The Precusor to HPV – ViroLIEgy

In order to understand the deception of virology, it is important to trace every “virus” to its point of origin in order to see that there is no credible proof of any purified and isolated “virus” at any point in its history. Often times, this leads one to the investigation of so-called plant and animal “viruses.” In the case of the human “papillomavirus,” it’s point of origin is tied closely to what is known as the Shope “papillomavirus,” which is yet another invisible filterable “virus” that was assumed to be in the inoculant. This “virus” was “discovered” in rabbit warts by Richard Shope in 1933. If this name sounds familiar, it’s because Shope is the man responsible for “discovering” the swine flu in 1931 and unleashing the fraud of influenza upon the world.

Unfortunately for my home state of Iowa (Shope’s home base as well) and for all of humanity, Richard Shope’s finding of the “papillomavirus” in rabbits paved the way for the “discovery” of the human “papillomavirus” and its inevitable association with cervical cancer. His work created the necessary framework needed for the introduction of toxic vaccines for both the flu and HPV. Sadly, he is celebrated in Iowa for his role in this fraud:

Old Gold: UI Virus Expert Paved the Way for Today’s Vaccine

“Virologist Richard Shope helped discover the cause of the 1918 flu and lay the groundwork for life-saving vaccinations.

As people throughout the world roll up their sleeves to receive COVID-19 vaccinations, they can thank an alumnus of the State University of Iowa’s College of Medicine for his critical role in making such treatment possible.”

“Shope never forgot his Iowa roots. He returned to Iowa City on numerous occasions to speak about advances in virology-related research. His 1931 findings alone were significant, as were later contributions on papillomaviruses. In 1957 he received the esteemed Albert Lasker Clinical Medical Research Award, and in 1963 he was awarded an honorary doctorate degree by the UI.”

https://magazine.foriowa.org/story.php?ed=true&storyid=2058

As can be seen, Shope’s work is bragged about in Iowa. He was also credited in a 2009 New York Times article with helping to pave the way for the HPV vaccine. Amusingly, the article speaks of the many “pseudoscientific myths” which linked herpes, promiscuity, Jewish women not eating bacon, and foreskin smegma as causes of cervical cancer. Sadly, it left out the fact that Shope’s work and the HPV link to cancer is yet another in a long line of pseudoscientific myths:

How a Vaccine Search Ended in Triumph

“Species as different as birds and whales have their own papillomas. There are more than 100 human strains. Many are harmless. Some cause warts on hands, noses or genitals, and some cause cancer. As a result, blame has been laid on origins like toads, witchcraft and God’s anger at promiscuous women.

Against that background of superstition, the two newest vaccines use technologies that sound almost like science fiction.

Gardasil, made by Merck, uses a yeast to grow the proteins that form the outer shell of the virus; every batch of 360 proteins almost magically assembles itself into a soccer ball exactly mimicking the shell’s shape.

Its rival, Cervarix by GlaxoSmithKline, produces the same protein, with the same power, in an insect virus grown in a broth of caterpillar ovary cells.

But each step forward to those techniques was a triumph of hard science over the pseudoscientific myths that for centuries surrounded the disease.

The first was posited by a doctor in Florence in 1842. He noticed that prostitutes and married women died of cervical cancer, but nuns almost never did. Though he might have discerned that it was sexually transmitted, he was thrown off by another fact: nuns often died of breast cancer. His conclusion was that nuns’ corsets were dangerously tight.

One may laugh, but prominent American scientists made a similar error in the 1970’s, noting that many women with cervical cancer had a history of genital herpes. Instead of realizing that it was a coincidence, they erroneously concluded that the herpes virus was the cause. And they were closer to the mark than 1950’s researchers, who had blamed smegma, which builds up under the foreskin of men who do not wash.

Research that could have led them in the right direction was done in the 1930’s by Dr. Richard Shope of the Rockefeller University, who on a hunting trip heard a friend describe seeing rabbits with “horns,” which were actually large warts.

Dr. Shope asked his friend to send some of the horns. He then ground them up, filtered them through porcelain that let only tiny virus-size particles through, and injected the filtrate into other rabbits, which grew horns in turn.

“Incidentally, that’s where the jackalope myth comes from,” said Dr. William Bonnez, who was part of the University of Rochester’s vaccine development team. (Jackalopes, jackrabbits with antelope horns, are made by taxidermists and appear on things like postcards from Wyoming. But references to horned rabbits go back centuries, and their condition probably stemmed from papilloma infections.)

