Cancer. Discovered and Cured? Forgotten?
Patented Cancer Vaccine
DESCRIPTION OF THE INVENTION
This invention relates to chemical substances comprising a product of the growth of a microorganism of Order II, Actinomycetales Buchanan, as identified in Bergey's Manual of Determinative Bacteriology, Sixth Edition, 1948, p. 111, to method of preparing same and isolation thereof and to the chemotherapeutic treatment of animals and humans therewith. Published works have identified additional species and resulted in a classification of the organism under the order Actinomycetales. This order has been reclassified as follows:
ORDER--ACTINOMYCETALES
family--Progenitoraceae
genus--Cryptocides
species--
Cryptocides tumefaciens
Cryptocides sclerodermatis (sclerobacillus)
Cryptocides wilsonii
stains isolated from lupus erytheuratosis, rheumatoid, arthritis, periarthritis, nodosum, sarcoidoses (that is, from collogen diseases other than cancer and other such diseases specified elsewhere herein)
varieties: hominis, rodentii, avii, etc. A culture of Progenitor cryptocides was deposited in the American Type Culture Collection, Accession Number 31,874.
All of the species noted above have been observed to be interchangeable within the scope of this invention.
P. Cryptocides has been assayed and assigned the tentative formula, C.sub.30 H.sub.38 N.sub.2 O.sub.3 (certain products of Cryptocides were crystals extracted from cultures of urine from terminal cancer patients). Extracts also have been crystallized from the blood and urine of cancer patients and the crystals result from the presence of the organism.
The microorganism involved (Cryptocides) has been identified as a highly pleomorphic intermittently acid-fact micro-organism, with both a virus-like and a PPLO or L transitional phase. This organism is a great simulator, whose various forms may resemble micrococci, diphtheroids, bacilli, fungi, viruses, and host-cell inclusions. Cryptocides has the ability to change its form and may vary its appearance from that of a fungus to that of a cluster of virus-size pleuro-pneumonialike organisms (PPLO or Mycoplasma). Collagenophillic mycobacterium-like, which include the cancer organism, are able to change their forms. Cryptocides has filterable or extremely small forms (submicroscopic) similar to viruses, and rather large mycelia. There were some variations as to size and some differences in the kind of media or material in which it will grow. Certain strains of it ferment one kind of sugar, some others, and some can live with little (or no) oxygen, whereas some require more. It can be identified as a single agent. The microorganism undergoes many changes in morphology and some of those forms might be zoogleal or "L" Forms. Zoogleals are intermediate forms of microorganisms which ordinarily have cell walls, but, in which, under certain circumstances the cell walls are absent. Some of these forms can be passed through very fine filters that hold back the usual bacteria and allow only very small particles such as viruses and small L forms to pass through. Such filter-passing bodies can regrow to become bacilli (bacterial cultures). The microoganisms involved have many forms but they always grow up to be the same thing no matter how often they are cultured. Cryptocides is not a virus but is a pleomorphic bacterium.
Cryptocides is acid fast, that is, it retains the Ziel-Neelsen stain in the presence of acid. Cryptocides is related to the tuberculosis family of microbes. It is filterable through filters designed to hold back bacteria. It is sensitive to tetracycline, kanamycin, ampicillin and furacin, but occasionally resistant to pencillin, sulfa drugs and mycostatin. As for the pleomorphism, cryptocides exists as virus-sized bodies of 20 to 70 microns, as elementary bodies of 0.2 micron, and in coccoidal forms of 0.5 microns or larger. The latter are usually gram-positive and resemble common micrococci but are distinguishable by variation in size and the sprouting of filaments or spicules. The organism may also appear in amorphous mycoplasmalike forms, as rods or filaments of varying lengths, and in older cultures, as spores and hyphae.
Cultures made from animal tumors and fluid have great similarities with cultures derived from many types of fresh uncontaminated human tumors, from blood and other body fluids of patients who have advanced cancer.
The L-forms are bacterial forms without cell walls. They resemble pleuro-pneumonilike organisms (PPLO), also known as mycoplasma. However, the mycoplasma appear to reproduce continously under some conditions in the same stage, with the absence of cell walls, while other organisms have a tendency to revert more quickly to the more stable bacillary or coccal forms of origin. The L-forms are the link between bacteria and the viruslike minute bodies that are a stage in the life cycle of certain microorganism. Many viruses may actually be L-forms of microbes which, under certain conditions may be induced to return to their original forms. Previously the appearance of the both adult and L-forms led to the erroneous conclusion that there was a mixture of microorganisms, a contamination of pure strains with other nonrelevant microorganisms, but this was shown to be an erroneous misconception. Some true contaminants are readily recognized by their growth pattern but the Cryptocides is a great simulator of other organisms. It requires infinite patience to observe its growth pattern and to recognize its transition from one form into another.
The microorganism involved requires definitive bacteriologic media for its primary isolation, differential staining techniques for its identification, high power microscopic resolution, and the electron microscope to reveal its most minute forms. Specific cultures can be obtained on solid media used for the isolation of the tubercle bacillus. This mycobacterium-like organism is believed to be a primary etiologic agent in proliferative and degenerative diseases such as cancer and perhaps many other socalled autoimmune diseases. Cryptocides is believed to be the causative or infectious agent (microbial) of cancer, in all of its forms, Cryptocides would therefore be called an antigen. More specifically, the filterable forms of P. Cryptocides which are of virus size are the causative agents in human and animal cancers.
