly possible to show the benefit of second-look operation, since patients with resectable recurrences exhibited longer survival times compared with patients with similar carcinoembryonic antigen doubling times without treatment. Moreover, the introduction of monoclonal antibodies with increased specificity for malignant states, has facilitated the selection of patients for second-look operation because unspecific carcinoembryonic antigen elevations are less frequent and recurrent disease can be predicted more reliably due to the higher carcinoembryonic antigen increments associated with malignant growth.

[Granisetron, a antiemetic for treatment of cytostatic drug-induced vomiting: results of a practice-oriented study]. / Granisetron, ein Antiemetikum für die Behandlung der Zytostatika-induzierten Emesis: Ergebnisse einer praxisorientierten Studie.

The present multicentre Austrian investigation of the prophylactic intravenous administration of granisetron, a serotonin antagonist, routinely for control of cytostatic-induced nausea and emesis was carried out in 102 patients with cancer of various types undergoing different emetogenic cytostatic regimens (232 cycles of chemotherapy). A major therapeutic response, i.e. maximally one vomit over the first 24 hours, was achieved in 78-90% of patients undergoing a single or multiple day regimen of chemotherapy. Delayed emesis, experienced between day 1 and day 4 after chemotherapy, was observed in < 5% of the patients. However, particularly in single day regimens 25% of the patients showed only a moderate response to granisetron in suppressing delayed emesis. Tachyphylaxis to granisetron therapy was not observed in the first 3 consecutive cycles of chemotherapy. The individual global efficacy of emesis control by granisetron (day of chemotherapy over all cycles plus the following 7 days) was very good. An excellent therapeutic response was seen in 53-55% of all cases. The study also demonstrated the economic advantages of granisetron therapy. In the majority of patients (88/102) only a single dose of granisetron (3 mg) was required. The tolerability was also very good. The main adverse events reported were headache (7.8%) and constipation (4.9%).

Circulating carcinoembryonic antigen (CEA), a growth parameter in malignant disease.

In a series of experiments we measured tumor volume or tumor mass together with the serum CEA concentration in human adenocarcinoma-bearing nude mice. We observed a linear relationship between log CEA and time over a period of up to 30 days after transplantation of the tumors. In all experiments tumor mass-volume correlated well with concomitant increase of the circulating CEA concentration. A marked dissociation became evident when tumor growth rate slowed down. At that time histologic investigations indicated that necrosis of the tumor could be a potential factor for this phenomenon. A retrospective analysis of the CEA time courses in 114 patients with recurrent colorectal cancer gave evidence for similar CEA developments in 87 cases also exhibiting linear log CEA development with time. Individual CEA doubling times calculated from linear log CEA increases correlated well with observed survival in 36 patients with untreated visceral metastasis. Moreover, the CEA doubling time represented an objective parameter to rate the effect of various postoperative therapies when survival is expressed in multiples of individual CEA doubling times.

Growth of human colonic adenocarcinoma and development of serum CEA in in athymic mice. I: Strict correlation of tumour size and mass with serum CEA concentration during logarithmic growth.

The secretion of CEA into the blood of athymic mice was studied with 4 sublines of human colonic adenocarcinoma cell lines, HT 29 and SLu. Growth curves based on tumour volume (caliper measurements) or tumour mass (weight) correlated with a concomitant increase of serum CEA during the logarithmic growth phase, but showed a marked dissociation when the growth rate slowed down. In the logarithmic growth phase doubling times between 2 and 6 days were calculated and about 6-7 doubling times passed until the shift in the growth rate was observed, independently of the sublines transplanted. Constant increases of CEA between 0.03 and 0.45 microgram/l serum per mm3 increase of tumour volume, depending on the sublines, were recorded during the logarithmic growth phase. Sublines releasing high amounts of CEA in vitro (cell culture) retained this characteristic in vivo. Correlation between tumour volume and tumour mass or serum CEA showed correlation coefficients of 0.820-0.977 during the logarithmic growth phase.

Immunotherapy of tumor-bearing mice with chemically modified tumor membrane fractions of two syngeneic tumor cell lines.

The effects of chemical modification on the immunogenicity of plasma membrane fractions of virus-induced and chemically induced tumor cell lines were tested in syngeneic tumor-bearing mice. The immunotherapeutic effects were dependent on (1) the chemical nature of the modifying reagent, which also determines the molecular site of modification in the membrane components, (2) the degree of modification, (3) the immunizing dose of modified membranes, and (4) the time schedule of immunization. Best results were obtained when the immunizing membrane samples were modified by methylation or acetylation using immunizing doses of membrane equivalents corresponding to 10(3)-10(4) cells. Up to 30% of the animals remained tumor-free and the observed survival of the animals was different from that of the control when immunotherapy was started five days after tumor transplantation. A delayed start of immunotherapy, or simultaneous immunization with tumor transplantation, or immunizations with nonmodified membrane samples led to reduced observed survival. The therapeutic effects were specific for each individual cell line. Cross-protection was not observed when mice bearing chemically induced tumors were treated with modified membrane samples derived from a syngeneic virus-induced tumor cell line.

A new reliable method for evaluation of foot pad swelling as an experimental in vivo immune phenomenon.

Weight determination of replicas of the feet from living mice is described as a new method for evaluating the degree of the experimentally induced foot pad swelling in immunized mice. The new method gives objective, well documentable results and offers advantage to the commonly used assay with the dial calliper gauge when there is need for a good documentation or whenever there is a large number of animals to be tested within a narrow time margin.

Preferential induction of cell-mediated immunity by chemically modified carcinoembryonic antigen.

Modification of carcinoembryonic antigen (CEA) with various chemicals was investigated. Modification of CEA with dimethylsulfate or acetic anhydride resulted in derivatives which preferentially induced delayed-type hypersensitivity (DTH) against native CEA in mice. The strength of the DTH reaction was dependent on the number and chemical nature of modifying groups as well as on the immunizing dose. The strongest DTH reaction without detectable formation of antibodies was achieved by low dose immunization using heavily methylated CEA.

Immunogenicity of tumour cells modified with various chemicals.

Mouse tumour cells were treated with various chemical modifiers. The number of modifying groups per cell was determined with labelled reagents. The effects of the different modifying groups on the immunogenicity of the tumour cells was tested in syngeneic mice for tumour protection using a challenge dose of viable cells at 1000 or 10,000 time LD100. Best protection was obtained after immunization of animals with tumour cells modified with dimethylsulphate or acetic anhydride, or with glutardialdehyde-fixed cells treated with a carbodiimide and methylamine. Up to 40% of the animals remained tumour-free. The other animals exhibited a greatly increased mean survival time. The post-challenge sera showed no detectable amounts of antibodies against the tumour cells.