Dr. Shope’s work showed the cause was a virus, but it was not until the 1980’s that DNA amplification allowed a German researcher, Dr. Harald zur Hausen, to pin down papilloma as the cause.

“That was really the pivotal point,” said Dr. Douglas R. Lowy, chief of the cellular oncology laboratory at the National Cancer Institute and part of a team whose subsequent work led to the vaccine. “Before that, the field suffered from the boy-who-cried-wolf phenomenon, as in people would say, ‘Last year you said it was herpes virus, now you say it’s papilloma; why should we believe you?’

Below are highlights from Shope’s 1933 21-page SPV pseudoscience followed by a summary:

INFECTIOUS PAPILLOMATOSIS OF RABBITS

“Our attention was recently called to a disease occurring in wild cottontail rabbits in northwestern Iowa. Rabbits shot there by hunters were said to have numerous horn-like protuberances on the skin over various parts of their bodies. The animals were referred to popularly as “horned” or “warty” rabbits.

Warts from a naturally occurring case of the disease in Iowa were obtained and sent to the laboratory in sterile 50 percent glycerol. These glycerolated warts furnished us our original material for investigation. A little later, in a shipment of a dozen wild cottontail rabbits from southern Kansas, three were found to be affected with the same wart-like disease. To date, out of 75 wild cottontail rabbits received from Kansas eleven have been found to carry one or more warts. These eleven animals serve as the basis for our description of the naturally occurring disease.

Description of the Naturally Occurring Disease

In wild cottontail rabbits the presence of warts has caused no apparent discomfort in our experience and induced no demonstrable evidence of generalized illness. Most of the animals were sacrificed, shortly after their arrival, for pathological material, but four, kept under observation for 7 weeks or longer, at no time appeared ill and were in good physical condition when finally killed. The number of warts on individual animals in our series varied from one to ten in all cases except one. The exceptional animal was almost literally covered with warts and these, when removed at autopsy, were sufficient to fill a 200 cc. flask.”

Experimental Transmission

“No difficulty has been encountered in transmitting the condition to either domestic or wild cottontail rabbits when materials from naturally occurring cases have been employed. The method used is, in brief, as follows:

Either freshly removed warts or those that have been stored in 50 percent glycerol at refrigerator temperature are ground to a fine paste with sterile sand and physiological saline in a mortar. More physiological saline is added to make a 3 to 5 percent final suspension. Such a suspension is then centrifuged and the supernatant fluid, which is only slightly turbid, is removed and used for inoculation. Suspensions prepared in this way remain infectious for at least a month when kept at refrigerator temperature.

Inoculation by scarification was regularly performed in these experiments. Rabbits to be inoculated were shaved on the abdomen or sides and lightly scarified either by needle or by rubbing the shaven skin with a moderately coarse grade of sterilized sandpaper. To obtain discrete warts the former method was employed, while scarification with sandpaper was used when a confluent and massive growth of warts was desired. The scratches were made only deep enough to cause a barely perceptible oozing of blood-tinged fluid. A small amount of the infectious suspension was immediately applied by dropping it from a syringe, and this fluid was
rubbed well into the scarifications by means of a spatula or the flat handle of a scalpel. The area thus inoculated was allowed to become almost dry before the animal was released and put into its cage.”

“In spite of the great size of many of our experimentally produced wart masses, the animals showed no loss in weight and the entire course of the disease was free from any general clinlcal evidence of illness. In their gross appearance the experimental warts in both domestic and wild rabbits have been identical with those seen in the naturally occurring disease. Photographs of experimentally produced warts are given in Figs. 2 to 4.

Experimental warts, as well as those occurring naturally, appear to remain
stationary when they reach 1 to 1.5 cm. in height. One of our rabbits, however, at present, 6 months after inoculation, is carrying a large wart mass which in places is 3 cm. in height. With two exceptions, we have seen no warts retrogress in animals infected in the usual way. In the exceptional animals, one a wild and the other a domestic rabbit, warts developed slowly after an unusually long incubation period. They reached a maximum height of only 2 to 3 ram. between 30 and 40 days after inoculation and in 50 days had completely disappeared. Both of these animals were inoculated with the same infectious suspension and were the only ones so inoculated. In no animal in which growth of warts took place in the usual fashion and in which the lesions reached a thickness of 1 cm. or more have we seen any evidence of retrogression. To date we have had experimentally infected
animals under observation for 6 months only. While there has been no evidence Of retrogression of the papillomata except in the cases mentioned, there has also, so far, been no evidence that the lesions of prolonged standing are acquiring malignant properties. Animals are being held under observation to determine what the ultimate fate of the papillomata will be.

