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Background: Recent studies in prostatic tissue suggest that Propionibacterium acnes (P. acnes), a bacterium associated with acne that normally lives on the skin, is the most prevalent bacterium in the prostate and in men with benign prostatic hyperplasia. Its prevalence is higher in samples from patients subsequently diagnosed with prostate cancer. The aim of our study was to test whether circulating levels of P. acnes antibodies are associated with prostate cancer risk and tumour characteristics using plasma samples from a population-based case–control study. Methods: We measured plasma concentration of P. acnes antibodies for 809 cases and 584 controls using a recently developed ELISA assay. We compared antibody titres between cases and controls using unconditional logistic regression adjusted for batch and variables associated with the study design (i.e., age, year of selection and centre). The primary analysis included P. acnes titres in the model as a dichotomous variable using the median value for controls as the cut-off value. Results: P. acnes antibody titres for both cases and controls ranged from 1 : 16 (i.e., low concentration) to 1 : 65 536 (i.e., high concentration; median value=1 : 1024). The odds ratio for prostate cancer associated with titres at or above the median value was 0.73 (95% CI 0.58–0.91, P=0.005). The association appeared to be particularly strong for advanced prostate cancer (AJCC Stage grouping III–IV) for which the odds ratio was 0.59 (95% CI 0.43–0.81, P=0.001) but there was insufficient evidence that the association differed by tumour stage (p heterogeneity=0.07). Conclusion: These results need to be confirmed in prospective studies but they are consistent with the hypothesis that P. acnes has a role in prostate cancer.

The effect of adjuvant immunotherapy with a single neighbourhood injection of 2 mg C. parvum (CP) was investigated in a randomized study involving 43 patients with carcinoma of the cervix uteri, all of whom were treated by radical surgery. All patients had carcinoma confined to the cervix, the upper part of the vagina or the parametrial region. When the malignancy had spread to the parametrial region, additional postoperative radiotherapy was given. 22 patients received immunotherapy 10 days before surgery, whereas the remaining 21 control patients received no immune stimulation. Only minor side effects of CP were encountered. Follow-up shows a relapse rate of 5% in the CP treated group and of 29% in the controls (P less than 0.05). A further 15 patients with more advanced malignancies were added to our studies. In these, CP stimulation had no effect on relapse rates, but the relapse-free intervals were longer after immune stimulation: control 3.5 months (mean) +/- 1.5 (s.d.), CP 13.0 months +/- 7.0 (P less than 0.05). The number of peripheral T cells and the ability to become sensitized to DNCB were increased after CP stimulation. A decrease was found in the number of blood monocytes and the number of monocytes capable of transforming into active macrophages, indicating a possible sequestration of these cells in the tissues.

Cells from the spleen, lymph node, peripheral blood and peritoneal exudate of mice treated with C. parvum were tested for their ability to inhibit tumour growth in vitro. The peritoneal exudate cells from C. parvum treated mice were extremely effective in inhibiting tumour growth whereas the spleen and peripheral blood cells were only moderately so. In contrast, the lymph node cells caused only a modest inhibition of tumour growth at a very high effector to target cell ratio. Spleen cells from normal mice also exerted a moderate anti-tumour effect.

I.p. administration of 1.4 mg Corynebacterium parvum (C. parvum) 24 h before inoculation of Landschütz ascites carcinoma (LAC) cells significantly impaired growth of the tumour in MF1 mice. The injection of tumour cells caused a transient inhibition of the activity of the mononuclear phagocyte system (MPS) in both normal and C. parvum-treated hosts, as evidenced by impaired clearance of colloidal carbon from the bloodstream and reduction in hepatic phagocytosis of 51Cr-labelled sheep erythrocytes. Depression in Kupffer-cell activity was associated with a shift in particle distribution towards the spleen. The pronounced hepatosplenomegaly in response to C. parvum was significantly less in animals which also received tumour cells. Histological examination of liver and spleen revealed evidence of depressed MPS activity. Granuloma production in the liver in response to C. parvum was inhibited in tumour-bearing mice, and macrophage proliferation within the spleen was also reduced. Ascitic fluid showed similar inhibitory effects to those of tumour-cell suspensions, suggesting production by LAC of a heat-stable macrophage-inhibitory factor. Images Fig. 3 Fig. 4

The SAL rat leukaemia, which resembles acute myeloblastic leukaemia, appeared initially to be non-immunogenic since resistance to an i.p. challenge with as few as 100 cells could not be obtained using stimulation of the RES or by immunization with SAL cells exposed to x-rays, nitrogen mustard, iodoacetate or glutaraldehyde. However, immunization with SAL cells exposed to low doses of mitomycin-C slowed the growth of the challenge inoculum. Cells treated with high doses of mitomycin-C did not immunize. The results are interpreted in terms of rapid shedding of a tumour-specific antigen from the membrane of SAL cells.