Peyton Rous did not call his tumor filtrates (from chickens) viruses but instead "tumor agents." His material could be dried and held on a shelf at room temperature for months and then, mixed with saline, it could be reactivated to initiate fresh tumors. A true virus has been defined as a submicroscopic infectious unit that lives only in the presence of living cells and cannot exist even momentarily outside of them. Many have tried to find a virus implicated in some form of human cancer, but none has been found.
Applicants have grown the Rous tumor agent in sterilized beef broth that contained suitable nutrients for bacterial growth, and traced its growth pattern through all of the bacteriological stages. Applicants knew that the infectious agent passed through filters that permitted only the passage of so-called viruses. Applicants filtered the cultures, not the extracts of the tumors, through bacteria restraining filters and studied these with a electron microscope. Applicants kept the cultures in which there did not appear to be any visible form of life, incubated them at 37.degree. C. and from these seemingly clear broths with the agent in them, there arose the bacterial and fungal stages of the cryptocides. Applicants ruled out contamination that might account for the bacterial growth on incubation by repeating the experiment dozens of times. It was a tedious process but proved that this so-called virus could and did convert to a bacterium that had not only submicroscopic forms but also bacillary, coccal or round forms, and that could also develop funguslike stages and spores. (On studying the growth of the tubercle and lepra bacilli these stages were entirely comparable with the Cryptocides.)
When applicants examined the cultures obtained from human cancers, there was no discernible difference in the growth pattern. The growth pattern of the chicken cancer isolates and that of man were the same. They grew in the same kind of broth in the same way and they appeared the same in chicken and human tissues. They had the same staining properties with the Ziehl-Neelsen dye. Applicants did sheep immunization studies in which they found significant cross-agglutination between the Rous sarcoma, fowl leukosis and various strains of human cancers. When applicants injected the isolated cultures into mice, the characteristic disease and lesions developed. The Rous isolates had to be readapted to chicken tissues by passage on the allantoic membrane of fertile eggs and then replanted into young chicks. Applicants also carried on immunization of rabbits with the leuokosis agent and used the antiserum to cure chickens dying of fowl leukosis. In every way the Rous agent appears to be a prototype for human cancer.
The above studies led to the cultivation of the same kind of microorganisms from other animal tumors. These invariably grew and appeared similar to the Rous and human strains. Sometimes there were differences in size or different sugar or oxygen requirements for cultivation, but essentially they were the same basic type or organism.
The cancer organisms (cryptocides) appear to resemble mycoplasma, organisms that exist without cell walls, expecially since the cytosinguanine ratio of their mucleic acid, DNA, is similar to that of certain mycoplasma. However, the usual mycoplasma tend to remain in their state of existence without cell wals but the Cryptocides may pass rapidly through the stage without walls to the form of true bacteria. Perhaps all mycoplasma could be induced to become bacteria but this is still a disputed point.
Dr. Robert Huebner, head of the National Institute of Allergy and Infectious Diseases, Bethesda, Md., has stated that cancer is a viral infection. Of the various agents suspected, he stated that the C-particle is the most likely agent. It has been called by this name because the round bodies found in cancerous tissues often appear in a C shape. However, the comparison of the C-particles in mouse leukemia with the filted Cryptocides isolates examined under the electron microscope show them to be similar in size and shape. In preparations from cultures the round forms are often seen to split and assume the C form. It would seem that this splitting into a C is characteristic, but not necessarily a method of identification. All the other methods are necessary as well.
The cancerous growth itself is not the entire disease. The small coccuslike granules which can be seen dividing in cancer cells represent the intracellular parasite that is the causative agent. The parasite within the cancer transforms the normal cell into a sick cell that cannot mature by differentiation. It is the filtered material from tumors and other cancerous growths, as well as the cells themselves, which transmit cancer from one species to another species. Or, in other words, the cancerous agent can cross species barriers and infect other species.
P. Cryptocides not only causes cancer but a number of other ailments that effect man. The infectious nature of arthritis, of some kinds of heart, liver and kidney impairment, and most recently of diabetes is known. The patterns of these diseases point to their latent infectious nature. Cryptocides is an infectious agent. But, the tumors are only a part of the resultant disease. In addition to tumors, there are cheesy lesions or areas resembling tuberculosis, which can involve any one of the essential organs such as the liver, kidney heart or lung. These organs might show changes in the connective tissue, called collagen, which can lead to degeneration as seen in the chronic human degenerative diseases. The organism may assume a latent form and be inactive as long as the body's defense mechanisms are adequate, but when they are not, disease results. The exact kind of disease depends on the age of the host and its state of resistance, as well as the strain of the organism.
All cancerous bloods examined have revealed the cryptocides organisms. Applicants have made a film of untreated blood from a terminal cancer patient in which the parasites are seen in Brownian motion in the red cells. The parasites stay inside the cells of patients who are holding their own against the disease, but in advanced cases, the numerous cells rupture, releasing the organisms.
The microorganism is apparently ubiquitous in nature, existing in a reservoir in soil and water, and is found in all classes of animals, including man. It can exist as a latent infection in host tissue without causing apparent ill effects. However, when the immunologic barriers are lowered it can invade the host in prodigious numbers and involve any or all of the host tissues, causing various kinds and degrees of pathologic change equilibrated between the ferocity and numbers of the invader and the ability of the host to resist them. Not only is the organism pleomorphic but the pathologic changes induced in experimental animals show varying degrees of disease ranging from the lethal through the semi-immune, neoplastic and degenerative stages. Hyper-immune and degenerative stages may be relatively quiescent but can become slowly and progressively fatal.