Filtrability of the Wart-Inducing Agent

Warts to be used as a source of infection in the filtration experiments were removed from the 50 percent glycerol in which they had been stored and were washed in three changes of sterile physiological saline. They were then minced with sterile scissors, ground in a mortar with sterile sand, and suspended in sufficient physiological saline to make an approximately 5 percent suspension. Suspensions thus prepared were cleared by centrifugation. The decanted supernatant fluid was usually almost water-clear with only a faint opalescence, and for this reason was rapidly filtrable. 1 cc. of a broth culture of B. prodigiosus was added to each 15 to 20 cc. of fluid just before it was passed through Seitz or Berkefeld filters. The resulting filtrates were tested for sterility in 1.5 cc. amounts. All filtrates recorded were bacteriologically sterile.

The results of the filtration experiments are summarized in Table I. Warts produced by filtrates, recorded in Table I as positive, were as extensive and characteristic as those in the control animals which had
been inoculated with unfiltered suspensions. Furthermore, when domestic rabbits were used as the test animals, filtration, especially through Berkefeld V or N candles, instead of prolonging the incubation period as might be expected because of some possible removal of the filtrable agent by absorption on the filter surface, usually had either no effect or shortened the period. In wild rabbits, from the limited data at hand, it would seem that filtration resulted in a slight prolongation of the incubation period. From the data recorded in Table I it can be concluded that the etiological agent causing warts in rabbits readily passes Berkefeld filters, of V, N, or W porosity but does not regularly pass a Seitz filter when two pads are employed. Filtration through a Seitz filter, using one pad, allowed not only the virus to pass but also B. prodigiosus.

No extensive attempts to cultivate visible microbial forms from filtrates of proven infectivity were made. However, during the investigation active filtrates have been cultured repeatedly in plain and blood broth and on plain and blood agar and such cultures have remained sterile both as regards the test organism, B. prodigiosus, or any other visible bacterial form. While no special media have been employed in these attempts to demonstrate the bacteriological sterility of active filtrates, the results obtained using the media mentioned above, considered with the fact that sections of actively growing warts or films of active unfiltered infectious suspensions have failed to reveal the presence of any constant perceptible microbial form, would seem clearly to indicate that no visible organized agent is etiologically essential to the wart production.”

Routes of Infection

Only the method of inoculation by scarification has yielded constant results in our hands. Inoculation intravenously with infectious Berkefeld filtrates, after first abrading an area of the skin of the abdomen with a sterile needle, led to infection of the abraded areas in two out of four cases. Of the two positive animals, one, a wild rabbit, developed only a single wart; while the other, a domestic rabbit, developed four warts on the abraded area and two on the back of the neck. The incubation period in both of these cases was over three times as long as that of the control animals infected by scarification. At autopsy, all four intravenously inoculated animals were free from visceral pathology ascribable to the wart-inducing agent. Inoculations of either wild or domestic rabbits intraperitoneally, subcutaneously, intratesticularly, or intracerebrally, with filtrates of proven infectivity on scarification, have yielded entirely negative clinical and pathological results. About 50 percent of the intradermal inoculations resulted in wart formation although in these instances the warts appeared not at the point where the inoculum had been deposited but at the point where the needle had pierced the epidermis and where some of the inoculum had leaked from the needle tract. The incubation period of warts produced in this way was always longer than when infection had been accomplished by scarification.

Attempts to Transmit the Wart-Producing Agent in Series through Rabbits

In all, twenty-six domestic and wild rabbits have been inoculated in the usual way with suspensions prepared from experimentally engendered domestic rabbit warts ranging in age from 1 to 116 days. Not only did all such inoculations yield negative results but the animals, when subsequently tested, were found to be still fully susceptible to infection with the wart-producing agent from wild rabbit papillomata. On the other hand, either naturally occurring or experimentally produced warts from wild rabbits proved readily transmissible to either wild or domestic rabbits. Warts from nine naturally occurring cases of the disease in wild rabbits have been tested and all found to be infectious for both wild and domestic rabbits. In like manner, experimentally produced warts from nine wild rabbits have been tested for infectivity. Eight of these proved infectious for either domestic or wild rabbits while the warts from one proved to be non-transmissible.