Using a direct radioimmune antiglobulin technique and a competitive double-antibody radioimmune assay, we have demonstrated the presence of appreciable amounts of host immunoglobulins on the surface and in extracts of cell suspensions from freshly excised solid tumours. IgA appeared to have the greatest concentrations from freshly excised solid tumours. IgA appeared to have the greatest concentration, followed in turn by IgM congruent to IgG2a greater than IgG1 congruent to IgG2b greater than IgG3. The amount of immunoglobulin appeared to be influenced by the tumour under investigation and its mode of maintenance. It could also be increased by the administration of C. parvum but was not significantly influenced by the T-cell status of the host.

In view of the relatively poor prognosis for patients after surgery for locally invasive colorectal cancer a trial of repeated systemic infusions of Corynebacterium parvum (CP) has been carried out. It is in this group of patients, with a high risk of recurrence from small residues of cancer left by the surgeon, that immunotherapy should have its optimum chance of success. A total of 92 patients were included in a randomized controlled study. The two groups were comparable in terms of tumour stage at presentation, operation performed and mean age of patients, but the control group had a preponderance of male patients. The study was carried out over 54 months. Treatment resulted in greater side effects than had been predicted, and as a result many patients could not be considered for inclusion.

Administration of C. parvum alone did not improve the survival of C57BL/6 mice injected with the EL4 lymphoma. The anti-tumour effect of anti-EL4 Ig was however increased by C. parvum treatment, and the combination therapy of anti-EL4 Ig and cytotoxic drugs was even more improved. However, C. parvum only had this effect when given by the same i.p. route as the tumour cells, and the effect was greater when C. parvum was injected 5 days before than 1 day after tumour cells.

The anti-tumour activity induced by glucans (lentinan, yeast cell walls, pseudonigeran, dextran, DEAE-dextran and dextran sulphate) and fructosans (levan and carboxymethyl-levan) was compared with the activity of C. parvum. The following effects on tumour systems in CBA mice were assayed: (a) adjuvant activity on the immune response against tumour-specific transplantation antigens (TSTA) with a methylcholanthrene-induced fibrosarcoma; (b) cytostatic activity of peritoneal macrophages against radiation-induced leukaemia cells; and (c) inhibition of tumour nodule formation in the lungs following i.v. injection of fibrosarcoma cells. All the polysaccharides induced cytostatic macrophages, but the dextrans and levans did so only after i.p. and not i.v. injection. Only lentinan, yeast cell walls and pseudonigeran were active in the lung-nodule inhibition test; and only lentinan and dextran sulphate showed slight adjuvant activity for TSTA. It is concluded that the anti-tumour activity induced by these polysaccharides is predominantly non-specific macrophage-mediated and much weaker than that found with C. parvum.

Differences were seen in the ability of 2 strains of C. parvum to augment cytotoxicity attributable to NK cells towards a rat lymphoma. Furthermore, 2 batches of the same strain of C. parvum prepared by different methods also differed in their ability to augment cytotoxicity. Other factors influencing cytotoxicity were dose, route of inoculation and time after injection at which the assay was performed. Although all preparations of C. parvum augmented the cytotoxicity of peritoneal-exudate cells when injected i.p., only the most stimulatory preparation consistently augmented splenic cytotoxicity when given by this route. I.v. administration of 1 mg of C. parvum produced peak levels of splenic cytotoxicity 2-3 days later, but this response was strictly dose-dependent, since 1 microgram depressed splenic cytotoxicity. This dose-dependent effect also extended to ADCC, since 1 mg stimulated cytotoxicity towards antibody-coated P815 cells, whilst 1 microgram depressed it in a manner similar to its effect on natural cytotoxicity. Whilst the cytotoxic cells of stimulated rats closely resembled the NK cells of normal rats, BN rats responded differently to C. parvum from W/Fu or WAG rats, in that marked lysis of P815 or RBL-5 cells was observed, though these targets are usually resistant to lysis by rat NK cells in short-term assays.

Sadler and Castro (1976) reported that a single dose of C. parvum vaccine given i.p. or i.v. to mice 4 days before excision of subcutaneous transplants of Lewis lung carcinoma significantly reduced the incidence of lung metastases in the operated mice. In similarly designed experiments, using 2 different carcinomas of spontaneous origin in our own inbred mouse colonies, we were unable to demonstrate any influence of C. parvum vaccine on the incidence or latent period of secondary disease in operated mice. We discuss possible reasons for our failure to reproduce the findings of Sadler and Castro.

