Quantitative investigations into the elimination of in vitro-obtained spores of the non-pathogenic Clostridium butyricum strain CNRZ 528, and their persistence in organs of different species following intravenous spore administration.

Before the Clostridium tumour assay can be applied to the diagnosis of cancer, we sought to investigate--within the framework of a biopharmaceutical safety test--the organ persistence of test spores of Clostridium butyricum CNRZ 528. We found that non-pathogenic spores obtained in vitro, like pathogenic native spores, escape phagocytosis in various organs up until about 2 years, as tested by anaerobic cultures. The elimination of spores depended on the species of animal, the spore dose and the organs investigated. In rabbits, one week after injection, we recovered clostridial spores from blood and spleen cultures more rarely than from liver and lung. The half-life of blood clearance in patients was one day or, at half the spore dose, two days. That deep tissues of healthy animals are not normally sterile became evident in rabbits after sporadic isolation and characterization of non-administered saccharolytic and proteolytic clostridial species. During a 10-year observation period, the rate of obtainment of viable spores by in vitro cultures lessened; however, for administration of the spores in clinical phase I and phase II studies, the spore quality was acceptable.

Application of microbiological cancer test to cattle infected with bovine leucosis virus.

A microbiological cancer test, previously verified in men and dogs using a clostridium strain (Clostridium butyricum CNRZ 528), was applied to cattle infected with bovine leucosis virus (BLV). An extended period of time was allowed to pass after infection with BLV, which had been checked up through specific serological and virological examinations. The cattle belonged to different age groups and stages of infection (with and without haematological alterations [preleukosis], with incipient tumour development [swelling of externally visible and palpable lymph nodes]). Controls included BLV-infected cows as well as test animals to which isotonic saline had been applied or healthy BLV-free cattle in which the clostridium strain had been used. The serological investigation was carried out in a blind test. 3 of 6 BLV-infected spore-treated heads of cattle responded positively to the cancer test, while the other 3 were negative. The 3 cows with positive cancer test were haematologically and serologically leucosis-positive animals with clinically detectable enlargement of lymph nodes. The 3 negative ones of this group, also serologically and haematologically leucosis-positive, were younger animals without signs of tumorous process. 3 spore-treated BLV-free cows and 2 BLV-infected animals, treated with isotonic saline, were cancer test-negative, as well. Finally, 4 BLV-infected and 2 BLV-free cattle, all of them without spore injection, were completely cancer test-negative. 1 cow of the BLV-infected group did not produce spore antibodies after spore treatment, while 1 cow of the BLV-free untreated control group developed spore antibodies.

The role of cell population kinetics in the efficacy of penicillin--an experimental analysis and stochastic modeling of the tumour tetanus and wound tetanus of the mouse.

When investigating tetanus lethality summation curves of mice under comparable quantitative conditions following a temporarily limited administration of penicillin, the curves obtained can be calculated by the kinetics of tumour cells or wound fibroblasts. In particular, it has been shown that the optimal efficacy of penicillin, after short-time usage as compared with a long-time administration schedule, is determined by the generation time of the tetanus rods as a function of the mitotic cycle of the "pace-making" tumour cells or wound fibroblasts. Further variables of the mathematical model imply the pharmacokinetics of penicillin and the recovery process of the "hit" tetanus rods. From these results some basic experimental and clinical tetanus issues can be elucidated; thus, the mitosis theory of tetanus is being verified for the stage of incubation and of clinical manifestation, while the classical necrosis theory of the pathogenesis of tetanus infection should be valid only for the final stage.

Methodological aspects of a serodiagnostic Clostridium tumour test--experience with spontaneous canine tumours.

The paper describes the development of appropriate antigen and method combinations for the microbiological cancer test using the non-oncolysing strain Clostridium butyricum CNRZ 528 in dogs with spontaneous tumours. The diagnostic rod antibodies could be determined quantitatively by the complement fixation test if a short-time warm fixation and a long-time cold fixation procedure were combined in separate runs and if two different antigens, a rod corpuscular antigen and a rod surface antigen, were used. Since complement-fixing antibodies were not always detected in cases of malignant tumours, we additionally used the passive haemagglutination method after pre-absorbing the sera with cross-reacting clostridial antigens. The efficiency of the microbiological cancer test could not be substantially increased, however, by the method combination the reliability of the evaluation of low seropositive titres was improved.

Antibacterial immunity and its modeling in experimental tumor tetanus and wound tetanus of the mouse.