We have not yet attempted to pass the wart-producing agent through a long series of wild rabbits but in the course of obtaining fresh infectious material it has at present reached its third serial passage. In spite of the fact that the agent cannot be propagated in series through domestic rabbits, it is probable that it can be passed indefinitely in series through wild rabbits and that any of these serial wild rabbit passages can be used in infecting domestic rabbits.

No attempt has so far been made to transmit the domestic rabbit warts by means of tissue grafts, although in a small number of experiments freshly prepared cell-containing suspensions of young actively growing papillomata from domestic rabbits have yielded negative results when inoculated intracutaneously or subcutaneously into domestic rabbits. Instead, it has seemed best to study the rabbit papillomata first as an infectious process caused by a filtrable agent and to determine, if possible, why this agent should be readily transmissible in series when inducing warts in wild rabbits and non-transmissible when inducing similar growths in domestic rabbits.

That the degree of maturity of the warts in domestic rabbits at the time that attempts were made to transmit them in series was not a determining factor is indicated by the fact that warts taken at intervals of 6 to 8 days, from their first appearance until they were 116 days old, yielded no successful infections.

Domestic rabbit warts glycerolated for varying periods of time were
repeatedly tested for infectivity to determine whether or not glycerol storage has an activating effect on the agent as it does on herpes virus of low activity (3-5). The results of these experiments were all negative.

In a series of experiments conducted before the presence of neutralizing antibodies in the blood serum of wart-bearing animals had been demonstrated, it was found that when an inactive domestic rabbit wart suspension was mixed with an equal part of a suspension prepared from wild rabbit warts of known infectivity, the resulting mixture was either completely non-infectious or the incubation period was prolonged and the resulting warts few in number as compared with control animals. This suggested the presence in warts from domestic rabbits of an inhibitory substance similar to that found by Sittenfield, Johnson, and Jobling (6) and Murphy, Helmer, Claude, and Sturm (7) in fowl tumors. In the light of subsequent experiments in which the sera of wart-bearing rabbits were found to neutralize partially or completely the wart-producing agent, it seems possible that the inhibitory properties observed in non-infectious domestic rabbit wart suspensions might in reality have been due to contained humoral antibodies. A point of argument against this belief is that, while humoral antibodies were demonstrable in the sera from both infected wild and domestic rabbits, only the domestic rabbit warts possessed demonstrable inhibitory properties. We have as yet made no systematic attempt to render experimental domestic rabbit warts infectious by removal of a hypothetical inhibitory substance. We have tried, however, to infect rabbits with inactive experimental domestic rabbit wart suspensions that had been heated to 60°C. for 30 minutes in the hope that that temperature might inactivate the possible inhibitor without affecting the wart-producing agent, with suspensions prepared from domestic rabbit wart cells that had been washed repeatedly and sufficiently to remove all freely soluble humoral antibody, and with Berkefeld filtrates of inactive wart suspensions. All three of these procedures yielded completely negative results. Both the Iowa and the Kansas strain of the disease were used in these attempts to transmit warts in series through domestic rabbits.

DISCUSSION

The absence of significant visible bacterial forms in highly active wart-producing suspensions together with the ready filtrability of the etiological agent and the inability to cultivate, on lifeless media, any visible microbial form from demonstrably active filtrates; the agent’s ability to transmit in series through wild rabbits; its glycerol resistance; its ability to induce in its hosts an immunity which is constant although of variable degree; and its apparent tropism for one type of tissue place this agent in the filtrable virus group.

The non-transrnissibility of the agent in series through one of its demonstrably susceptible hosts, the domestic rabbit, is not a characteristic of most of the known virus diseases. An analogy, however, is to be found in the group of filtrable fowl tumors. Des Ligneris (8), working with Rous Sarcoma 1 of chickens, has found that while both turkeys and guinea fowls are susceptible, transmission through these two alien species is limited to two successive serial passages. Similarly, Andrewes (9) has found that while Rous Sarcoma 1 will produce fatal metastasizing tumors in its first pheasant passage it cannot be transmitted in its characteristic form from pheasant to pheasant. It seems probable that the domestic rabbit (genus Oryctolagus) is sufficiently distantly related to the wild cottontail rabbit (genus Sylvilagus) to behave towards infection with a filtrable new growth of wild rabbit origin in much the same manner as do turkeys, guinea fowls, and pheasants towards infection with a filtrable chicken tumor.

