Eosinophilia is primarily a T-cell-dependent phenomenon, related to helminthic infections. Using a rat model (+/rnu), it has been shown that keyhole limpet hemocyanin (KLH) in complete Freund’s adjuvant (CFA) induced blood eosinophilia, but only after pretreatment with cyclophosphamide (CY). CY alone caused an eosinophilopenia which was reversed by CFA or KLH-CFA treatment. Eosinophilia was also induced in congenitally athymic rnu/rnu rats after CY pretreatment and KLH-CFA stimulation, the response being of a similar order as that in +/rnu animals. It is concluded that a non-parasite antigen can induce blood eosinophilia after pretreatment with CY, the response being thymus-independent. This model may be appropriate to study the biological significance of the eosinophil response.

Verschillende karakteristieken van BCG bestemd voor immunotherapie bij de behandeling van kanker werden onderzocht. Dit rapport beschrijft de resultaten verkregen van BCG-RIV lot no.603. In dit rapport wordt de methode van bereiding beschreven alsmede het aantal kweekbare deeltjes, het drooggewicht, metabole activiteit, steriliteit, virulentie, stabiliteit en resultaten van veiligheids-onderzoek in diermodellen. Tevens werd de immunostimulerende activiteit en de anti-tumor werking bepaald. Geconcludeerd kon worden dat deze BCG-batch niet afwijkt ten opzichte van voorafgaande BCG batches.

Naakte ratten kunnen, in tegenstelling tot hun thymus-houdende controles, niet goed geimmuniseerd worden tegen Listeria monocytogenes. De fagocytotische activiteit van macrofagen in naakte ratten is wel intact, en zelfs hoger dan in controles. Dit lijkt op te gaan zowel voor systemische als pulmonale weerstand tegen deze infectie. Dit diermodel is hierdoor geschikt voor het onderzoek naar onderliggende mechanismen van effecten van oxiderende gassen op de afweer tegen pulmonale infecties: De effecten van expositie van thymus-houdende dan wel naakte ratten, al dan niet tegen Listeria monocytogenes geimmuniseerd, op de uiteindelijk gemeten weerstand, zal een belangrijke indicatie geven waar uiteindelijk oxiderende gassen op het afweer mechanisme zullen ingrijpen.

In this study, the distribution of immune spleen cells was investigated after adoptive transfer of immunity in inbred strain 2 guinea pigs. Spleen cells obtained from line 10 immune donor animals became specifically restimulated in vitro with 3 M KCl-extracted line 10 soluble proteins, but not with 3 M KCl-extracted line 1 or liver proteins. After 4 days culture in vitro, these specifically restimulated immune spleen cells retained their antitumor activity in vivo after adoptive transfer. The specifically restimulated immune spleen cells were radiolabeled with [3H]thymidine, 1 X 10(8) viable cells were adoptively transferred in tumor-bearing guinea pigs, and their distribution was investigated. As controls for the specific localization of the immune cells at the line 10 tumor, the presence of labeled cells was studied in the contralateral transplanted line 1 hepatoma as well as in cellular inflammatory reactions elicited by injection with incomplete Freund's adjuvant (IFA) and complete Freund's adjuvant (CFA). A significantly higher localization of the labeled immune spleen cells in the line 10 tumor and the first and second draining lymph nodes of the line 10 challenge site were found when compared to the influx of these cells in the line 1 tumor and the nontumor antigen-related inflammatory reactions. Because our immune donor animals were immunized with a mixture of line 10 cells and BCG, these animals are immune to both. Line 10 immune spleen cells were restimulated in vitro with PPD and were radiolabeled. These PPD-restimulated immune spleen cells showed no preferential localization at the line 10 tumor challenge site but, as expected, a tendency for localization at the CFA (H37Ra) injection site. Furthermore, PPD-reactive spleen cells from BCG-immunized guinea pigs showed a significantly higher accumulation at the CFA injection site compared to the IFA injection site and the line 10 and line 1 tumor challenge site. From the results, it is concluded that line 10 tumor-immune and BCG-immune spleen cells are two distinct cell populations, and that the existence of cross-reacting antigens between BCG and the line 10 hepatocarcinoma are of no importance for the rejection of the line 10 tumor by immune spleen cells.