Man and/or animal can be a latent carrier of the Cryptocides. Many of applicants' experimental animals that have surived cancer have developed interstitial collagen disease as a result of their inoculations with applicants' bacterial isolates of Cryptocides, and also developed heart lesions. When baby mice born of infected mothers died, the autopsy showed destruction of heart muscle. These lesions contained the acid-fast organisms (Cryptocides) in the heart muscle. Also, a number of research people in England have reported strange microbic bodies previously unrecognized in the hearts of people who died of coronary disease. One of the applicants had cancer of the forehead treated successfully with radium fifteen years previously, but was a latent carrier of the Cryptocides. That applicant was treated after that period of time with an autogenous vaccine, has had a new vaccine prepared every year, and has continued treatment.
"Mycobacterial Forms in Myocardial Vascular Disease", Virginia Wuerthele-Caspe Livingston and Eleanor Alexander-Jackson, (1965) proposes the theory that there are microbic bodies in the lesions of heart diseases and that they are especially numerous in the areas where the blood vessels have ruptured. Until recently the theory has been the coronary blood vessels of the heart are narrowed due to arteriosclerosis, and that fatty deposit in the wall of the vessels, and overweight are the determining factors in this type of heart disease. Now the medical researchers are becoming aware of the fact that the blood bessels themselves are often not involved so much as the supporting tissues and muscles of the heart so that the heart vessels rupture due to extrinsic factors outside the vessel rather than from intrinsic disease. This is particularily true of patients with collagen diseases such as scleroderma and lupus erythematosis. Vascular and myocardial pathology is related to chronic low-grade infection by the mycobacterium-like organisms (Cryptocides).
Degenerative changes occur in coronary heart disease in the presence of the invasive mycobacterial parasite cryptocides.
Postmorten heart sections of 6 patients with coronary and aortic disease were stained by the Fite modification of the Ziehl-Neelsen technique (for demonstrating Lepra bacilli in sections) using Kinyoun's carbon-fuchsin, and compared with sections of the same involved areas stained with conventional H and E. Eight predominant types of lesions were observed in the myocardium
1. PERIVASCULAR CHANGES AROUND THE SMALL CORONARY VESSELS. In the loose connective tissues numerous small acid-fast bodies can be seen.
2. CELLULAR INFILTRATION. This is frequently seen not only around the vessels but between the muscle fibers as well. These cells consists almost entirely of mononuclear types, predominantly lymphocytes, while large mononuclear phagocytes laden with organisms plasma and other mononuclear cells are present in relatively large number.
3. FIBROBLASTIC INFILTRATION. The presence of these organisms appears to stimulate the formation of fibroblasts. In some areas, the muscle fibers and interstitial tissues appear to be replaced by fibroblasts.
4. INFARCTION. Where there has been an infarct, there may be a softened central area with numerous small acid-fast cocci and coccobacilli present in the collagenous hemorrhagic softened area. 5. NECROSIS. Necrotic changes may involve the blood vessels. Striking degenerative changes of the vessel walls are observed as illustrated not only by the sections of coronary vessels but also by the sections of in involved aorta. Proliferative changes may involve the endothelium, with invasion of the endothelial cells, and are accompanied by thickening and narrowing of the wall. Hairlike filaments of the organisms were seen protruding into the lumen. These changes are also present in the vasa vasorum of the aorta.
6. THROMBOSIS AND RECANALIZATION. Some areas of recanalization were observed in heart, liver, and spleen.
7. CHANGES IN THE ELASTIC LAYER OF THE AORTA. The elastic fibrils have lost their identity and have become collagenized with loss of structure. As scar tissue forms, cholesterol-like plaques occur. It seems possible that deposity may be derived in part from the fatty envelopes of these organisms. In other tissue where masses of the organisms have proliferated, polyhedral crystals resembling cholesterol have been observed.
8. CHANGES IN THE HEART MUSCLE. Individual nuclei of the heart muscle are frequently parasitized, and replaced by small acid-fast globoidal bodies. The muscle fibers themselves appear in a state of gradual digestion and disintegration by both minute and larger acid-fast forms.
All of the above can be treated and detected by this invention
Neoplastic changes have been shown by Diller and Diller (Intracellular acid-fast organisms isolated from malignant tissues, Trans. Amer. Micr. Soc., 84:138-148, 1965), to arise in tissue culture as the result of exposure to this specific invading microorganism.
The Journal of the American Medical Association, July 28, 1969 Vol. 209, No. 4, contains a summary of the work of K. A. Bisset, New Scientist, June 12, 1969, who speculates that many diseases like leukemia and arthritis could be caused by Mycoplasma or by forms of this elusive bacteria and wrote that the fact that mycoplasm can break down into viruslike particles, easily identifiable on electron-microscope examination and similar to those found in blood of leukemia patients, leads to a strong suspicion that Mycoplasma may be a culprit in the development of certain malignant processes.
Dr. Florence B. Seibert, Veterans Administration Research Laboratory at Bay Pines, Fla., has reported immunologic studies with the organisms. Labeled antiglobulin, which was specific for an isolate from a human breast, adenocarcinoma induced specific fluorescence in the white blood cells of patients with leukemia and myeloma, demonstrating an immunologic relationship.