The presence of Fc receptors on the surface of cell suspensions obtained from a transplanted isogeneic methylcholanthrene induced murine fibrosarcoma has been investigated by determining the capacity of such cells to form rosettes with antibody coated SRBC. These studies indicate that a large percentage of cells in the tumour had Fc receptors on their surface. The proportion of such cells was increased by reducing the number of cells transplanted, by administering cyclophosphamide to the host, and on occasions by the i.p. injection of C. parvum. It was largely unaffected by the route of tumour cell transplantation or by T cell depletion of the host before transplantation but appeared to decline in older (i.e. larger) tumours. Both phagocytic and non-phagocytic cells had Fc receptors on their surface. The phagocytic population appeared to be affected most by procedures which altered the overall percentage of Fc receptor bearing cells. The Fc receptor bearing tumour cells were separated from those devoid of Fc receptors on the basis of their adherent properties. Upon transplantation to isogeneic hosts both populations gave rise to tumours containing a high percentage of Fc receptor bearing cells. These studies suggest that many of the Fc receptor bearing cells in our tumour are probably infiltrating cells of host origin. Their significance in relation to tumour growth remains to be established. Images Fig. 2 Fig. 3 Fig. 4

I.p. or i.v. administration of Corynebacterium parvum (CP) to MF1 mice induces a generalized inflammatory response, associated with marked hepatosplenomegaly and accompanied by a pronounced granulomatous response in the liver. Injection of the Landschütz ascites carcinoma (LAC) 24 h after CP substantially reduced the intensity of the inflammatory response, and decreased both the frequency and size of the hepatic granulomas, as revealed by morphometric analysis of histological sections. The difference in cellular composition of the granulomas between the experimental groups, as revealed by light microscopy, was further emphasized and characterized by ultrastructural studies. These revealed the predominance of macrophages within the granulomas in tumour-bearing mice, in contrast to the predominance of epithelioid cells in the lesions which developed in mice given CP alone. Our experimental findings show that the inhibitory effect of the growing LAC on granuloma formation in response to CP cannot be ascribed to (a) sequestration of the microorganism within the growing tumour, (b) a nonspecific inflammatory stimulus, (c) diversion and seqestration of mononuclear phagocytes in the growing tumour or (d) the presence of lactate dehydrogenase-elevating virus in either the host or tumour cells. The inhibition of liver granuloma formation is consistent with an effect mediated by soluble, heat-stable tumour-associated factor(s). Images Fig. 4 Fig. 3 Fig. 6

The effects of C. parvum on RFM/UN myeloid leukaemia were studied. Mice inoculated with 7.0 mg but not 0.7 mg C. parvum i.p. survived significantly longer than untreated leukaemic mice (P less than 0.001). Administration of silica abrogated the effects of C. parvum, whilst polyvinyl pyridine-N-oxide prevented the inhibitory effects of silica. These studies demonstrate that a single large dose of C. parvum, either before or after leukaemic-cell passage, can significantly prolong the survival of RFM mice bearing myeloid leukaemia. The effects of silica and PVNO on C. parvum suggest a critical role for macrophages in C. parvum effects on myeloid leukaemia. Images Fig. 2 Fig. 3

Intravenous injection of Corynebacterium parvum (C. parvum) 4 days after s.c. inoculation of 5 X 10(5) cells derived from the immunogenic fibrosarcoma FSA/R induced tumour growth inhibition over a period of 21 days in syngeneic C3H/Buf mice. This was not accompanied by a change in the proportions of host cells within the tumour, but the activation state of tumour-infiltrating macrophages was increased following C. parvum therapy. Two macrophage subpopulations were identified in FSA/R tumours after fractionation by unit gravity velocity sedimentation. After i.v. C. parvum therapy the tumour-infiltrating macrophage subpopulation which sedimented between 1 and 6 mm h-1 was consistently activated as determined by measurement of Fc receptor avidity. Other intra-tumour macrophages were generally unaffected by C. parvum treatment. We have previously shown that the host cell fraction sedimenting between 1 and 6 mm h-1 is enriched with monocytes and the data presented in this paper suggest that these cells may enter the tumour in a pre-activated state following intravenous C. parvum therapy.

