Antitumor effect, cardiotoxicity, and nephrotoxicity of doxorubicin in the IgM solid immunocytoma-bearing LOU/M/WSL rat.

Antitumor activity, cardiotoxicity, and nephrotoxicity induced by doxorubicin were studied in LOU/M/WSL inbred rats each bearing a transplantable solid IgM immunocytoma. Animals with a tumor (diameter, 15.8 +/- 3.3 mm) were treated with iv injections of doxorubicin on 5 consecutive days, followed by 1 weekly injection for 7 weeks (dose range, 0.015-4.0 mg/kg body wt). Tumor regression was observed with 0.5 mg doxorubicin/kg. Complete disappearance of the tumor was induced with 1.0 mg doxorubicin/kg. Histologic evidence of cardiotoxicity scored as grade III was only observed at a dose of 1.0 mg doxorubicin/kg. Light microscopic evidence of renal damage was seen above a dose of 0.5 mg doxorubicin/kg, which resulted in albuminuria and very low serum albumin levels. In the group that received 1.0 mg doxorubicin/kg, the serum albumin level decreased from 33.6 +/- 4.1 to 1.5 +/- 0.5 g/liter. Ascites and hydrothorax were observed simultaneously. The same experiments were performed with non-tumor-bearing rats, in which no major differences were observed. In conclusion, antitumor activity, cardiotoxicity, and nephrotoxicity were studied simultaneously in the same LOU/M/WSL rat. Albuminuria due to renal damage led to extremely low serum albumin levels, so ascites and hydrothorax were not necessarily a consequence of the observed cardiomyopathy.

T-cell involvement in adoptive transfer of line 10 tumor immunity in strain 2 guinea pigs.

Several aspects of adoptive transfer of tumor immunity were studied in the line 10 hepatocarcinoma in the syngeneic Sewall-Wright strain 2 guinea pig. In particular, the need for cooperation between donor and recipient T-cells was investigated. Donor immune spleen cells remained immunologically capable of inducing tumor rejection for at least 160 days after adoptive transfer. Irradiated (1,000 rad) or mitomycin-treated immune spleen cells lacked tumor-rejection activity, which is indicative of the necessity for in vivo proliferation after adoptive transfer of immunity. Furthermore, adoptive transfer of tumor immunity was abrogated after treatment of the line 10 immune spleen cells with rabbit anti-guinea pig-thymocyte serum (ATS) plus complement. The role of recipient T-cells was investigated in strain 2 guinea pigs which were T-cell depleted by thymectomy, irradiation, and bone marrow reconstitution (T-XBM animals). Severe suppression of T-cell activity was present at 2 and 6 weeks after irradiation and bone marrow reconstitution. At 10 weeks nonspecific T-cell activity was partially restored. The induction of antigen-specific responses, measured by delayed-type hypersensitivity skin testing in vivo and antigenic stimulation in vitro, was suppressed at 2 weeks after irradiation and bone marrow reconstitution. Additional in vivo treatment of T-XBM animals with a rabbit ATS improved the T-cell depletion only moderately. Tumor growth and tumor rejection after adoptive transfer of immunity were equal in normal and T-cell-deprived recipient animals, thus indicating that recipient T-cells are not needed for tumor rejection after adoptive transfer of line 10 tumor immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-course study on doxorubicin-induced nephropathy and cardiomyopathy in male and female LOU/M/Wsl rats: lack of evidence for a causal relationship.