Koch's law is the foolproof method of proving the cause of a disease. It is as follows:
1. The microorganism must be present in every case of the disease.
2. It must be possible to cultivate the microorganism outside the host in some artifical media.
3. The inoculation of this culture must produce the disease in a susceptible animal.
4. The microorganism must then be reobtained from these inoculated animals and cultured again.
Applicants have fulfilled Koch's law using pure, uncontaminated cultures of cryptocides. Pure cultures were obtained repeatedly from the various proliferative and neoplastic diseases of both men and animals. Then they were injected into animals capable of being infected. Gradually diseased areas developed which resembled those from which the cultures were obtained. Then the pure cultures were reisolated from the infected animals. Thus Koch's postulates were fulfilled.
A blood specimen of a terminal cancer patient was cultured, the culture was extracted and the extract produced tumors in mice. This demonstrates the growth factors.
In one attempt to produce antibodies and antiserum in sheep that would be beneficial in the treatment of human cancer, sheep were immunized with an attenuated or weakened culture. Twenty sheep were examined and found to be free of disease. Some of the stock cultures applicants had on hand such as cultures from human breast cancer, from a sarcoma of a young boy, from a human leukemia, from the Rous chicken tumor, from arthritis, and from fowl leukosis, were attenuated. Applicants injected two sheep with each strain. After about four weeks, some of the sheep became sick. Attenuated vaccines from the cancer cultures were used weekly for immunization. Several ewes aborted their young. The fetuses were macerated. Some of the sheep developed very swollen painful joints and could scarcely graze. Others looked poorly because emaciated. Applicants realized that the vaccines which were attenuated were still alive and had not fully immunized the sheep but had diseased them. The sheep were bled in order to assay their serum for antibodies. Sera was obtained but the sheep had to be destroyed. Although the sheep had to be destroyed applicants learned that the fowl leukosis serum agglutinated in high dilution the cultures from the boy's sarcoma, that the breast cancer serum reacted with the human leukemia isolates, and that the Rous sarcoma serum reacted with all of the cultures. This meant that the cultures from the human cross-reacted with one another strongly and with the animal sera, showing that tumors are not tissue or species specific.
Three chickens having fowl leukosos, a cancerous disease, and which could no longer stand up, were taken to applicants' laboratory and in a short time they were dead. Applicants made cultures from their heart's blood. These grew to be the same kind of cultures as those derived from all of the other tumors experimented with by the applicants.
A pure, selectively grown bacterial culture of the type described above, obtained from urine and blood using sterile precautions, contains chemical substances related and/or identical to the actinomycin group. To test this point, a phenolized pure culture was acidified with HCl to pH 2 (Congo red/thymol blue) and was left standing overnight after short boiling. A mixture of n-butanol/conc. NaCl, equal parts by volume, containing a few drops of glacial acetic acid, was used for extraction. After gentle shaking for about 15 minutes a dark cherry-red layer of the solvent was separated for further processing. This crude mixture gave peak absorption at 440/450 mn and 410/425 mn values which compare favorably except for a third absorption peak at 240 m.mu. obtained by Waksman (Waksman, S. A., and Lechevalier, H. A.: The Actinomycetes, vol. III, The Williams & Wilkins Co., Baltimore, 1962, p. 168) which was missing. From an ascending paper chromatogram a reddish zone was eluted with ethylacetate and an acetone-ether mixture, both gave upon evaporation some microscopic crystals (red plates). The controls containing broth and 2% phenol but no organism gave upon extraction a barely yellow-tinted layer of the solvent. This exploratory separation technique was then repeated with several 25-hour urine specimens obtained from terminal cancer patients. After separation of the organic layer from the urine specimens again a more or less pronounced color was present ranging from dark honey-brown to cherry red; controls taken from healthy persons did not contain such colors. The presence of these dark colored compounds seems to be most pronounced in terminal cases. All crude mixtures isolated from cultures and/or urine were subjected to further separation by chromatographic techniques.
METHODS FOR THE ISOLATION AND IDENTIFICATION OF A PLEOMORPHIC INTERMITTENTLY ACID-FAST ORGANISM FROM NEOPLASTIC DISEASES, AND THE PREPARATION OF CULTURES
Isolation from Urine (Crofton Method)
Obtain a midstream clean-catch specimen of urine in a sterlized screw-top glass container.
Make up DiFco's brain-heart infusion agar: 37 grams of the agar base are added to a liter of distilled water, heated to melt and mix, and distributed into flasks or bottles of 95 ml amounts, and autoclaved. Five percent (5%) human blood (outdated bloodbank blood may be used) is added when the melted agar has cooled down to 45-50 degrees C., and the mixture is poured into sterile Petri dishes.
Streak the surface of the blood agar plate with a sterile swab which has been dipped in the urine. Incubate plate to 37 degrees C. and examine after 24 hours. If growth has appeared, note types of colonies, make duplicate smears, and stain one by Gram's stain and the other by Alexander-Jackson's modified Ziehl-Neelsen technique: flood side with Kinyoun's carbolfuchsin for 3 to 5 minutes in the cold, wash, decolorize briefly with 70% alcohol containing 1 to 3% HCl as these organisms decolorize more readily that M. tuberculosis, counter stain by flooding slide with Loeffler's methylene blue and add 6 to 8 drops of normal (4%) sodium hydroxide. Tilt slide to mix, and wash after 30 seconds.
APPEARANCE OF THE MICROORGANISMS ON BLOOD AGAR
Colonies--usual types of growth obtained
1. white discoidal, often hemolytic, and with a raised center--having a fried egg appearance, but usually larger than classic PPLO colonies grown on PPLO agar.
2. grayish muccoid, often confluent.
3. pigmented: yellowish, occasionally pinkish coral.
4. wrinkled intermediate SR worm-casting type resembling M. tuberculosis colony.