Detailed serological studies have been undertaken in a small group of cancer patients receiving nonspecific immunotherapy with Corynebacterium parvum (C. parvum). These patients included 4 cases of recurrent malignant melanoma, 2 of stomach cancer and 2 of recurrent breast cancer. They all received an initial i.v. infusion of 20 mg of a formol killed suspension of C. parvum followed by 2 mg (i.m.) at weekly intervals for 10-11 weeks. This protocol consistently resulted in an increase in the circulating IgG levels of all patients but had a variable effect on their IgA, IgM and IgE levels. Increases in the concentration of all 4 IgG subclasses contributed to the overall increase in IgG levels and these changes ranked IgG2 greater than IgG1 greater than IgG3 = IgG4. It also had an inconsistent effect upon the levels of alpha-macroglobulin in pregnancy but the levels of normal serum alpha2-macroglobulin were virtually unchanged. Pre-existing antibodies to C. parvum were noted in all the patients. Titres rose appreciably following C. parvum administration and remained at high, though fluctuating levels, throughout the 100-day period of observation. Absorption studies suggested that the development of antibodies to C. parvum accounted in part for the increased IgG levels noted following this form of therapy. The significance of these changes in relation to the possible anti-tumour effect of C. parvum is discussed.

The relative efficiency of active nonspecific or specific immunotherapy of developing methylcholanthrene induced fibrosarcomata with C. parvum was compared. For nonspecific immunotherapy, mice were challenged with tumour cells s.c. or i.v., and 2 days later injected i.v. with dilutions of C. parvum. The only significant effect was a retardation of s.c. tumour growth by the highest concentration of C. parvum (350 mug). However, active specific immunotherapy, using mixtures of C. parvum and irradiated or living tumour cells in the footpads, suppressed tumour growth when given at 2 or 6, but not 10, days after tumour challenge. Successful therapy required: sufficient tumour cells (greater than or equal to 5 X 10(4)); an optimal dose of C. parvum (5-120 mug, increasing with the number of tumour cells); an intact T cell system; the same tumour cells for challenge and treatment. The specificity was confirmed in a protection system in which treatment was given 7 days before tumour challenge. No protective immunity could be achieved with mixtures of C. parvum and foetal cells. Thus in this system C. parvum potentiates protective immunity only to the tumour unique TSTA.

The level of peripheral blood platelets was determined after i.v. injection of Corynebacterium parvum in normal C57BL mice and in those bearing the Lewis lung carcinoma. Twenty minutes after injection of a formalin-killed active strain (CN6134, (CN6134, which inhibited tumour metastases) or a killed inactive strain (CN 5888, which did not inhibit metastases) the number of circulating blood platelets was reduced by 50%. The level of platelets returned to control values by 8 h after the active, and by approximately 3 days after the inactive strain. The active strain alone caused a second and prolonged fall in platelet numbers, from approximately 16 h to 21 days after injection. Heparin given 3 X weekly to these mice restored the platelet count to normal values by 10 days after injection of active-strain C. parvum. The level of platelets in tumour-bearing mice was essentially similar to that in normal mice. Possible causes of the thrombocytopenia and the significance of platelets in metastasis are discussed.

The murine dose of i.v. C. parvum (466 microgram) was compared with a single, low, human-equivalent dose of 70 microgram and with repeated weekly low doses. All treatments increased the antibody titre against C. parvum (CP). However, repeated doses stimulated a much higher titre than single doses. In all treated animals spleen weight peaked at 2 weeks and then fell. A single low dose caused a 3-fold increase, a single high dose or multiple low doses a 6-fold increase. Liver weight changes followed a similar pattern. Hepatosplenomegaly was prolonged by multiple doses. The effects of these treatments on Lewis tumour metastases were studied. A single high dose and a single low dose on the day of tumour implantation (Day 0) were equally effective at inhibiting pulmonary metastases. Repeated low doses starting on Day 0 were no more effective than a single dose. The effect of CP on survival after primary-tumour excision on Day 10 was observed. Low dose CP on Day 7 doubled the harmonic mean of survival time. Repeated doses were no more effective than a single dose. Low-dose prophylaxis up to 2 weeks before tumour significantly inhibited metastases. However, when repeated low-dose prophylaxis was combined with a single low dose on Day 0, the anti-metastatic effect was abrogated. This neutralization of the anti-metastatic effect of CP given on Day 0 was found to persist after a 13-week treatment-free interval. Possible mechanisms for this phenomenon are discussed.

The effects of operation (lower-limb amputation) on the growth of the Lewis lung tumour and its metastases were studied. The role of C. parvum in counteracting these effects was investigated. Anaesthesia alone or with amputation did not affect primary tumour growth. C. parvum depressed this growth. Anaesthesia did not affect the number of pulmonary metastases, but amputation caused a significant increase. C. parvum inhibited metastases and completely counteracted the effects of operation on them. Large doses of cortisone acetate significantly increased metastases but small doses had no effect. Experiments with adrenalectomized mice suggested the effects of operation were due to non-specific stress.