In a previous study on doxorubicin-induced cardiotoxicity in LOU/M/Wsl rats, severe nephropathy has been observed; therefore, the question was raised whether nephropathy adds to or even might be responsible for doxorubicin-induced cardiomyopathy in rats. For elucidation of this question, the temporal relationship between the onset of doxorubicin-induced cardiomyopathy and nephropathy was studied. In addition, examination was made of whether modifications of the treatment schedule could circumvent nephrotoxicity. Because preliminary studies had shown that female LOU/M/Wsl rats developed less doxorubicin-induced albuminuria, both male and female LOU/M/Wsl rats were treated with an iv dose of 1 mg doxorubicin/kg (body wt)/rat on five consecutive days and then weekly. Saline-treated animals served as controls. Albuminuria, serum albumin, and serum creatine levels were assessed weekly. For histologic examination, 5 male and 5 female rats were killed weekly. At day 14 and thereafter, doxorubicin-treated male rats showed albuminuria greater than or equal to 10 g/liter. Albuminuria of greater than or equal to 10 g/liter was not avoided by modifications of the treatment schedule. Female rats had on day 14 a urinary albumin level of 1.0-3.0 g/liter, yet reaching the level of greater than or equal to 10 g/liter at day 49. In male rats serum albumin levels decreased to levels below 10 g/liter (p less than .001 vs. finding for day 0); in contrast female rats maintained constant serum albumin levels till day 49. Serum creatine levels showed a tendency to rise, the values of male rats not being measured after day 28 due to hyperlipidemia; the levels of female rats increased from 37.8 +/- 3.0 mumol/liter to 53.7 +/- 2.5 mumol/liter on day 49 (P less than .001). At day 10 in male and female rats a grade 1-1.5 cardiomyopathy score, assessed according to the modified Billingham scoring system, was found, gradually increasing to grade 2.5-3 cardiomyopathy, both in males and females, on day 49. In male LOU/M rats the nephropathy developed steadily from day 14 and thereafter, whereas in females the rate of development of kidney damage was slower and at the end point of the study the severity of kidney lesions was less in comparison to that of the males. The onset of cardiomyopathy and nephropathy was simultaneous. It was concluded that cardiomyopathy observed in LOU/M rats is a phenomenon independent of nephropathy.

Immunotherapy by intralesional injection of BCG cell walls or live BCG in bovine ocular squamous cell carcinoma: a preliminary report.

Cows of the Dutch Frisian and Maas-Rijn-IJssel breed with histologically confirmed ocular squamous cell carcinoma showed complete regression of the primary tumor in 70 or 60% of the cases after intralesional injection of a BCG cell wall or live BCG vaccine, respectively. Recurrence of the tumor was observed in 57% of the animals treated with BCG cell walls and in 25% of the animals treated with live BCG vaccine. Spontaneous regression was seen in 20% of the untreated cows. In a second control group, radical surgery, the most successful treatment for primary stage I tumors in humans, resulted in a 90% cure. Influence of immunotherapy on metastases could not yet be fully evaluated. White blood cell counts were not changed after therapy. It was not possible to link a favorable response to BCG therapy with the intensity of the delayed type hypersensitivity (DTH) reaction to purified protein derivative of mycobacteriae (PPD) or the formation of antibodies to BCG as determined by a micro-enzyme-linked immunosorbent assay. However, in animals that showed tumor regression, the DTH reaction to PPD had a tendency to persist for a longer period of time. It was concluded that 1) block resection was the best method of treatment for this tumor, 2) a single intralesional injection of a BCG cell wall vaccine was as effective as live BCG vaccine in the induction of complete regression of the primary tumor, 3) in this preliminary study BCG cell wall vaccine was less effective than live BCG vaccine in the prevention of recurrence, and 4) this naturally occurring tumor model is well suited for the study of the influence of BCG immunotherapy in a primary stage I tumor.

Influence of lipid composition on the antitumor activity exerted by doxorubicin-containing liposomes in a rat solid tumor model.

The effect of changes in lipid composition on the antitumor activity of doxorubicin (DXR)-containing liposomes was studied in immunoglobulin solid immunocytoma-bearing Lou/M Wsl rats. Rats bearing a tumor with a diameter between 20 and 30 mm were treated i.v. with 2 mg/kg free DXR or different DXR-containing liposome types for 5 consecutive days followed by one injection more at day 11 after start of therapy. A similar pattern of tumor regression was observed for free DXR and DXR entrapped in "fluid" liposome types. However, DXR entrapped in "solid" liposome types expressed an antitumor activity which was significantly delayed; during the first 3 days after start of therapy solid DXR-containing liposomes were less effective in inducing antitumor activity than fluid DXR-containing liposomes. In order to gain more insight into the mode of action of DXR-containing liposomes, one of the solid liposome types [composed of distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and cholesterol (chol)] was compared with one of the fluid liposome types [composed of egg phosphatidylcholine, phosphatidylserine, and chol] with respect to distribution and integrity in vivo. Results obtained after i.v. administration of [3H]inulin-labeled vesicles to tumor-bearing animals suggested that a differential liposome uptake by the tumor was not relevant for the explanation of the delayed antitumor effect. To monitor the structural integrity of liposomes after i.v. injection, the liposomes were double radiolabeled with [3H]inulin as a marker of the aqueous phase and cholesteryl [14C]oleate as a marker of the lipid phase. The bilayer structure of both liposome types remained intact during their presence in the blood compartment. Intact liposomes were taken up primarily by liver and spleen with subsequent degradation of the liposome structure. The degradation rate appeared to be dependent on the lipid composition of the liposomal membranes; phosphatidylcholine/phosphatidylserine/chol liposomes were degraded much faster than distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/chol liposomes. The difference in degradation rate was manifested more clearly in the spleen than in the liver. In vitro investigations on uptake and processing of liposomes by liver macrophages indicated that the difference in degradation rate between liver and spleen was caused by intrahepatic reutilization of [14C]oleate liberated from the liposome structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Adoptive immunity to the guinea pig line 10 hepatoma and the nature of in vitro lymphoid-tumor cell interactions.