Another property of the wart-producing agent that is unusual among viruses causing diseases in animals is its resistance to heat. Suspended in 0.9 percent NaC1 solution it proved capable of withstanding a temperature of 65°C. for 30 minutes in sealed ampoules without apparent damage to its wart-produclng properties. Virus heated to 67°C. for 30 minutes, while still active, produced, in our limited number of experiments, warts which either developed scantily or retrogressed after a few days’ growth. We are aware of no other animal virus which will withstand so high a temperature in the moist state; most are completely inactivated at much lower temperatures. However, among the plant viruses, which are on the whole as susceptible as animal viruses to the effects of heat, there are several which withstand heating to 65°C. or’more (10). The virus of tobacco mosaic is an example of a typical plant virus that is relatively heat resistant (11). For this reason it does not seem necessary to consider seriously the possibility that the unusual heat resistance of the wart-producing agent eliminates it from classification as a virus.

The not infrequent shortening of the incubation period in animals inoculated either with virus heated to from 45-65°C. or with virus that had been filtered through Berkefeld V or N candles cannot be explained. Removal of an inhibiting agent by these two procedures is suggested by the data.

In the gross and histologically, the warts of rabbits described in this paper are typical of virus-produced papillomata (12-15) as known in
man, cattle, and dogs. It has not been previously observed in studies of mammalian warts of this kind that an epithelial neoplastic process of identical gross and histological appearance can be induced in two animal species, in one of which the condition is not only transmissible in series, but transmissible by cell-free filtrates, and in the other of which it is not transmissible at all. Here then in what is certainly a single clinical entity are examples of the two extremes of neoplastic processes considered from the standpoint of transmissibility. In the wild rabbit the papillomata can be initiated by inoculating the animal with a filtrable agent and they are transmissible in series by inoculation with filtered or unfiltered virus. From an etiological standpoint, then, the wild rabbit warts are analogous to the chicken tumors which by some
are not considered as true representatives of neoplastic processes simply because they are transmissible by cell-free filtrates. Thus the wild rabbit papilloma represents the one extreme of a tumor induced by an infectious agent which can be separated from the cells and some of whose properties can be studied.

The other extreme is exemplified by the papillomata induced in domestic rabbits which, while initiated by the same virus, have so far resisted transmission either to domestic or wild rabbits. These are thus analogous to many of the tumors of mammals which cannot be transmitted in series by the usual methods of transplantation. No objection to the eligibility of the domestic rabbit warts for consideration as neoplastic processes could be raised on the grounds that a causative agent distinct from the proliferating cells can be discriminated. A study of this epithelial new growth in domestic rabbits without knowledge of its causation would probably lead an investigator to classify it as one of that large group of so called “spontaneous” mammalian tumors that are non-transmissible. It would not even be suspected that the papillomata had been caused by a filtrable virus of wild rabbit origin.

The question which is naturally brought to mind by the experiments of des Ligneris and Andrewes with fowl tumors and our own with rabbit warts is whether certain “spontaneous” non-transmissible or not readily transmissible tumors may not originally have been caused by viruses which produce transmissible tumors in some other species. A careful study, from this point of view, of the causes underlying the non-transmissibility of these various tumors may bring to
light new knowledge of the etiology of neoplastic processes in general,
especially in the group of mammalian tumors which are either entirely non-transmissible or transmissible only by viable cell-containing grafts.

SUMMARY

A papilloma has been observed in wild cottontail rabbits and has been found to be transmissible to both wild and domestic rabbits. The clinical and pathological pictures of the condition have been described. It has been found that the causative agent is readily filtrable through Berkefeld but not regularly through Seitz filters, that it stores well in glycerol, that it is still active after heating to 67°C. for 30 minutes, but not after heating to 70°C., and that it exhibits a marked tropism for cutaneous epithelium. The activities and properties of the papilloma-producing agent warrant its classification as a filtrable virus.

Rabbits carrying experimentally produced papillomata are partially
or completely immune to reinfection and, furthermore, their sera partially or completely neutralize the causative virus. The disease is transmissible in series through wild rabbits and virus of wild rabbit origin is readily transmissible to domestic rabbits, producing in this species papillomata identical in appearance with those found in wild rabbits. However, the condition is not transmissible in series through domestic rabbits. The possible significance of this observation has been discussed. The virus of infectious papillomatosis is not related immunologically to either the virus of infectious fibroma or to that of infectious myxoma of rabbits.

doi: 10.1084/jem.58.5.607.