The experimental and theoretical analysis of the tumor-tetanus phenomenon has provided us with new insights into the pathogenesis of tetanus infection. Our theoretical model of clostridial propagation in the proliferating tissue is based upon the principle of mitosis-controlled rod division (hit and cloning model). It has lent itself to the description of early growth stages of the clostridial rod population in our experiments of tumor tetanus and of wound tetanus of the mouse. However, the later course of the tetanus lethality curves under antitoxin protection, about a week following injection of the tumor cell-spore or CaCl2-spore suspensions, reveals a pronounced delay in clostridial propagation. Based on our model we can explain this process by a humoral immune reaction directed against the clostridial rods taking into account the variability of elimination of the heterologous tetanus antitoxin applied. The experimental results are in good agreement with those obtained by computer simulation. The theoretical knowledge resulting from these studies can be used for the interpretation of the serodiagnostic tumor test with apathogenic clostridia as well as for the quantitative assessment of the malignancy of neoplastic growth.

The serological clostridium tumour test--application of discriminance analysis.

The microbiological tumour test is based on the detection of humoral antibodies to clostridial rods following intravenous injection of apathogenic clostridial spores. As serological methods we used the complement fixation test with a simultaneous short-time warm and long-time cold incubation as well as the passive haemagglutination test after absorption of clostridial group-specific antibodies. The evaluation of titre dynamics was rendered difficult by normal clostridial antibodies, by serological cross-reactions and, above all, by border-line reactions. The serological test results from 189 dogs investigated were evaluated in a multivariate analytical procedure. We determined the adequate combination of methods and antigens, thus improving the reliability of the test results and objectifying the predictive value of low sero-positive titres. On the basis of the computer-calculated individual weighting factors and the corresponding reference value it has become possible to evaluate and classify actual serological data emerging from the previous overall investigation.

Conception and stages of development of the microbiological cancer test.

Pathology and experimental bacteriology have, in scientific medicine, created fundamental methodical presuppositions for the description and recognition of etiologic entities. With the tumour-tetanus phenomen, two pathogenic principles--cancer growth and tetanus infection--coincide in a so far unknown manner. Analytical experiments have led to the novel idea of the mitosis-stimulated anaerobic tetanus infection. This specific pathomechanism and its mathematical model formed the basis for the development of the serological Clostridium-tumour test with nontoxic, apathogenic Butyricus Clostridia as test microbes. As viable diagnostic agents the test spores are subject to the 12th Regulation of the Drug Act of the GDR. Only the histopathological judgement allows for the detection of micro-tissue alterations following i.v. overdoses of spores. The test spores were confirmed as safe and effective, and the microbiological cancer test approved to be appropriate for a clinical trial.

[Evaluation of thrombocyte-induced immunomodulation in the tetanus-infected mouse]. / Prüfung thrombozytär bedingter Immunmodulation an der tetanus-infizierten Maus.

The infection model of the mouse-tetanus assay was utilized in experiments to test for immunomodulating effects of thrombocyte preparations. It has been established that the substances of platelets cause alteration of tetanus reactivity in mice which occurs in two phases: a non-specific early effect starting only a few hours after application displaying a drastic increase of resistance of the animals to sporangium activity (possibly through enhancement of phagocytosis or bypass operation). A late phase of effectiveness develops after weeks, visible by an increase of morbidity and mortality following a repeated challenge which may be due to a decrease of a specific immune response. The immune modulating action is that of an inducer acting in xenogeneic systems and altering also the immune response to transplantation antigens.

[The doctrine of immunity in the light of Robert Koch's concepts]. / Die Immunitätslehre im Lichte der Vorstellungen von Robert Koch.

Robert Koch's epochal discovery of the tubercle bacilli provided the first complete evidence for the specific nature of the causative agents of the most important human infectious disease. It was on this basis that Koch developed his ideas about the doctrine of immunity. The high specificity of "Koch's basic experiment" became the cornerstone of the specific early recognition and prophylaxis of tuberculosis and provided a pattern of specific immune diagnostics in general. Basing on the antitoxin experiments of Behring and Ehrlich against diphtheria and tetanus, Koch presumed humoral immune mechanisms to exist also for tuberculosis. These investigations were abandoned after long-lasting efforts. The concept of Robert Koch and his school about the specific humoral immunity of infectious diseases was dominating immunological research for half a century until the elucidation of the cellular nature of the tuberculin reaction became a general asset of modern immunology. Only thereafter this discipline gave full swing from the immunochemical to the immunobiological approach. Robert Koch's merits as a physician and as an investigator reside in his strictly causal-analytical way of thinking and working which was adopted also in the search for the causes of cancer. In studying the immunological virus-host relationship one arrives from the "whence" of the disease at the "why" of its pathological form. This may be conceived of as a conceptual model for problems concerning the biological evolution and differentiation in modern pathology.

[The tumour-tetanus phenomenon as a basis for computation of cell kinetic parameters of tumour growth (author's transl)]. / Das Tumor-Tetanus-Phänomen als Grundlage für die Berechnung zellkinetischer Parameter des Tumorwachstums.