Adoptive transfer of spleen cells from specifically immunized donors to nonimmunized recipients was used to study tumor immunity in vivo to the syngeneic line 10 guinea pig hepatoma. Hepatoma cells cultured as monolayers on fibronectin-coated surfaces served as targets for immune splenocytes in a 3H release cytotoxicity assay in vitro. An antigenically distinct syngeneic guinea pig hepatoma (line 1) was used to study the specificity of adoptive systemic immunity and of the cytotoxicity in vitro. The protection afforded by adoptive immunization against challenge with hepatoma cells was tumor line specific, while in most cases cytotoxicity in vitro was not. The in vitro cytotoxic effect was abolished after absorption of the immune spleen cells with monolayers of either line 10 or line 1. In contrast, the in vivo tumor-specific rejection activity of line 10 immune spleen cells was depleted after absorption with line 10 but not with line 1 or other control monolayers. These studies revealed that the immune cells mediating cytotoxicity in vitro were functionally distinct from those conveying adoptive protection in vivo. Immune cells possessed receptors for tumor-specific rejection antigens on hepatoma cells, and their interaction did not lead to destruction of the neoplastic cells in vitro.

In vivo targeting of OV-TL 3 immunoliposomes to ascitic ovarian carcinoma cells (OVCAR-3) in athymic nude mice.

Specific binding of immunoliposomes to target tumor cells was investigated in a xenograft model (athymic nude mice) of i.p. growing human ovarian carcinoma (OVCAR-3). For the first time, quantitative evidence is presented that attachment of a tumor-specific antibody (OV-TL 3) dramatically enhances the association of liposomes with i.p. growing OVCAR-3 cells. The OV-TL 3-mediated binding of liposomes to the OVCAR-3 cells was rapid; 30 min after i.p. injection approximately 70% of the injected dose of OV-TL 3 immunoliposomes was associated with the OVCAR-3 cells while for unconjugated liposomes a value of only approximately 3% was obtained. At 2 h after injection, a maximal binding level of 84% was achieved in case of the OV-TL 3 immunoliposomes whereas the binding level of unconjugated liposomes was still about 3%. Twenty-four h after injection about 83% of the injected dose OV-TL 3 immunoliposomes still was associated with the OVCAR-3 cells, compared to about 10% of the injected dose of unconjugated liposomes. Accordingly, unconjugated liposomes disappeared from the peritoneal cavity much faster than the OV-TL 3 immunoliposomes. By comparison with immunoliposomes bearing irrelevant antibody, the specificity of the binding of the OV-TL 3 immunoliposomes to the OVCAR-3 cells was demonstrated. In addition, it was observed that the sustained high OV-TL 3 immunoliposome levels found in the peritoneal cavity are the result of both reduced particle clearance from the peritoneal cavity and the tenacious binding of the immunoliposomes to the tumor cells. Finally, data are presented showing that the degree of binding of OV-TL 3 immunoliposomes to OVCAR-3 cells in vitro and in vivo correlates positively with the antibody (Fab') density on the liposomes.

Antitumor activity, induction of cross-resistance, and nephrotoxicity of a new platinum analogue, cis-1,1-diaminomethylcyclohexaneplatinum(II) sulfate, and of cis-diamminedichloroplatinum(II) in an immunocytoma model in the LOU/M rat.