5. dull granular surfaced, irregular edges, often hemolytic, and resembling B. subtilis, but virulent for mice. Motile forms transferred tp A-J broth produce a white or grayish white soft rim or pellicle, and a toxin-like substance.
This organism tends to resist emulsification to some degree when a loop of culture is rubbed with a drop of water on a glass slide to make a smear.
MICROSCOPIC APPEARANCE
The cancer isolate is either Gram positive or Gram variable. The Gram stain is not the stain of choice, but should be used to eliminate true Gram negative organisms, which show no Gram positivity at all such as B. coli, proteus or pseudomonas.
Acid-fast forms may or may not be found, as this organism is intermittently acid-fast rather than more consistently so as in M. tuberculosis or other classic mycobacteria. However, if possible, careful search for acid-fast forms is desirable, as they are hallmarks of this mycobacterium-like organism. Slender filaments, sometimes with lateral branching, and sometimes acid-fast, help to distinguish it from common micrococci. The rods may be slender and diphtheroidal, or thicker and subtillis-like. The latter sometimes contain tiny brightly acid-fast bodies surrounded by a colorless vacuole-like area or capsule. This appearance plus virulence for mice and guinea-pigs distinguish them from subtillis rods. The main morphologic forms are:
1. tiny acid-fast elementary-type bodies, often refractile;
2. Coccoidal forms of varying sizes with or without threads or protruding filaments;
3. rods as described above; X, Y, and V forms commonly seen, filaments may become very long and wide;
4. cyst-like bodies of various sizes from 3 to more than 10.mu. and often containing smaller bodies which may be brightly acid-fast;
5. L or PPLO forms consisting of a lightly stained matrix containing more deeply stained bodies of various sizes, or tangled branching threads and ring-forms. These are revealed by Alexander-Jackson's Triple Stain modification of the Ziehl-Neelsen technique;
6. spore forms of oval shape seen in old cultures;
7. sub-microscopic bodies 20-70 m.mu. revealed by the electron microscope, and of virus size.
The product of the growth of the specific Actinomycetales organism is a chemical substance which is obtainable on recrystallization (Wolter)--of a suspension of the isolate with conventional paper chromatography annular separation procedures, and identifiable as comprising a formation of
(a) red crystals identical with those described by Waksman as having the absorption peaks indicated, supra;
(b) yellow crystals, similar to an actinomycin D fraction;
(c) formation of small placques of crystals similar in appearance to gramicidin;
(d) a waxy, higher-alcohol formation, and
(e) a brownish, foul aromatic residue.
CHROMATOGRAPHIC IDENTIFICATION
To an acidfied (pH 5) sample of urine in which the organisms are grown, (phase I) and kept in the refrigerator, is added one-fourth volume of n-butyl alcohol and the mixture is shaken for one-half hour. The mixture is refrigerated until the layers separate. Separation is done by decantation first and then by using a separatory funnel. The butanol layer has attained a reddish brown color and in some cases a yellowish color. This procedure results in an aqueous phase (II) and a butanol phase (III). The aqueous phase is extracted once or twice more in the same manner with n-butanol, so long as the color appears in the extract.
A portion of the combined extracts is evaporated in an evaporator at 35.degree.-40.degree. C. The dried residue is dissolved in a small amount of methyl alcohol, solution (IV). Layers of a silica gel preparation, MM-SGel-HR for thin layer chromatography, Machorey Nagel and Co. 516 Durem, Germany, is spread on glass plated, prepared, dried and stored in a dessicator. By means of a small pipette, a spot of solution (IV) is placed on the silica gel layer of one of these plates near one edge and near this spot at the same distance from this edge and in the same way, another spot of actinomycin D, (Merck, Sharp and Dohme), is placed in the same manner. Both spots appeared bright yellow. Other pairs of spots of these two solutions are placed on the same row using 5 m.mu., 10 m.mu. and 15 m.mu.. (One m.mu. equals one microliter.) Of the solvents tried for developing the chromatograph, the most effective was butanol-methanol-water in the ratios of volume of 6:1:3. 60 ml. of butanol, 10 ml methanol and 30 ml of distilled water are mixed and put into a thin layer chromatography chamber. The paper lining the walls of the chamber is wet by swirling the solution in the chamber. Then the plate with the spots is placed on edge in the chamber with the row of spots parallel to and near the bottom but above the surface of the solution. A cover is placed over the top of the chamber sealing it. The chamber is kept in the dark during the process of separation since a better yield elution is obtained. In previous runs each actinomycin D spot travelled as a single spot leaving nothing in the pathway by visible or ultraviolet light, and phase (III) left material spread from the top streaking down along the pathways. However the top was always at a level with and the same color as the actinomycin D. Subsequently, after drying the plates, the actinomycin D spots and those portions from the spots of phase (III) on a level with the actinomycin D spots are cut out and separately eluted with methanol, as are other portions from the spots of phase (III), since these may contain other actinomycins than actinomycin D. Visible ultraviolet and infrared absorption spectrograms are made of the different elutes and compared. The TLC method is based and adapted from methods reported by Cassani et al, J. Chrom. v. 13, 1964, 238-239.
ACTINOMYCIN BIO-ASSAY
Blood and tissue cultures and urine samples obtained in accordance with the foregoing procedures were extracted with and equal volume of butanol, water, acetic acid (4:5:1) and the upper phase taken to dryness. The residues were taken up into two 5 ml. portions of ether and evaporated at 35.degree.-40.degree. C. Paper discs were dipped into ether solutions of extract, the ether evaporated and the discs placed on standard actinomycin assay plates. A standard preparation of actinomycin D was also run. The zones of inhibition are shown in Table I; zones above 15 mm. in diameter fall within the standard range and are calculated in terms of actinomycin D equivalent.