A verbal description of a mathematical model is given which theoretically explains the basis of the biological specificity of the tumour-tetanus phenomenon. The main postulate of the model concerns a mitosis-controlled clostridial growth. It is based on the experimental and mathematical demonstration that clostridia are stimulated by the dividing tumour cells. In particular, we have shown that the parameters which describe the kinetics of the pacemaker cells of the host can be deduced from the experimental tetanus lethality curves of the animals. Using these mathematical parameters one obtains stimulated curves which excellently fit the experimental test results. The pacemaker mechanism is the prerequisite of a specific clostridium-tumour assay utilizing serological methods with atoxic non-oncolytic clostridial strains as test microbes.

[Investigations on the characterization of a clostridial strain for tumour diagnosis (author's transl)]. / Untersuchungen zur Charakterisierung eines Clostridienstammes für die Krebsdiagnostik.

Careful delineation of a clostrial strain for human tumour diagnosis as against oncolytic tissue-active clostridial strains should be obligatory. We investigated the possibility to differentiate Clostridium butyricum CNRZ 528 (Bergère) against three different strains kept in our institute by means of biological, biochemical and by phage typing procedures. In addition, consistency in the control of half-technological spore production is required. It could be confirmed by this study that oncolytic properties of clostridial strains are biologically testable with adequate sensitivity in the hamster A Mel 3 tumour model. By means of typing procedures, we were able to clearly differentiate between Clostridium butyricum CNRZ 528 on one hand and Clostridium oncolyticum M 55 as well as Clostridium butyricum Jena H 8 on the other. The results of the tests are unsuitable for identifying Clostridium butyricum CNRZ 528 or Clostridium butyricum McClung 1672 A, respectively, nor can we distinguish Clostridium oncolyticum M 55 from Clostridium butyricum Jena H 8.

[Application of the serological clostridium assay in tumour diagnostics (author's transl)]. / Der Einsatz eines serologischen Klostridientestes in der Tumordiagnostik.

The suitability for serologic tumour diagnostics of the non-oncolysing strain Clostridium butyricum CNRZ 528 has been investigated. By introducing the transplantable Brown Pearce tumour of the rabbit and spontaneous tumours of the dog as test models the antibody production against clostridial rods--indicating malignant growth as had been proved with the Möse strain Cl. oncolyticum M55--has been evaluated. It could be established--utilizing the complement fixation test--that one serological method alone is not sufficient for obtaining clear-cut test results in anyone case. It is to be recommended that the indirect hemagglutination and the complement fixation tests ought to be combined. Our studies on specific clostridial antigens and on optimization of techniques will be continued.

[Host range testing of tumour-selective strains of Clostridium butyricum by Bacteriophage 5 (author's transl)]. / Wirtsbereichstestung von tumorselektiven Clostridium-butyricum-Stämmen mit dem Phagen 5.

Subcultures of Clostridium oncolyticum M55 and in a slightly modified manner of an oncolytic strain of Cl. butyricum H8 are differentiated by lysotypic reaction of the bacteriophage 5 from several tumour-selective non-oncolytic strains and subcultures of Cl. butyricum.

[The microbiological cancer test--further studies with experimental mouse tumor tetanus and wound tetanus]. / Der mikrobiologische Krebstest - weiterführende Untersuchungen mit dem experimentellen Tumor-Tetanus und Wund-Tetanus der Maus.

A re-evaluation of the specificity of the tumour-tetanus assay of the mouse was performed by analysing the wound-tetanus assay under comparable test conditions. This was achieved by injecting 1 X 10(6) viable Ehrlich carcinoma cells admixed with 1 X 10(2) tetanus spores subcutaneously, in a 0.1 ml dose volume or, 1 X 10(2) tetanus spores suspended in 5% CaCl2 solution, respectively. By comparison, these two groups of mice developed about the same tetanus mortality rates, however, following tetanus antitoxin therapy with 3 doses of 100 IU each day on days 0, 4 and 7 after infection, clinical signs of late tetanus exclusively belonged to tumour bearing animals. This typical tetanus behaviour may be explained by a spatial-temporal association between growing tetanus rods and proliferating cells of warm-blooded animals. In this manner tumour tissue can be differed from wound granulomatous tissue by way of permanently cloning stem cells.

[Experimental studies on the pathogenicity of tetanus spores regarding eucaryotic cell systems (author's transl)]. / Versuche zur Pathogenese von Tetanus-Sporen unter Berücksichtigung eukaryoter Zellsysteme.

After injection of known numbers of viable tumour cells admixed with tetanus spores into the subcutaneous tissue between the shoulder blades the sigmoidal tetanus lethality curves of mice can be quantitatively analysed following the underlying growth of tetanus clostridia and tumour cells. We suggest that the "driving force" for exponential clostridial growth and toxin synthesis are oxygen-consuming clones of tumour cells. Our experimental data are in good accordance with a stochastic mathematical model which allows for computing the cloning probability of one single spore on the basis of a 1-mitosis-1-clostridium division-principle.