A newly synthesized platinum analogue, cis-1,1-diaminomethylcyclohexaneplatinum(II) sulfate (TNO-6), was compared with cis-diamminedichloroplatinum(II) (cis-DDP) for antitumor activity and nephrotoxicity. Antitumor activity was determined in an IgM immunocytoma model in the LOU/M rat. Tumor cells were inoculated on the left flank, and therapy was started when a tumor diameter of 10 to 30 mm was reached. At the start of the therapy, the primary tumor had already metastasized to the draining lymph node and liver. Both platinum compounds, dissolved in 5% glucose water, induced an almost complete tumor regression within 10 to 14 days (average, 84% tumor load reduction) and prolonged survival, compared to that of nontreated animals. The antitumor activity induced by repeated i.p. administration of cis-DDP and TNO-6 reached its maximum at a dose of 1.0 mg/kg body weight (twice a week for 7 weeks). This treatment regimen resulted in a highest tolerable dose for cis-DDP of 1.0 mg/kg and for TNO-6 of 2.0 mg/kg. However, when rats were treated with a 2.0-mg/kg dose of TNO-6, no increase in antitumor activity was obtained. For both platinum compounds, tumor recurrence occurred in almost all animals within 2 to 7 days after the maximum tumor load reduction. Tumors that recurred were found to be cross-resistant to both platinum compounds tested but were sensitive to treatment with doxorubicin (Adriamycin). With regard to toxicity, repeated administration of TNO-6 (1.0 mg/kg twice a week for 7 weeks) induced less decrease of body weight than did cis-DDP. For TNO-6, even in the highest dose investigated (2.0 mg/kg twice a week for 7 weeks), no nephrotoxicity was observed on histological examination of kidney and blood urea and creatinine values, whereas for cis-DDP nephrotoxicity was still present in the lowest dose investigated (0.5 mg/kg). From the comparison of the antitumor activity and nephrotoxicity of TNO-6 and cis-DDP, administered i.p. in 5% glucose solution, it is concluded that both drugs have comparable antitumor activity and potency. In contrast to the effects of cis-DDP, no nephrotoxicity was observed with TNO-6; thus, TNO-6 might be a good alternative to cis-DDP in avoiding nephrotoxicity during platinum therapy.

Reduced cardiotoxicity and nephrotoxicity with preservation of antitumor activity of doxorubicin entrapped in stable liposomes in the LOU/M Wsl rat.

LOU M/Wsl rats inoculated s.c. with 10(4) immunoglobulin immunocytoma cells have a palpable tumor at Day 17. Doxorubicin (DXR) has been entrapped in negatively charged liposomes (lip- DXR) composed of egg phosphatidylcholine, cholesterol, and phosphatidylserine and in positively charged liposomes (lip+ DXR) composed of phosphatidylcholine, cholesterol, and stearylamine. DXR, lip- DXR, and lip+ DXR were administered i.v. (0, 0.25, 0.5, 1.0, and 2.0 mg/kg) at Day 17 for 5 consecutive days and then weekly. Control animals showed progressive tumor growth leading to death 27 days after inoculation. Antitumor activity for all three preparations was dose dependent. DXR and lip- DXR showed the same antitumor activity; lip+ DXR had less antitumor activity. The overall survival of tumor-bearing animals treated with 2.0-mg/kg lip- DXR was significantly prolonged (p less than 0.05) in comparison to the animals treated with 2.0-mg/kg free DXR. Grade III cardiomyopathy was observed 47 days after treatment with free DXR; treatment with lip- DXR resulted in Grade I cardiomyopathy. In animals treated with 1.0-mg/kg and 2.0-mg/kg free DXR urinary albumin concentrations of 10 g/liter were observed. Treatment with 1.0-mg/kg lip- DXR and 1.0-mg/kg lip+ DXR resulted in urinary albumin concentration of less than 3.0 and less than 1.0 g/liter, respectively. Free DXR, 1.0 mg/kg, resulted in a decline of serum albumin concentration from 27.8 +/- 3.2 g/liter to 9.6 +/- 4.2 g/liter. No such decline was observed after treatment with lip- DXR and lip+ DXR. Treatment with a 1.0-mg/kg dose of free DXR resulted in severe glomerular and tubular lesions which were not found after treatment with 1.0-mg/kg lip- DXR and 1.0 mg/kg lip+ DXR. Administration of lip- DXR resulted in lower DXR levels in cardiac and renal tissue compared to administration of free DXR. After administration of lip+ DXR, very low tissue and tumor DXR levels were found. In conclusion, treatment with lip- DXR or lip+ DXR resulted in a prolonged survival, less albuminuria, and higher serum albumin levels. Also, fewer lesions in heart and kidney were found, correlating with lower DXR levels in these organs. Only lip- DXR had the same antitumor effect as free DXR.