In mice, i.v. C. parvum induces intravascular coagulation. This is a prolonged reaction lasting up to 7 days. It results in thrombosis in hepatic vessels with consequent hepatic necrosis, and thrombosis in pulmonary and splenic vessels. This may be important in the assessment of the tumour-inhibitory activity of C. parvum. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

The anti-inflammatory agent sodium aurothiomalate appears to act upon mononuclear phagocytes, inhibiting their lysosomal enzyme activity. Evidence is presented that gold salts can increase the number of lung tumour nodules that develop following intravenous injection of tumour cells and pretreatment can enhance the take of a subcutaneous tumour inoculum. In contrast, they do not affect the later growth of tumour. Gold salts can also suppress the action of systemically administered C. parvum in inhibiting the growth of subcutaneous tumours. These results are taken as supporting the evidence in favour of a fast acting nonspecific anti-tumour mechanism, probably macrophage mediated, that can be inhibited by gold salts and enhanced by C. parvum. The effect of gold salts upon other biological changes induced by C. parvum is examined, including its adjuvant action, and the results are discussed in the context of the mechanisms underlying the immunotherapeutic action of this organism.

An investigation was made into the effect of Corynebacterium parvum therapy on cerebral tumours in mice. I.v. C. parvum caused a slight but significant increase in the survival of BALB/c mice injected intracerebrally (i.c.) with not more than 50 Meth A cells. C. parvum was most effective if given on the same day or 5 days after tumour. If this interval was increased there was no effect. Multiple i.v. injections were no more effective than a single dose. I.v. C. parvum had no influence on the survival of C57BL mice injected i.c. with Lewis tumour cells, and had little effect on the induction of i.c. or s.c. tumours by methylcholanthrene. It was concluded that C. parvum therapy was of little use in the treatment of cerebral tumour in mice. The clinical implications of these findings are discussed.

With progressive growth of syngeneic sarcomata in rats there was a rise in serum levels of lysozyme which correlated with their immunogenicity and their macrophage content. By an examination of lymph/blood differences in normal and in tumour bearing rats and of the production of lysozyme by cells obtained from the tumours and maintained in vitro, it is apparent that the macrophages resident in a tumour mass make a massive contribution to the elevation in serum lysozyme concentrations. Tumour cells did not release detectable lysozyme activity. Tumour amputation led to a rapid fall in lysozyme levels. Irradiation of the host rats abolished the lysozyme response and the subsequent development of metastases in these rats was associated with a rise in serum lysozyme. The serum concentration of this enzyme reflects the macrophage content of a tumour mass and the draining lymph nodes. We conclude that under well defined conditions serum lysozyme activity may be a useful marker of macrophage mediated host responses to a tumour.

I.v. injection of Corynebacterium parvum (CP) into C57BL and BALB/c mice caused profound coagulation changes, featuring thrombocytopenia, decreased fibrinogen, increased fibrin/fibrinogen degradation products, and a concomitant microangiopathic haemolytic anaemia. These changes were greatest on the 9th day after CP, with recovery by Day 21. I.p. injection caused similar effects but s.c. injection was ineffective. Radiolabelled-platelet kinetics and distribution after i.v. CP indicated disseminated intravascular coagulation with rapid fibrinolysis; EACA treatment exacerbated the thrombosis. The coagulopathy correlated with hepatosplenomegaly, and both were dose dependent. Splenectomy did not effect the coagulopathy, but indomethacin totally abrogated the changes, suggesting that prostaglandin biosynthesis is involved in the pathogenesis.

Morphological changes are described in pelvic lymph nodes excised 10 days after C. parvum (CP) treatment of patients with cervical carcinoma. Guinea-pig popliteal and inguinal lymph nodes were investigated from Days 1 to 10 after an injection of 70 micrograms CP into the footpad. Eosinophils were detected from the first few hours after stimulation, initially in the marginal sinus, then in the medullary sinuses and subsequently in the efferent lymphatics. From Day 2 to Day 6, histiocyte accumulations with the appearance of epithelioid cells were found mainly in subcapsular and interfollicular areas, and small granulomas were also seen in the paracortex. The granuloma formation in the lymph node was considered as an indication of the activation of histiocytes. Besides small granulomas in the paracortex, activated interdigitating cells, surrounded by scattered lymphoblasts and eosinophils, were also present. We considered this lymphoblastic response and eosinophilic accumulation as likely to be due to blastogenic factor and eosinophil stimulation promotor. Eight to 10 days after CP stimulation, the macrophage lymphoblast eosinophil response was replaced by a B-cell reaction: germinal-centre activation and medullary plasma cells. Such a B-cell reaction was also found in the human pelvic nodes removed at operation, but this reaction could not be attributed to CP treatment alone, since cervical-carcinoma patients not treated with CP also showed such reactions. In contrast, pelvic lymph nodes removed at necropsy from females killed in traffic accidents showed no predominance of either B- or T-cell stimulation. Images Fig. 2 Fig. 3 Fig. 4