TABLE I
UV ABSORPTION CURVES
0.2 ml. of each ether solution was evaporated to dryness and the residue taken up in 1 ml. of methanol and UV absorption curves were recorded. End absorption at this level of purity prevented measurement of 440 mm. absorption. Small peaks, typical of trace amounts of actinomycin, were found with samples 1-6 inclusive and sample 8 with a slight response present with sample 17. Definitive biochemical tests such as the cytosine-guanine percentage of the DNA have helped to classify the cryptocides microbial isolates.
Further concerning cryptocides, these organisms, which appear primarily as small acid-fast granules in young cultures, and which tend to become non-acid-fast in the larger forms present in older cultures, may exhibit a number of morphologic phases with intermediate transitional forms. These include (1) filterable and submicroscopic bodies; (2) larger granules readily visible under the light microscope an often resembling ordinary micrococci; (3) larger globidal cystlike bodies and thin-walled sacs containing the smaller forms; (4) PPLO or L type zoogleal symplasms without cell walls; (5) rods of various sizes capable of developing a characteristic motility; (6) long filaments and threads which may show lateral branching; and (7) thick-walled spore-like bodies. The lesions produced by these organisms in experimental animals were generally pseudocaseous, degenerative in type, occasionally neoplastic, and occurred principally in the liver, kidneys, and lungs although at times, there was involvement of the heart, spleen adrenal glands, stomach, lymph nodes, and omentum.
Dr. Afton Munk Livingston, and Dr. Virginia Livingston, Transactions of the New York Academy of Sciences, May 1972, report the recognition of the P. cryptocides organisms in the blood of cancer patients compared with the blood of healthy individuals, of which a summary follows. Examinations by darkfield microscope of fresh blood, and also by brightfield microscope using supravital stains serving as a diagnostic and prognostic tool in following the course of the cancerous disease in the patient in conjunction with several other microbiological evaluations.
PREPARATION OF SLIDES FOR BLOOD EXAMINATION
The patient's finger is immersed in 70 percent alcohol and air dried. A steril lancet is used to puncture the finger, a small drop of free-flowing blood is placed on a sterile clean slide and covered with a sterile covership. Care is taken that the blood does not flow beyond the edge of the coverslip. Using a small weight for approximately one minute, light pressure is applied to the coverslip to spread and separate the blood cells. The preparation is then examined under darkfield at .times.750 and .times.1350 magnification. For lightfield examination, the same method is followed and in addition, a small drop of 1 percent aqueous sterile crystal violet, freshly prepared and filtered, gently applied to the preparation. If the number of organisms, to the blood as well as the motility of the various stages are to be evaluted, then the blood is diluted 1:100 with sterile distilled water using a sterile red-blood-cell diluting pipette. The pipette is then thoroughly shaken and a few drops are expelled from the pipette into a sterile Petri dish. A small measured amount of 1 percent aqueous crystal violet is added. This mixture may then be used to flood a blood counting chamber. This method provides a quantitative estimate of the numbers of the organism as well as their motility, which may last as long as fifteen minutes. However, for the usual brightfield examination of the blood with crystal violet, the blood drop is placed directly by the slide and the small amounts of crystal violet is added before the coverslip is placed over the preparation and light pressure applied.
DARKFIELD EXAMINATION OF UNSTAINED FRESH BLOOD PREPARATION
A number of interesting observations may now be made by the darkfield, pulsating orange bodies in the red cells may be observed. In the background, there are bright dancing forms which appear to be small L-forms of the organism. In several infected hosts a number of motile rods may be observed. Spheroplasts and mesosomes both large and small are present. These may have many fine delicate vibrating forms in their periphery. Forms resembling a medusa or a octopus with waving filaments may be present. Organisms may bud from the surface of the red cells and from fine hairlike filaments which resemble the handle of a tennis racquet. There may also be numerous threadlike filaments free in the serum, varying in size, some 10-15 microns in length. These are motile and appear to wind in and out around the red cells. There are also long tubular structures 50 microns or more in length, and about 10 microns in width that are milky white, highly luminescent, containing numerous refractile granules. The tube in some cases appears to arise from a coalescence of the L-forms or to bud from a spheroplast. It is transparent since cells can be seen through it. When the tube wall disintegrates the refractile bodies are released in the serum and may enter fresh red cells. There are also large round milky white forms appearing to be protoplasts about 20 to 60 microns in diameter which contain granules resembling spheroplasts or mesosomes. The protoplasts may have budding forms at the periphery and may release rather large vesicular refractile bodies resembling the spheroplasts or mesosomes. At times, the extruded mesosomes are large enough to be mistaken for red blood cells, but they do not have the bluish tinge of red cells seen in darkfield. Rather, minute dancing particles may later appear within them.
In addition, shrunked red cells with a ground-glass appearance spiculated at the periphery may be observed. We have termed these structures "spent cells" since they appear to be red cells that have been consumed by the parasites. They are lighter and smaller than normal erythocytes and have a tendency to be pushed to the periphery of the blood drop when it is prepared for examination. Changes in the character of the leukocytes are also apparent. Many leukocytes in the advanced stages of diseases appear smudged, inactive and only dimly luminescent whereas normal leukocytes have vigorously active granules and active amoeboid movements. Under some circumstances great numbers of fine spicules occur in the dark field. These are very delicate and appear to arise from minute L-forms. They are not thrombocytes. At times they appear to shed from the surface of the protoplasts. Why they should be more numerous at one time than at another is not understood but their appearance may be related to the pH of the blood. Orange crystalline forms of the organisms as well as free crystals may also be seen in and around the microbial clusters in the plasma. They apparently arise from the waxy-secretions of these mycobacteriumlike organisms. These are the crystals that have been extracted from pure cultures and urines of terminal cancer patients and that have been used for various types of bioassay.