In Summary:

• Iowa virologist Richard Shope helped discover the cause of the 1918 flu and lay the groundwork for vaccinations
• His 1931 findings alone were considered significant, as were his later contributions on “papillomaviruses”
• Against that background of superstition, the two newest vaccines use technologies that sound almost like science fiction
• Gardasil, made by Merck, uses a yeast to grow the proteins that form the outer shell of the “virus;” every batch of 360 proteins almost magically assembles itself into a soccer ball exactly mimicking the shell’s shape
• Cervarix by GlaxoSmithKline, produces the same protein, with the same power, in an insect “virus” grown in a broth of caterpillar ovary cells
• But each step forward to those techniques was considered a triumph of hard science over the pseudoscientific myths that for centuries surrounded the disease
• In the 1870’s, a doctor in France noticed that prostitutes and married women died of cervical cancer, but nuns almost never did
• His conclusion was that nuns’ corsets were dangerously tight
• Prominent American scientists made a similar error in the 1970’s, noting that many women with cervical cancer had a history of genital herpes
• Instead of realizing that it was a coincidence, they erroneously concluded that the herpes “virus” was the cause
• 1950’s researchers blamed smegma which builds up under the foreskin of men who do not wash
• Research that could have led them in the right direction was done in the 1930’s by Dr. Richard Shope of the Rockefeller University
• Shope received horns from rabbits, ground them up, filtered them through porcelain that let only tiny “virus-size” particles through, and injected the filtrate into other rabbits, which grew horns in turn
• Dr. Shope’s work was said to show the cause was a “virus,” but it was not until the 1980’s that DNA amplification allowed a German researcher, Dr. Harald zur Hausen, to pin down papilloma as the cause
• According to Dr. Douglas R. Lowy, chief of the cellular oncology laboratory at the National Cancer Institute: “Before that, the field suffered from the boy-who-cried-wolf phenomenon, as in people would say, ‘Last year you said it was herpes virus, now you say it’s papilloma; why should we believe you?’

• Warts in rabbits from a naturally occurring case of the disease in Iowa were obtained and sent to the laboratory in sterile 50 percent glycerol
• These glycerolated warts furnished the original material for investigation

Quick Detour #1:

Glycerol is not a harmless solution for animals and the warts used for the study were immediately placed in a 50% solution. Presented here are three sources on glycerol toxicity:

Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

“The concentration- and temperature-dependence of glycerol toxicity was determined by exposing the cells to a range of glycerol concentrations at two temperatures, 21°C and 37°C. To decouple toxicity and osmotic damage, the cells were brought to the peak glycerol concentration using multiple steps as necessary (see S1 Supporting Information). Fig 2 depicts the time-dependent cell yields for glycerol exposures at 21°C and 37°C. In general, cell yield decreased as glycerol exposure time increased, as glycerol concentration increased and as temperature increased. Indeed, statistical analysis by 3-way ANOVA revealed that all of these factors (i.e., exposure time, concentration and temperature) had statistically significant effects on the cell yield (p < 0.0001). An exponential decay model was fit to the data to determine a cytotoxicity rate constant k for each glycerol concentration and temperature. Although the data has high variability and deviates from the exponential decay model in some cases, the results show that the rate of cell death increases with both glycerol concentration and temperature.

These trends are even more noticeable when the best-fit toxicity rate constants are plotted as a function of concentration, as shown in Fig 3. The toxicity rate is higher at 37°C than 21°C, and the toxicity rate increases with glycerol concentration.”

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142828

Inhibition of cell proliferation by glycerol

“The effect of glycerol on proliferation of BHK, CHO, HBL, MCF-7, and human glioma cells was studied. Cell proliferation was significantly decreased in all the cell lines at glycerol concentrations of 2–4% in the culture medium. The inhibition was dose-dependent, complete suppression of proliferation occuring at a glycerol concentration of 4% for the MCF-7 cell line and 6–8% for the BHK, CHO and human glioma cells. Studies on [³H]thymidine incorporation correlate with the effect on cell proliferation. The viability of the cells was not significantly affected until higher concentrations of glycerol (12%+) were present. Recovery studies with BHK cells indicated that replacement of the glycerol medium with glycerol-free medium resulted in full recovery following exposure to 4% glycerol and only partial recovery (65%) of proliferation rate following exposure to 10–12% glycerol. It is concluded that glycerol, a substance that is normally present in tissues, can serve as a potent inhibitor of cell proliferation.”

https://www.sciencedirect.com/science/article/abs/pii/002432059190275G

Glycerol Intake, Blood Cholesterol Level and Anemia in the Guinea Pig
and Rabbit.