The effectivenss of maltose tetrapalmitate (MTP) as an antitumour immune adjuvant was verified by its comparison with other known immunopotentiators, namely BCG, Corynebacterium parvum, levamisole and pyran copolymer. Copenhagen x Fisher 344/CRBL F1 hybrid male rats inoculated s.c. with the Dunning R3327A prostatic adenocarcinoma were used as the test system. All animals treated with immunoadjuvants showed a delay in tumour appearance and inhibition of early tumour growth. MTP was found to be the most effective, followed by levamisole, BCG, pyran copolymer and C. parvum in order of decreasing efficacy. Intratumoral treatment of small or large s.c. tumours with BCG, MTP and C. parvum was ineffective in our cases. However, this treatment was effective with MTP and BCG if they were used against a differentiated form of R3327 tumour. MTP and levamisole were found to be equally effective when given orally in drinking water. Experiments involving surgical excision of tumours followed by MTP therapy in two s.c. implanted animal tumour models (viz. a poorly immunogenic ascites mammary carcinoma 13762 in Fisher 344/CRBL rats, and an SV40 virus-induced sarcoma of low immunogenicity in Syrian hamster) showed beneficial effects of MTP on local tumour recurrence and tumour growth. Pre- and postoperative MTP treatment was at least as effective as postoperative MTP treatment alone.

Large differences in tumour necrosis factor (TNF) productive ability 05337257erved among various strains of mice. DDY, CD-1, ICR and DBA/2 mice could produce a high titre of TNF activity, whereas Balb/c, C3H/HeJms and A/J mice produced a low titre of TNF activity. Administration of 200 micrograms/mouse of LPS to some strains of mice, i.e. DDD and C57B1/6J resulted in good production of TNF. ICR nu/nu mice produced the highest TNF activity among the nude mice. Balb/c nu/nu and DDD nu/nu mice exhibited very low titres of TNF activity. Nude mice required a rather higher dose of the priming agent, Propionibacterium acnes, than heterozygote littermates. Although it is commonly accepted that dual stimulation is necessary for TNF production, TNF activity was detected without the priming agent in SD rats and Golden hamsters by single injection of LPS. In these animals, much higher TNF production was observed after Propionibacterium acnes treatment than after a single injection of LPS. Large differences in TNF productive ability also existed among strains of rats. Although all animals receiving priming agents revealed hyperplasia of reticuloendothelial system, the sensitivity of the animals to LPS is considered to be the most important factor in their TNF productive ability.

Assays for the capacity of peripheral-blood monocytes (a) to mature in vitro into macrophages, (b) to reduce nitro-blue tetrazolium (NBT) and (c) to lyse antibody-coated human Group A red cells, were applied to a group of 82 patients with histologically proven malignant melanoma. In patients with micrometastatic disease there was an enhancement of red-cell lysis and NBT reduction, suggesting that their monocytes are in some way "activated", whereas NBT reduction was suppressed in those with overt dissemination. Monocyte maturation in vitro was impaired in all patient groups to an extent which correlated with overall tumour burden. Corynebacterium parvum was administered i.v. to 12 patients with disseminated disease and by the intradermal route to 24 patients with micrometastatic disease. The 3 monocyte functions were significantly enhanced by C. parvum.

The effects of the host's immune response on metastatic spread was investigated by observing the numbers of pulmonary metastases that developed from an s.c. implant of the Lewis lung carcinoma in C57BL mice in which different cell populations had been suppressed. Macrophage function was impaired by treatment with silica (Si), cortisone acetate (CA), or trypan blue (TB). T-cell function was depressed by adult thymectomy and sublethal irradiation, or by treatment with antilymphocyte serum (ALS). Metastasis was significantly increased and phagocytic activity decreased by Si and CA, but were unaffected by TB. Thymectomy and irradiation had no effect on metastases, whereas ALS when given before, but not after tumour growth, reduced their number. The antimetastatic action of the immunopotentiating agent C. parvum was investigated in these immunologically impaired mice. It was unaffected by Si, CA or TB. However, the inhibiting effect of these agents on phagocytic activity was overcome by treatment with C. parvum. Its antimetastatic action was unaffected in mice which had been thymectomized and irradiated, but could be abrogated by ALS. However, ALS was only able to prevent this activity if given before tumour growth, it was ineffective if given after tumour growth. This study showed that metastatic spread was inversely related to phagocytic activity. The antimetastatic effect of C. parvum appears to be mediated through macrophages in concert with a subpopulation of T lymphocytes, which were considered to be necessary in the sensitization arm of the response as opposed to the effector arm of this response.