BRIGHTFIELD EXAMINATION OF SUPRAVITALLY STAINED FRESH BLOOD PREPARATIONS
On a blood preparation stained with crystal violet and examined by the brightfield method a clear white light and a magnification of at least .times.1000 microbial forms are revealed that are not seen in the darkfield. There are large branching fungal forms that are not luminescent in the darkfield. These fungal forms may extend over a considerable area involving several microscopic fields. Some of these are branching and appear to have conidial or frutting bodies attached to their branches. Microcolonies may be clearly seen surrounding individual red cells and some appear to arise from parasites extruded from the cells. These microcolonies appear to develop into a network of interlacing branching fungal filaments which act as bridges between the red cells and cause them to adhere in clumps. The number of fungal forms which hold the erythrocytes together or adhere to their surfaces may be directly related to the sedimentation rate. The greater the adherence of the erythrocytes due to the mycelial forms, the more rapid the sedimentation rate. The red cells become separate and free as the number of both intra-and extracellular parasites diminish. The stained preparations in the counting chamber have L-forms, which appear much more numerous than in the darkfield, and occur in clusters, which have marked Brownian movement. These clusters agglutinate and become motionless after ten to fifteen minutes. Introduction of gamma globulin or specific antiserum under the coverslip of the counting chamber caused instant agglutination and cessation of motion. By this method, antibody activity of blood serum can be roughly estimated. Other dyed microbial forms in the brightfield may be compared with those in the darkfield. The vibrating orange bodies in erythrocytes in the darkfield appear as violet bodies in lightfield. The brightly luminous tubles take on a light violet color with deep purple granules.
The same comparison between darkfield and stained brightfield preparations may be drawn by examining blood cultures grown in broth. Hanging drops of cultures sealed with sterile vaseline are preferable to ordinary wet perparations since they are safer to handle and can be preserved for a longer period of time. Conventional staining of slide preparations appears to break up many of the delicate microcolonies and interlacing fungal forms. Wet supravitally stained preparations in hanging drops also indicate the degree of motility of many of the microorganisms. Other dyes have been used which penetrate to some extent but do not provide sufficient contrast. They are Sudan black, saffron yellow, Congo red, May Grunwald, toluidine blue, gentian violet, as well as several others.
All cancerous patients yielded L-forms as well as other pleomorphic stages on blood culture which, on further cultivation, developed the typical acid-fastness of the Progenitor cryptocides group as previously described. However, the cancer patient even in the advanced stages of the disease is usually afebrile. Comparable numbers of microorganisms other than the Progenitor cryptocides groups might be expected to produce an acute febrile reaction. There undoubtedly can be a mild or transitory bacteremia in blood due to relatively nonpathogenic bacteria such as some of the diphtheroids. However, with the previously described methods, the great numbers of the Progenitor group as a silent but lethal bloodstream infection may be readily demonstrated. Advancing infection of the bloodstream with P. cryptocides is relatively asymptomatic until large numbers of the organisms are present and there is a concomitant breakdown of the immunological and dextoxifying system.
The autogeneous vaccine is known to exist by disc-saturated inhibition on culture plates of extracts and also from the serum of cancer patients.
Administration of the autogenous vaccine should be initiated by high dilutions of the lowest order of dosage at twice weekly intervals, with gradually increasing dosages until overdosage symptoms occur. Preferably, subcutaneous injection of the autogenous vaccine in a suitable pharmaceutical carrier, such as sterile water or saline solution may be employed, although oral administration of the product in a suitable carrier also may be employed.
Use of the autogenous vaccine of this invention may prove to be of value in the palliative treatment of animals and humans afflicted with various forms of neoplastic diseases, as indicated by treatment thereof with autogeneous vaccines made from a suspension of the isolates in 2% phenol. In preparation, the vaccine is allowed to stand overnight at room temperatures, centrifuged and further diluted with 0.5 phenol or saline.
Subcutaneous injection is initiated with the highest dilution of 1 million organisms per ml and 0.1 ml twice weekly, until overdosage symptoms occur. Therapy is continued with higher concentrations, e.g., 10 million and 100 million organisms/ml. Oral administration of the same dosage can also be employed.
A study of one hundred random blood samples, taken in the office of a physician who specialized in allergy and immunologic disease, showed that all tumor-bearing patients, in comparison to office personnel used as controls, gave positive cultures for the cryptocides organism. A number of patients with chronic degenerative disease were also positive. While many patients who had reached a healthy old age were negative, several "tired" young people without apparent disease were positive.
A reddish brown material has been extracted from the tissue, urine and blood of cancer patients in increasing amounts as they became terminally ill, and (this material has not been found in normal controls. It is carcinogenic for mice, increasing the incidence of pulmonary tumors. The biological effects have been assayed in preliminary studies with tissue-culture systems and with tumor-genesis in mice.
The applicants have found the presence of actinomycin-like crystals in body tissues and in cultures.
