Experimental screening of BCG preparations produced for cancer immunotherapy: safety and immunostimulating and antitumor activity of four consecutively produced batches.

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. General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch. The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG. Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases. In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.

Listeriosis in humans and animals in the Netherlands (1958-1977).

During the past twenty years 793 strains of Listeria monocytogenes were isolated from human beings in the Netherlands; of these 193 were taken from neonates and babies up to two months old. Isolations from adults came from patients (242) as well as clinically healthy people (358). Septicaemia appeared to occur to an equal extent in men and women (20 and 29 respectively), whereas meningitis was found more often in men than in women (50 and 22 respectively). A clear predisposition for L.m. develops after administration of immunosuppressive treatment and also in cases of liver disorders. Among veterinary surgeons listeriosis has been observed as an occupational disease. Isolations from animals have shown that L.m. may cause infections in every species of warm-blooded animal. Next to meningitis and abortus chronic and atypcial symptoms of the disease may be observed in animals. Epizootic spread of the disease has hardly ever been observed in the Netherlands. In clinically healthy humans and animals both the haemolytic and the non-haemolytic type of L.m. have been isolated from feces; as regards the latter type it is very questionable whether it has any pathogenic significance. When inoculating 10 days old hen's eggs the haemolytic strains will kill all embryo's within 4 days whereas all embryo's inoculated with non-haemolytic strains will survive. Both types of strains have also been isolated from waste- and surface waters. As regards epidemiological and epizootiological aspects the conclusion is warranted that continued research will be needed to get a clear picture of the course of infection of L.m. When isolating strains from contaminated material the so-called cold enrichment icubation at 4 degrees C continues to be of great value; in the course of our experiments the nalidixic-acid trypaflavine serum agar proved to be a very good selective medium. A number of stable biochemical reactions of L.m. are rather characteristic (salicine+, galactose-) but provide no clue to distinguish between haemolytic and non-haemolytic strains.

Effect of BCG and C. parvum on in vivo Listeria clearance and tumor growth. Comparative studies in normal and congenitally athymic (nude) mice.

Clinically, it is important to know whether agents used for immunostimulation require the presence of functional thymus-derived (T) cells. Previous studies showed that both BCG and C. parvum induced a macrophage-dependent enhanced in vivo listeria clearance and in vivo inhibition of a fibrosarcoma induced by 20-methylcholanthrene. These two models were re-evaluated in congenitally thymusless (nude or nu/nu) B10LP mice lacking functional T cells. In nu/nu mice, (1) BCG failed to enhance listeria clearance, and when given simultaneously with tumor cells, had no effect on tumor growth; (2) C. parvum enhanced listeria clearance and simultaneous administration of C. parvum and tumor cells exerted an inhibitory effect on tumor growth. The general conclusion was that under the experimental conditions chosen functional T cells are required for an effect of BCG, but not for that of C. parvum. It is not clear whether these observations have any clinical significance.

Proposals for quality control methods of bacterial vaccines for immunostimulation. III. Effect of BCG and C. parvum on in vivo Listeria clearance and tumor growth.

Two quality control methods for BCG and C. parvum are described. First, in vivo macrophage dependent-spleen clearance of Listeria monocytogenes in inbred B10LP mice. BCG and C. parvum were administered intravenously prior to listeria inoculation (a prophylactic model). Conditions for enhanced listeria clearance including dose, route and time interval were described for each vaccine. Next, a tumor model was developed: i.e. a fibrosarcoma, chemically induced by 20-methylcholanthrene in inbred Balb/c mice. Increase in footpad thickness was used as a criterion for tumor growth. Optimal dose, route and time interval were established both in prophylactic and therapeutic situations, including intratumoral application. Depending on the conditions chosen, both inhibition and enhancement of tumor growth were observed. General conclusions of these studies are: (a) both models yield information on the effect of bacterial vaccines for immunostimulation; (b) statements on the effectiveness of any product should be based on careful studies including all variables.

Salmonella in effluent from sewage treatment plants, wastepipe of butcher's shops and surface water in Walcheren.

In the frame of the "Walcheren-project" in which the epidemiology of salmonellosis is studied in a certain area, effluent from sewage treatment plants, wastepipe's of butcher's shops and surface waters, which receive the effluent were studied for the presence of salmonellae. From 160 samples of effluent 150 (94%) contained salmonellae. The most common serotype was S. typhi murium (35%) followed by S. panama and S. infantis. 14 butcher's shops' wastepipes were sampled 54 times. 14 (26%) times salmonellae were found, but only twice was the type isolated from the butcher's shop the same as found in the effluent on the same day. With regard to the presence of salmonellae in surface waters receiving effluent it was shown that from the immediate vicinity of the plant to 250 m downstream from the site of drainage of effluent the number of salmonellae per 100 ml remains almost constant. After 1.5-4 kilometers Salmonella could not be isolated from any of the samples examined. The results underline the hypothesis that salmonellae multiply in the sewage system and/or plant. The spread of samonellae by effluent seems to be limited to the plant itself and of the nearest vicinity. Proposals are brought forward to interupt contamination cycles by decontamination measures.

Effect of pretreatment with Bacillus Calmette-Guerin on the course of a Listeria monocytogenes infection in normal and congenitally athymic (nude) mice.

The effect of pretreatment with BCG on the course of a Listeria monomcytogenes infection was studied in nu/nu mice and in their heterozygous littermates (+/nu). First, evidence was presented that the nu/nu mice used lacked functional T cells, since BCG treatment resulted only in skin reactivity to tuberculin in +/nu mice and not in nu/nu mice. Acquired resistance to Listeria (based on lower spleen counts) was only obtained in BCG pretreated +/nu mice. Evidence was presented that BCG pretreatment in nu/nu mice failed to increase non-specific resistance, both in terms of spleen counts and survival rate. These results seem to imply that functional T cells are required for this type of non-specific resistance to heterologous antigens. In this connection attention has been drawn to the possible implication of BCG treatment in man.

Occurrence of Listeria monocytogenes in feces of pregnant women with and without direct animal contact.

Women under policlinic control during their pregnancy were divided into 3 groups, viz. women living on farms with animals, women having pet-animals in their home and women without any direct contact with living animsla. 5-7 feces samples were controlled for L. monocytogenes during pregnancy and puerperium, together with one sample of meconium of the neonate. Of women from farms, with pet-animals and women without living animal contact, respectively 32%, 39% and 34% of the samples were positive. From a total of 1337 samples 116 (8,8%) were positive. From 87 meconium samples all but one were negative. The results obtained support the hypothesis, that L. monocytogenes is an ubuquitous organism without typical zoonotic characteristics, although infection through animals may occur.

Effect of Corynebacterium parvum on the course of a Listeria monocytogenes infection in normal and congenitally athymic (nude) mice.

Spleen counts of Listeria monocytogenes in normal mice challenged with various numbers of organisms were generally lower in mice injected 3 days previously with killed Corynebacterium parvum. This increased resistance was unexpected since C. parvum generally inhibits T cell-mediated immunity, the functional immunity against Listeria being T cell-dependent. The inhibitory effect on T cells seemed to be compensated by the stimulatory effect of C. parvum on macrophages. Therefore, the experiments were repeated in congenitally athymic (nude) mice (B10LP) lacking functional T cells. C. parvum pretreatment of nu/nu mice prolonged the time between challenge and death, the number of survivors in the pretreated and non-treated mice being approximately equal. It was concluded that C. parvum exerted a stimulatory effect on macrophages resulting in a higher resistance against Listeria.