The effect has been examined of various host treatments (C. parvum injection, immunization, thoracic irradiation, cyclophosphamide injection, and anticoagulation) on both lung colony formation and clearance of radioactive cells from the lungs after i.v. injection of tumour cells. Two tumour-host models have been used: the non-immunogenic KHT tumour in C3H/Km mice, and the immunogenic EMT6 tumour in BALB/c/Ka mice. Even for the at most weakly immunogenic KHT tumour, the number of artificial pulmonary metastases could be modified by a factor of up to 10(4) by different host treatments before i.v. inoculation of tumour cells. For all pretreatments except immunization, the shape of the curve of loss of radioactivity from the lungs vs time was biphasic, with an initial steep portion representing intravascular death of the tumor cells, followed 1--2 days after tumour-cell injection by a shallow exponential curve. It was concluded that the shallow slope represented spontaneous death of tumour cells in the perivascular tissues. Essentially all the injected tumour cells lodged initially in the lungs, and this was unaffected by the different host treatments. Furthermore, except for specific immunization, cell death in the perivascular tissues was also unaffected by host treatment. However, the survival of the tumour cells during the 24 h after injection (before they became extravascular) was extremely dependent on the particular host pretreatment. It would appear from these studies that host treatments such as C. parvum injection or anticoagulation can markedly affect the number of blood-borne pulmonary metastases, but they will only be effective if given before the tumor cells arrive in the lung vasculature.

The i.v. administration of Corynebacterium parvum (CP) to patients who had recently undergone resection of colorectal tumours was found to have the following effects: 1. Polymorphonuclear leucocyte counts were raised 24 h after CP administration, while both lymphocyte and monocyte counts fell during this period. Polymorph and lymphocyte counts had returned to pre-infusion levels at one week, but monocyte counts were significantly increased at this time. 2. The lymphocyte mitotic response to PHA was reduced during the 24 h after CP infusion. 3 The spontaneous, antibody-induced, and PHA-induced lymphocyte-mediated cytotoxicity against a nucleated target cell fell significantly 3 h after CP infusion, but these functions recovered by 7 days. 4. A rise in serum lysozyme was found 3 and 24 h after CP administration. However, these increased levels were not maintained beyond 24 h.

Local injection (i.e. injection at the site of tumour inoculation) of strains of C. Parvum which have a significant anti-tumour effect when given systemically (i.e. intravenously or, in the case of subcutaneous tumour transplant, intraperitoneally) strongly inhibits the growth of isogeneic transplants of a fibrosarcoma in intact CBA mice but has little or no effect on subcutaneous transplants of the same tumour in T-cell deprived mice. The anti-tumour effect of local injection of C. parvum, unlike that of systemic injection in this particular tumour system, thus appears to be T-cell dependent.

Growth of isogeneic transplants of a spontaneous murine adenocarcinoma, which is virtually devoid of tumour-specific transplantation antigens, is inhibited by i.v. injection of C. parvum 3 days after tumour inoculation, or by mixing a small dose of C. parvum with the tumour inoculum. Moreover, the therapeutic effect of cyclophosphamide, followed by i.v. or i.p. injection of C. parvum 5 days later, on established transplants of the same tumour is greater than that of cyclophosphamide alone. These findings are consistent with the hypothesis that in both situations (i.e. before the appearance of a palpable tumour and after reduction of an established tumour transplant with cyclophosphamide) the effect of C. parvum is largely due to activation of macrophages or macrophage precursors. They have the important practical implication that adjuvant therapy with C. parvum may be of value, even with tumours which are devoid of TSTA.

A randomized trial comparing routine follow-up with a treatment regimen aimed at increasing specific anti-tumour immunity has been carried out in 95 patients after total surgical excision of lung cancer (not small-cell). Treatment consisted of inoculation with an autologous irradiated suspension of tumour cells combined with a small dose of C. parvum given intradermally during convalescence. Although treatment was associated with virtually no side effects, there has been no apparent benefit and input to the trial has now stopped.