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)

To evaluate the functional significance of triphenyltin hydroxide (TPTH)-induced lymphopenia and lymphocyte depletion in thymus-dependent areas of spleen and lymph nodes, various immune function studies were carried out after 3 or 4 weeks TPTH exposure. Weaned male rats were fed a diet containing 25 mg TPTH/kg, a concentration that did not influence food intake and weight gain. TBTO exposure was continued during the course of the function tests. As parameters of the cell-mediated immunity in 2 experiments the delayed-type hypersensitivity reactions to ovalbumin and tuberculin were significantly suppressed. No effect was observed on allograft rejection, splenic clearance of Listeria monocytogenes at days 5 and 6 after infection, and responsiveness of thymocytes to different T-cell mitogens. In contrast, the response of splenic lymphocytes to the T-cell mitogen phytohaemagglutinin was significantly suppressed. As TPTH treatment reduced the number of spleen cells, mitogenic response calculated per whole spleen was significantly depressed. Regarding the humoral immunity, no effect was observed on serum IgM and IgG levels, on the thymus-independent IgM response to E. coli lipopolysaccharide (LPS), and on the primary and secondary IgM and IgG response to the thymus-dependent antigen tetanus toxoid. Also, no effect was found on phagocytic and killing capacity of macrophages as demonstrated by unaltered splenic clearance of L. monocytogenes at days 1 and 2 after infection. Slightly enhanced mortality of TPTH-treated animals was observed in a L. monocytogenes mortality assay. Finally, TPTH did not increase the susceptibility of rats to endotoxin (LPS).

Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models.

Immunity to Trichinella spiralis infection in laboratory rodents has been regarded as thymus (T)-dependent. Both expulsion of adult T. spiralis worms from the intestinal tract and the number of muscle larvae in the striated musculature have been described as being under the control of T-cell-mediated immunity (Gore et al., 1970, J. Parasitol. 56: 122; Ruitenberg and Steerenberg, 1974, J. Parasitol. 60: 1056-1057; Ruitenberg et al., 1977, Immunology 33: 581587). The same holds true for serum antibody production (Ruitenberg et al., 1977, loc. cit.). Also the inflammatory reaction around the encysted muscle larvae was found to be absent in T-cell-depleted mice (Walls et al., 1973, Clin. Exp. Immunol. 13: 231-242). However, our studies in congenitally athymic (nude) mice of B 1 OLP origin showed both a minor cellular reaction, including IgA containing plasma cells and eosinophils in the small intestine, and a minor local increase of mononuclear cells, mast cells and eosinophils in the striated musculature around encysted larvae (Gustowska et al., 1980, Parasite Immunol. 2: 133-154). Because these athymic (nude) mice lack functional T-cells we postulated that the described reactions, being minor as compared to those observed in euthymic mice, were T-independent. Because we had no evidence for any functional significance of these observations we studied the possible protective effect of a primary oral T. spiralis infection upon a subsequent challenge in this strain of athymic mice. For the experiment we used male SPF B 1 OLP nu/nu mice, approximately 9 wk of age at day 0 of the experiment, obtained from the Central Institute for the Breeding of Laboratory Animals T.N.O., Zeist, The Netherlands, where these mice are produced by continual back crossing with strain B10 OLP. During the experiment the animals were housed in a laminar flow cabinet. The T. spiralis strain used was isolated in 1960 from a pig in Poland and since maintained in our Institute in Wistar rats and from 1972 onwards in Swiss mice. Larvae used for oral infection were obtained from mice infected for 7 wk by artificial digestion for 2 hr (K6hler and Ruitenberg, 1974, Bull. WHO Org. 50: 413-419). To prevent crowding of adult worms, the intestinal phase of the T. spiralis infection was abbreviated with an aqueous solution of 90% v/v of 2-(beta-methoxyethyl) pyridine (Promintic?, Laboratoire Roger Bellon, Neuilly-Seine, France) which was subcutaneously administered at one dose of 5 mg per mouse. The effective dose for killing adult T. spiralis worms is known to be 6 mg, but at this dose our nude mice died within 3 hr after administration. Administering 5 mg per mouse resulted in survival of all treated mice, except one; however, not all adult worms were killed. The time interval of 28 days was chosen on the basis of the kinetics of the IgA cellular reaction in the gut of the athymic mice, found previously (Gustowska et al., 1980, loc. cit.). At the end of the experiment the numbers of adult worms and of muscle larvae were determined. The small intestine was slit lengthwise, rinsed in tap water, and cut into pieces of about 1 cm, which were placed in a Baermann funnel with physiological saline at 37 C for 4 hr. Counting was performed at a magnification of X25. The carcass was cut into small pieces and digested in tap water with 1% HC1 and 1% pepsin (E. Merck, Darmstadt, W. Germany) at 37 C overnight. Counting was performed by dilution and at a magnification of X25. Student's t-test was used to calculate twosided significances of differences between the groups. The experimental design was as follows: the primary infection was given at day 0, abbreviated at day 28 by drug treatment, and parasitological examinations were performed at day 35. The challenge was given at day 35, and parasitological examinations were performed at day 70 (Table I). The results, summarized in Table II, are as follows. Promintic treatment abbreviated ade-