“A short report of Orekhovich and Plotnikov, indicating that the serum cholesterol level of rabbits which had received glycerol by stomach tube rose dramatically, prompted the following experiments. Ten guinea pigs and 4 rabbits received a 50% glycerol solution in saline or saline alone either by stomach tube or from a drinking cup daily for 30 to 40 days. The plasma cholesterol levels of all animals receiving glycerol, the red blood cell cholesterol levels of the rabbits and the red blood cell counts of the guinea pigs were compared with the respective values of the animals receiving saline only.

Results and discussion. Guinea pigs given more than 5 ml of a 50% glycerol solution daily by stomach tube died with acutely toxic symptoms. Rabbits tolerated 10 ml daily. The amount of glycerol solution consumed by the animals could not be established accurately. It very probably was more than that given by stomach tube.”

https://doi.org/10.3181%2F00379727-111-27875

As can be seen, glycerol is toxic to cells and cell death increases due to exposure time, concentration and temperature. Glyceeol is considered a potent inhibitor of cell proliferation with cell viability affected with concentrations of 12% or higher. The concentration used in Shope’s study was 50% which was shown in later studies to cause dramatic serum cholesterol level increases in rabbits and toxicity in guinea pigs ultimately leading to their deaths. It is therefore plausible to conclude that the wart material used to “infect” the rabbits were altered and toxic due to the concentration of glycerol used, not from some imaginary “virus.”

End Detour # 1.

• In wild cottontail rabbits the presence of warts caused no apparent discomfort and induced no demonstrable evidence of generalized illness
• Four rabbits that were not initially killed for material at no time appeared ill and were in good physical condition when finally killed
• Either freshly removed warts or those that had been stored in 50 percent glycerol at refrigerator temperature were ground to a fine paste with sterile sand and physiological saline in a mortar
• More physiological saline was added to make a 3 to 5 percent final suspension
• Such a suspension was then centrifuged and the supernatant fluid, which was only slightly turbid, was removed and used for inoculation

Quick Detour # 2:

It is assumed that physiological saline is harmless yet this has been shown not to be the case in several studies:

Disastrous physiological effects of saline on the cell membrane demonstrated in cardiac cells

“Objective: Saline is not an ideal storage solution. It has a low pH, no buffering capacity, and lacks other ions and nutrients. The objective was to explore the effects of storing cardiac muscle in saline.

Results: Saline caused transient hyperpolarization of the resting potential (-140 mV), prolonged duration of the action potential, and increased contraction amplitude, which was later reversed. The membrane resting potential depolarized after a few minutes to about -15 mV and the preparations became unexcitable. The depolarized preparations remained slightly contracted. Upon reperfusion both papillary muscles and cells became unstable and spontaneously active. Storing myocytes in saline for only 2 h resulted in excessive cell death.

Conclusion: Saline is disastrous for the function of the heart muscle and leads to depolarization, sustained contraction and unexcitable tissue. Saline should not be used as a storage medium, even for short periods of time.”

https://pubmed.ncbi.nlm.nih.gov/15513314/

0.9% saline is neither normal nor physiological

“Over the years, the name has morphed into what is more commonly called “normal saline” or “physiological saline” despite no additional evidence or rationale for the relabeling. The implied normalcy and physiological property have perpetuated indiscriminate use of saline in medical practice.”

“Despite its name, saline is neither “normal” nor “physiological”. Compared to human serum, saline has a nearly 10% higher Na concentration and 50% higher Cl concentration.”

“Evidence suggests that high Cl-containing fluids (such as saline) are associated with multiple adverse effects including an increased occurrence of kidney dysfunction with possible reduced survival. Although large randomized controlled trials comparing saline and balanced fluids are necessary, the time has come to at least recognize that saline is neither normal nor physiological. Saline administration should be considered to have the same status as a drug prescription. Strengthening knowledge of saline management would likely result in improvement in patient outcomes.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794509/

The above results are from studies on human tissues but they make the point clear that “physiological” saline is not an inert substance. This drug-like substance was added to the ground up warts which also had toxic glycerol which Shope then applied to the scarified skin of the rabbits.

End Detour # 2.