The basic requirement for formation of the cancer cell is the causative microorganism; all other factors such as coal-tar irritants, other microorganisms, the aging process, any chronic irritants leading to poor local resistance and giving rise to immature, succeptible reparative cells, may prepare the living matter, e.g., for the multiplication of the cancer organism and its penetration into the cyptoplasm and nucleus of the host cell.
Apparently the organism cryptocides can invade both cytoplasm and nucleus of host cells in any type of host tissue when body defenses are lowered. In experimental animals it can cause lesions that appear as necrotic abscesses, granulomas, fluid-filled cysts of neoplasms. The type of lesion apparently depends on specific and nonspecific immunocompetence and the age of the host.
Certain chemicals can have a tremendous effect upon the entire hormonal system. One of these substances is actinomycin produced by several of the actinomyces organisms and probably by many of the Actinomycetales. There is a whole array of chemicals and biologic produced by this group of microbes, which have been used as antibiotics and antineoplastic agents in some cases. The actinomycins even in very high dilution of one part in a billion or more may have a profound effect upon the entire business of life. The important thing to remember is that no funtcion of the body is exempt from this toxic material which is produced by these microorganisms belonging to the Actinomycetales. Not only are the normal functions of the host's hormonal system deranged but there are "false or counterfeit hormones" produced with further throw the body off balance. There is a practice of castrating men and women to arrest the growth of cancer. If castration is successful in prolonging life, then adrenalectomy and pituary gland removal might be done when the effects of tumor inhibition from the castration have worn off. This hormonal ablation presents a grim picture to say the least. Applicants believe that the hormonal stimulation of the sex glands, the adrenals and the pituitary are the result of toxic materials, hormonal derangers and counterfeit hormones, such as, phytosterols produced by the Cyptocides, that upset the balance of the patient's hormones not only by inhibitory effects by production of pseudo or counterfeit hormones that act on the physiologically controlled, normal glands causing abnormal response. Also various kinds of cell poisons and inhibitors destroy the efficacy of the lymphocytes to attack the cancer cells. The cancer cells themselves are prevented from reaching maturation by these cell poisons They are sick cells unable to reach a normal maturity and normal function, whereever they are located and whatever tissue they may be whether glandular, interstital, bone or blood.
The most important thing is to try to destroy the microbes that were producing the aberrant cell inhibitors and false hormones. However, it has been reported that low testosterone levels have been induced in patients with cancer of the prostate by treatment with diethylstilbestrol, a synthetic hormone; and amino-glutethimide, a powerful inhibitor of adrenal corticosteroid biosynthesis, with patient improvement. Furthermore, an immunological mechanism appears to be involved; the inhibition of steroid biosynthesis. By removing the lympholytic effect of corticosteroids, there is produced a marked hyperplasia and increase in the number of circulating lymphocytes which potentiate the immune response. The presence of lymphocytotoxic antibodies has been reported in patients with prostatic cancer. Perhaps this steroid is a "false steroid" and antagonism by the amino-glutethimidine and diethylstilbestrol may permit an increase in the production and circulation of normal lymphocytes capable of attacking the cancer cells.
The role of steroids in chronic diseases was demonstrated by Edward Kendall and Philipp Hench in their studies in rheumatology for which they received the Nobel Prize in 1950. It is true that the steroids do not have an inhibitory effect on these diseases but at the expense of suppressing immunity and permitting the underlying latent infection to continue or to increase in its growth potential.
It is known that a bacterium belonging to the Actinomycetales was able to produce unlimited amounts of steroids from the Mexican yam.
It is stated that some steroids decrease the numbers of circulating lymphocytes as well as blocking immunocompetence. Perhaps the "false steroids" are really responsible for this action. It has been shown that certain toxic antigens prevent the lymphocyte from maturing and become immunocompetent. Leukemia, or an accumulation of large numbers of cells, either lymphocytes or polymorphonuclear leukocytes, may represent a blocking of the pathway to maturity by a toxic agent such as a steroidal or actinomycinlike compound produced by the cryptocides. Perhaps the blocking factor may be related to a protective mechanism directed toward making the cryptocides insusceptible through some biochemical fraction that blocks the immune reaction of the lymphocyte.
The present invention is useful in the treatment of man and/or animal. Safety and effectiveness of the present invention has been demonstrated in animals and has been indicated in the treatment of humans by administration of the aforesaid vaccine form.
Among the various neoplastic diseases (often termed diseases of unknown etiology) subject to palliative treatment are cancer, tumor of the lymphoid tissues, Hodgkin's disease, reticulo-endothelial tissues, arthritis, lymphosarcoma, the broad spectrum of epidermoid cancer, scleroderma, adenocarcinoma, fibrosarcoma, liposarcoma, myosarcoma, acute glomerulonephritis, leimoya sarcoma, osteogenic sarcoma, chondro sarcoma, myeloma, rous chicken sarcoma, coal-tar-induced cancer, fowl leukosis, animal tumors such as Rous, Walker, Sprague-Dawley, Shope and Sarcoma 180, and the like. Many of the foregoing are degenerative and antoimmune diseases.
Malignancy is a neoplastic infection, which depends on the number and virulence of the invading organism, the susceptibility of various organs to it, as well as the natural immunologic components of the host.
The reddish-brown crystalline substances extracted from broths containing organisms within this invention are antibiotic to the bacteria and to all strains of the producing organisms themselves.
http://www.rexresearch.com/livingstoncancer/livingston.html
https://www.medicinacomplementar.com.br/biblioteca/pdfs/Cancer/ca-3167.pdf
https://juniperpublishers.com/jtmp/pdf/JTMP.MS.ID.555563.pdf