The effect of pre-treatment with Corynebacterium parvum on the growth in vivo of a range of experimental mouse tumours with differing characteristics has been investigated. Varying degrees of protection were observed which were generally greater with the more immunogenic tumours. Administration of C. parvum 7 days before immunization with irradiated tumour cells diminished the protective effect which could be obtained by immunization alone. The possible basis for these seemingly conflicting influences is considered.

Lack of immunological and anti-tumour effects of orally administered Corynebacterium parvum in mice

The effects are reported of a combination therapy of i.v. C. parvum and cyclophosphamide on the survival time and immune responses of patients with inoperable squamous-cell carcinoma of the bronchus. The immune status of the patients was evaluated by determining the antibody response to C. parvum, the E and EAC rosettes, the PHA response of blood lymphocytes, the skin-test reactivity to Candida and PPD, the response to DNCB and the chemotaxis and NBT-dye reduction capacity of neutrophil leucocytes. The survival time of patients treated with the combination therapy was found to be significantly shorter than that of untreated patients and of those receiving cyclophosphamide only. Severe side effects were observed after C. parvum infusions, with no decrease on repeated administration. The effect of C. parvum on the different immune parameters of cyclophosphamide-treated patients was negligible, though there was a normal antibody response to C. parvum.

Serum complement levels were assayed in 26 patients with disseminated cancer, who received immunotherapy with infusion of C. parvum. Complement activation, indicated by the consumption of C3 or C4 or both, was found in 46% of the patients. Serum samples showed direct correlation between decreased C3 and conversion of C3 proactivator, whereas such conversion did not occur when C4 alone was decreased. It is concluded that the bypass (properdin) pathway was activated in patients in whom C3 consumption was detected, while the classical (C1) pathway was activated in the patients with C4 consumption unaccompanied by C3 decrease. Direct correlation was observed between delayed cutaneous hypersensitivity reactions to recall antigens and the incidence of C. parvum-associated complement activation.

The present studies in mice and cancer-bearing patients, treated with C. parvum (CP) immunotherapy, were to determine the effects of CP on the production of immune complexes (IC) and associated disease. Using the Clq-binding assay, circulating immune complexes were detected in mice given a single high dose of CP (466 microgram) and repeated human-equivalent doses (70 microgram). All mice treated with CP developed proliferative glomerulonephritis, the severity of which was dose-related. The histological and immunofluorescent patterns of the nephritis were those attributed to immune-complex disease. The mice had haematuria but were not in renal failure. Fifty patients with inoperable lung cancer were studied. All received radiotherapy. Twenty-two had no other treatment (controls) and 28 were treated with infusions of CP. Using 2 immune-complex assays (Clq binding and monoclonal rheumatoid-factor binding) IC were found in 10/22 control patients but these did not develop haematuria or proteinuria. Twenty-four of the 28 patients treated with CP developed transient haematuria and/or proteinuria with red-cell and hyaline casts, the changes resolving over 5 days. Immune complexes were detected in 5 of these 28 patients before CP treatment. Although 16/28 had IC at the time of haematuria and proteinuria, these findings were difficult to interpret because IC may occur in response to the tumour, the radiotherapy, or the CP. Although no patient developed renal failure, we believe that those treated with CP should have regular assessment of their renal function. Images Fig. 2 Fig. 3 Fig. 4

Effect of C. parvum and active specific immunotherapy on intracerebral transplants of a murine fibrosarcoma

Corynebacterium parvum was labelled by growing live bacteria in the presence of [3H]thymidine. The bacteria were killed by formalin, washed thoroughly and resuspended at a concentration of 7 mg dry weight/ml. An activity of 1-6 X 10(5) ct/min/0-1 ml was obtained. The biological properties (inhibition of tumour growth and hepatosplenomegaly) of the labelled C. parvum were compared with those of commercially available vaccine, and were found to be similar. Labelled C. parvum was injected i.v., i.p., or s.c. into normal C57BL mice and the localization of activity determined at 4 h and 1,3,7 and 14 days after injection. After i.v. or i.p. injection, highest counts were recorded in the liver. Moderate activity was found in the spleen, lungs and small gut. After s.c. injection, the majority of radioactive label was detected at the site of injection and little found in other tissues. The distribution of injected C. parvum was also studied in mice bearing Lewis tumour, and was found to be similar to that in normal mice. Moderate amounts of labelled C. parvum were recovered from tumour. There appeared to be no relationship between the antitumour effect of C. parvum given by a particular route of injection and the concentration of C. parvum recovered from the tumour. Images Fig. 7 Fig. 8