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.

The anti-parasite response was investigated after oral infection of athymic nude (rnu/rnu) rats and heterozygous (+/rnu) littermates with 1000 muscle larvae of Trichinella spiralis. No IgM, IgG and IgE antibodies were detected in serum of rnu/rnu rats. Expulsion of adult worms from the small intestine was prolonged (worms were nearly all expelled at days 14 and 91 in +/rnu and rnu/rnu rats respectively). The yield of muscle larvae in the carcasses of nude rats at day 91 was 33 times higher than in +/rnu rats. In contrast to the strong inflammatory reaction in the parasitized tongue of +/rnu rats, no infiltration was observed in rnu/rnu rats. Using an immunoperoxide method with monoclonal anti-rat T-cell antibody, no T cells were identified in spleen, mesenteric lymph node and Peyer's patches. These data support earlier studies that the nude rat lacks functional T cells. As the counts of connective tissue mast cells (CTMC), intestinal mast cells (IMC) and globule leucocytes (GL) in small intestine of uninfected rnu/rnu rats were equal or higher than in +/rnu rats, it is concluded that the origin of these cells is thymus-independent. In contrast to +/rnu rats, infection of rnu/rnu rats induced no increase of CTMC, IMC or GL. Thus, these cells depend on T cells to undergo proliferation. Finally, results of this study were inconclusive whether IMC are precursors for GL, or that they represent independent cell populations.

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.

MAST-CELL MEDIATORS, including histamine, exert a variety of pharmacological effects, which can be explained by assum- ing two kinds of receptors, designated Hi

Two BCG preparations produced according to different principles for use as an adjunct to surgery in a comparative clinical trial in melanoma patients have been examined for immunostimulating, toxic, and antitumor activity in various murine and guinea pig models and for immunostimulation in vitro of human lymphocytes. One BCG preparation was prepared in the classic way, i.e., grown as a surface culture and subsequently homogenized in a ball mill, while the other preparation was grown as a dispersed culture.

Four preparations of Bacillus Calmette-Guérin (BCG), cultured either homogeneously dispersed or as a surface pellicle, were compared with reference to their immunomodulating capacities and antitumor effects. The BCG preparations included two vaccines that originated from the same seed strain, but one had subsequently been produced in each way. The immunological assays included in vivo stimulation of lymph nodes in a mouse model, in vitro stimulation of murine spleen lymphocytes, and in vivo stimulation of macrophages in a Listeria monocytogenes clearance model in the mouse. The antitumor effect was determined in a non-immunogenic, non-metastasizing murine fibrosarcoma. The results indicated that vaccines produced as a homogeneous culture in general induced a higher lymphocyte stimulation both in vivo and in vitro. In the Listeria clearance model a markedly enhanced clearance was established with three of the four preparations, the phenomenon being related to the number of culturable particles administered. This difference was not attributed to the production method, but to other factors, including the actual composition of the vaccine.

Transplants of experimental keratoacanthomas induced in skin grafts which were in the growth phase of the hair follicle cycle (anagen phase) were carried out in immunocompetent and immunoincompetent receipients ("nude" mouse, nu/nu). No differences in gross graft observations were noticed. More than 80% of all keratoacanthomas disappeared postgrafting. This percentage was the same for both groups of recipients. These data are in keeping with a nonimmunological regression of experimental keratoacanthomas. A possible correlation with the hair follicle cycle is suggested.