Platinum concentrations and DNA adduct levels in tumors and organs of cisplatin-treated LOU/M rats inoculated with cisplatin-sensitive or -resistant immunoglobulin M immunocytoma.

Female LOU/M rats, bearing either a cisplatin (cisDDP)-sensitive or -resistant IgM immunocytoma, were sacrificed at 1 or 24 h after cisDDP administration (i.v., 10 mg/kg of body weight). Platinum levels, determined with atomic absorption spectroscopy, were in the order kidney much greater than liver greater than tumor greater than spleen in the 1-h samples. In the 24-h samples, more platinum was found in spleens than in tumors; the levels in the kidneys were the same as those measured at 1 h, in the spleens they were higher, and in livers and tumors they were lower than at 1 h after the injection; the greatest decrease occurred in the resistant tumor. cisDDP-DNA adducts were detected after chromatography of digested DNA samples isolated from these tissues and from blood cells. The quantitation of the four cisDDP-DNA adducts (Pt-G, Pt-AG, Pt-GG, G-Pt-G, the same as found previously in cisDDP-reacted DNA) was performed with specific antibodies, in the competitive enzyme-linked immunosorbent assay. The cisDDP-DNA adduct levels in the various 1-h tissue samples showed the same ranking order as the platinum levels. The blood samples contained the lowest amount of adducts. Because of the high platinum level in the kidneys (26 mg/kg of wet tissue), the adducts in this organ also could be determined with atomic absorption spectroscopy (the four adducts comprised about 400 fmol/micrograms of DNA). Comparison of the atomic absorption spectroscopy and enzyme-linked immunosorbent assay data showed excellent agreement. Except for the kidney, all samples showed a decrease in adduct level between 1 and 24 h after cisDDP treatment. The data on the tumors indicated that the difference in susceptibility to cisDDP between the sensitive and resistant tumors is not due to a decreased platinum content or reduced DNA adduct formation in the resistant tumor.

Release of doxorubicin from peritoneal macrophages exposed in vivo to doxorubicin-containing liposomes.

Intracellular depot formation may be an important component of the mode of action of doxorubicin (DXR)-containing liposomes. In this paper it was investigated whether it is possible that DXR is released from macrophages which have taken up DXR-containing liposomes in vivo. Macrophages were harvested from the peritoneal cavity of LOU/M rats after i.p. administration of DXR-liposomes. Two different liposome types were used for this investigation. The amount of DXR associated with macrophages was determined by high performance liquid chromatography. In order to monitor DXR release from the macrophages, an in vitro tumor cell growth inhibition assay was applied. Peritoneal macrophages collected 24 h after an i.p. injection of DXR-liposomes (10 mg/kg body weight) caused considerable growth inhibition of tumor cells in culture. The cytostatic potential of macrophage monolayers in vitro depended on the type of injected DXR-liposomes and was directly related to the amount of macrophage-associated DXR. The DXR content of the macrophage monolayers was completely released from the cells into the culture medium during the cocultivation with tumor cells. Supernatants obtained from monolayers, which were cultivated in the absence of tumor cells, showed a high growth-inhibitory activity. DXR either free or in liposomal form was chemically stable for up to 26 h during incubation with lysosomal fractions isolated from rat liver homogenates. The results indicate that release of DXR from macrophages which have phagocytosed DXR-liposomes in vivo is a real possibility.

Design of immunoliposomes directed against human ovarian carcinoma.

Factors (protein/lipid ratio, pH of incubation medium, incubation time, anchor molecule density in the bilayer) affecting the covalent binding of anti-ovarian carcinoma Fab' to liposomes containing the anchor molecule MPB-PE (N-(4-(p-maleimidophenyl)butyryl)phosphatidylethanolamine) were explored. Standard experimental conditions were chosen and information on the relevant physicochemical parameters of the liposome dispersions was collected (mean particle diameter, size distribution, charge). The reproducibility of standard immunoliposomes prepared in subsequent batches in terms of Fab' binding, particle size and charge was established. In addition, preservation of immunoreactivity, no marker loss, and no aggregation/fusion was found for the standard immunoliposomes over a period of at least 3 weeks at 4 degrees C. In vitro up to 35,000 immunoliposomes were estimated to bind per human ovarian carcinoma cell. Internalization of the immunoliposomes could not be demonstrated. Electron micrographs showed binding of specific immunoliposomes to human ovarian carcinoma cells growing intraperitoneally in athymic nude mice.

Targeted delivery of diphtheria toxin via immunoliposomes: efficient antitumor activity in the presence of inactivating anti-diphtheria toxin antibodies.

Diphtheria toxin (DT) has attracted considerable attention for anti-cancer therapy. However, its extensive use is prohibited by (i) its non-specific action which can result in substantial toxicity, (ii) most patients have low serum levels of anti-DT antibodies (AT antibodies) which can inactivate DT and (iii) its immunogenicity will boost the circulating AT antibody level, thereby further compromising the antitumor activity. To overcome these limitations, we have developed a new approach for targeted delivery of DT utilizing immunoliposomes. In this approach, protection against the non-specific action of DT is combined with efficient antitumor activity even in the presence of inactivating AT antibodies.

Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma.

We investigated correlations between ozone-induced increases in inflammatory markers in induced sputum and in bronchial lavage fluid. Sixteen volunteers with intermittent asthma participated in a placebo-controlled parallel study with two exposures. Six days before and 16 h after the first exposure to ozone (0.4 ppm during 2 h) sputum was induced with hypertonic saline. This resulted in a significant increase in the sputum levels of eosinophil cationic protein (ECP; 1.8-fold; p = .03), neutrophil elastase (5.0-fold; p = .005) and the total cell number (1.6-fold; p = .02). After 4 weeks, a second exposure was randomized for air or ozone. Six days before and 16 h after the second exposure a bronchial lavage was performed. ECP values in sputum and in bronchial lavage fluid obtained after ozone correlated significantly (Rs = .79; p = .04), as did interleukin-8 (IL-8) values (Rs = .86; p = .01), and the percentage eosinophils (Rs = .89; p = .007). Moreover, the ozone-induced changes in percentage eosinophils observed in sputum and lavage fluid were highly correlated (Rs = .93; p = .003). In conclusion, changes in eosinophils, IL-8, and ECP markers induced by ozone and measured in sputum reflect the inflammatory responses in the lower airways of asthmatics, and may provide a noninvasive tool in epidemiologic studies on air pollution and asthma.

Long-term adjuvant immunotherapy in stage I high risk malignant melanoma, comparing two BCG preparations versus non-treatment in a randomised multicentre study (EORTC Protocol 18781).

The present study reports the results of a multicentre adjuvant trial with BCG (Bacillus Calmette-Guérin) in high risk patients (Breslow thickness > or = 1.5 mm, Clark level > or = III) with malignant melanoma, after surgical removal of their primary tumour. The trial was specifically designed in order to resolve the controversy and to provide some definite answers regarding the value of adjuvant BCG treatment in stage I malignant melanoma. Patients were randomised to either BCG RIV (108 patients) or BCG Pasteur (109 patients) for 3 years or to follow-up only (110 patients). The two vaccines used had greatly divergent properties regarding their mode of preparation, their composition and their immunomodulating activities. Of the 353 randomised patients, 23 were ineligible, 3 refused participation after randomisation and 327 were evaluable for final analysis. Median follow-up time was 6 years (range 0-10 years). The log-rank test comparison showed no statistical difference between the three arms regarding time to progression (P = 0.55) and duration of survival (P = 0.82). Treatment was generally well tolerated, with no major adverse events in either treatment arm. These findings confirm data with different BCG preparations and with stage II melanoma which also demonstrated no benefit regarding patient survival and time to relapse.

Reliability of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Trichinella spiralis infections in conventionally raised pigs.

An enzyme immunoassay with horse radish peroxidase as marker enzyme for detection of antibodies to Trichinella spiralis in pigs is described. In the enzyme-linked immunosorbent assay (ELISA) quantitation of specific antibodies is obtained by means of peroxidase labeled anti-species-immunoglobulin in antigen-coated tubes. The enzyme remaining in the tube after washing provides a measure of the amount of specific antibodies in the serum. A crude saline extract of T. spiralis muscle larvae served as antigen, 5 mug protein/ml being a satisfactory concentration. Lyophilization of antigen had no adverse effect on sensitivity. To decrease background staining the use of an optimal conjugate dilution was important. Adding bovine serum albumen to the conjugate was essential to decrease background reactions. Suitable substrate incubation times were studied. Washing was performed with tap water and Tween 20. In experiments with conventionally raised slaughter pigs infected with different numbers of T. spiralis larvae a positive correlation was found between initial dose of larvae and amount of antibodies detected by ELISA. Compared with immunofluorescence (IF) ELISA was more sensitive. IF yielded positive results in 11 out of 34 infected animals, whereas ELISA results were positive in 27. To evaluate ELISA under practical conditions extinction values of sera from infected and non-infected conventional pigs were compared with the highest extinction value in a group of 74 negative conventional pig sera. The relatively high background reaction of some of these negative sera decreased the number of positive practical ELISA results from 27 to 19 out of 34. In 1 out of 10 non-infected animals a false positive practical ELISA result was obtained.

Mechanization of the enzyme-linked immunosorbent assay (ELISA) for large scale screening of sera.

An on-line routing system (including dispensers, washing device, spectrophotometer, carts, elevators and identification device) for macro-ELISA is described. The system enables processing of 4,000 sera daily by 2 persons. Test results are presented on a data sheet as a combination of identification number and extinction value.

A helper T-cell epitope of the E7 protein of human papillomavirus type 16 in BALB/c mice.

The helper T-cell response to the E7 protein of human papillomavirus type 16 (HPV16) was studied using BALB/c (H-2d) mice. Twenty-two overlapping synthetic peptides spanning the HPV16 E7 protein were split into 6 groups. Mice were sensitized using mixtures of synthetic peptides corresponding to each of the groups. Lymph node cell suspensions were cultured with the corresponding mixture of synthetic peptides that was used for sensitization. Two mixtures induced a proliferative response. Analysis of the individual peptides from these mixtures showed that two (overlapping) peptides induced a proliferative response. This response was mediated by CD4+ cells. The common region of the two peptides was found to be a single epitope, and a minimal epitope was demonstrated (AHYNIVTFCCK). In conclusion, in contrast to others, we demonstrated a helper T-cell response in BALB/c mice. This may be due to the fact that we used synthetic peptides as immunizing agent. The helper T-cell epitopes in HPV16 E7 demonstrated previously are partly overlapping with the (minimal) epitope demonstrated here, underlining the 'public' nature of the epitope.

The effect of oral quercetin on UVB-induced tumor growth and local immunosuppression in SKH-1.

The effects of quercetin (4%) on UVB-induced carcinogenesis and immunosuppression were studied in hairless SKH-1 mice exposed daily to suberythemal UVB for 12/13 and 16/17 weeks. Macroscopic and microscopic examinations showed that quercetin did not affect the onset and growth of UVB-induced non-melanoma skin tumors. Quercetin prevented the UV-induced suppression of the contact hypersensitivity (CHS) and the reduction of the percentage of CD8-positive cells in spleen and lymph nodes. Other immunological parameters were not affected. Thus, the results indicate that oral intake of a high dose of quercetin does not prevent UVB-induced carcinogenesis, although it restores the skin-associated CHS response.

Application of immunofluorescence and immunoenzyme methods in the serodiagnosis of Trichinella spiralis infection.

To detect antibodies to T. Spiralis in sera, the IF methods with the cuticle of T. spiralis larvae (the tube test) was compared to the cryostat method. In the latter method, cryostat sections were prepared from isolated T. spiralis larvae or from tongue or diaphragm musculature in which encysted T. spiralis larvae were present. In this case, both cuticle and internal structures were employed as antigenic sites. The cryostat method proved to be more sensitive than the tube test. With the cryostat method, specific antibodies were detected in sera of experimentally infected mice 14 days after infection, whereas with the tube test, antibodies were detected on Day 24 postinfection and consistently thereafter. The enzyme-linked immunosorbent assay (ELISA) was then studied. Quantitation of specific antibodies was achieved with alkaline phosphatase- or peroxidase-labeled antispecies immunoglobulin in antigen-coated tubes. The enzyme that remained in the tube after washing provided a measure of the amount of specific antibodies in the serum. A saline extract of T. spiralis larvae served as the antigen. In the experimental models studied (T. spiralis-infected rabbits and pigs), ELISA proved to be more sensitive than IF. At Day 3 postinfection and thereafter, specific antibodies could be detected. ELISA was modified to satisfy requirements for routine application.