• Inoculation by scarification was regularly performed in these experiments
• Rabbits to be inoculated were shaved on the abdomen or sides and lightly scarified either by needle or by rubbing the shaven skin with a moderately coarse grade of sterilized sandpaper
• To obtain discrete warts the method with a needle was employed, while scarification with sandpaper was used when a confluent and massive growth of warts was desired
• In other words, the type of instrument used to scarify the skin was directly linked to wart growth…no “virus” necessary
• The scratches were made only deep enough to cause a barely perceptible oozing of blood-tinged fluid
• A small amount of the “infectious” suspension was immediately applied by dropping it from a syringe, and this fluid was rubbed well into the scarifications by means of a spatula or the flat handle of a scalpel
• In spite of the great size of many of the experimentally produced wart masses, the animals showed no loss in weight and the entire course of the disease was free from any general clinlcal evidence of illness
• With two exceptions, no warts retrogressed in animals infected in the usual way
• In the exceptional animals, one a wild and the other a domestic rabbit, warts developed slowly after an unusually long incubation period and reached a maximum height of only 2 to 3 ram. between 30 and 40 days after inoculation and in 50 days had completely disappeared
• While there had been no evidence of retrogression of the papillomata except in the cases mentioned, there had also been no evidence that the lesions of prolonged standing acquired malignant properties
• In other words, in two cases the warts disappeared on their own and in all cases the warts were not cancerous
• Warts used as a source of infection in the filtration experiments were removed from the 50 percent glycerol in which they had been stored and were washed in three changes of sterile physiological saline
• They were then minced with sterile scissors, ground in a mortar with sterile sand, and suspended in sufficient physiological saline to make an approximately 5 percent suspension
• The decanted supernatant fluid was usually almost water-clear with only a faint opalescence, and for this reason was rapidly filtrable
• 1 cc. of a broth culture of B. prodigiosus (a.k.a. serratia marcescen) was added to each 15 to 20 cc. of fluid just before it was passed through Seitz or Berkefeld filters
• The “control” animals had been inoculated with unfiltered suspensions
• From the data recorded in Table I Shope concluded that the etiological agent causing warts in rabbits readily passed Berkefeld filters, of V, N, or W porosity but did not regularly pass a Seitz filter when two pads are employed
• No extensive attempts to cultivate visible microbial forms from filtrates of proven infectivity were made
• The fact that sections of actively growing warts or films of active unfiltered infectious suspensions failed to reveal the presence of any constant perceptible microbial form, would seem clearly to indicate that no visible organized agent is etiologically essential to the wart production
• In other words, because they could not see another microbe, it wasn’t there and had no role in wart production…unlike the “virus” which they also could not see but ASSUMED was there and responsible for wart production
• Only the method of inoculation by scarification yielded constant results
• All four intravenously inoculated animals were free from visceral pathology ascribable to the wart-inducing agent
• Inoculations of either wild or domestic rabbits intraperitoneally (stomach), subcutaneously (skin), intratesticularly (testicles), or intracerebrally (brain), with filtrates of proven infectivity on scarification, yielded entirely negative clinical and pathological results
• About 50 percent of the intradermal (under the thin layer of skin) inoculations resulted in wart formation although in these instances the warts appeared not at the point where the inoculum had been deposited but at the point where the needle had pierced the epidermis
• Twenty-six domestic and wild rabbits were inoculated with suspensions prepared from experimentally engendered domestic rabbit warts ranging in age from 1 to 116 days
• Not only did all such inoculations yield negative results but the animals, when subsequently tested, were found to be still fully susceptible to infection with the wart-producing agent from wild rabbit papillomata
• In other words, their experiments showed wart material said to contain the “virus” from domestic rabbits was non-infectious while wart material from wild rabbits containing the same “virus” was somehow infectious…
• In like manner, experimentally produced warts from nine wild rabbits were tested for infectivity and eight of these proved infectious for either domestic or wild rabbits while the warts from one proved to be non-transmissible
• They did not attempt to pass the wart-producing agent through a long series of wild rabbits
• In spite of the fact that the agent could not be propagated in series through domestic rabbits, it was probable that it could be passed indefinitely in series through wild rabbits and that any of these serial wild rabbit passages can be used in infecting domestic rabbits
• No attempt was made to transmit the domestic rabbit warts by means of tissue grafts, although in a small number of experiments freshly prepared cell-containing suspensions of young actively growing papillomata from domestic rabbits yielded negative results when inoculated intracutaneously or subcutaneously into domestic rabbits
• Shope questioned why this agent should be readily transmissible in series when inducing warts in wild rabbits and non-transmissible when inducing similar growths in domestic rabbits
• Warts taken at intervals of 6 to 8 days, from their first appearance until they were 116 days old, yielded no successful infections
• Domestic rabbit warts glycerolated for varying periods of time were repeatedly tested for infectivity to determine whether or not glycerol storage has an activating effect on the agent as it does on herpes virus of low activity
• In other words, he assumed that glycerol itself has no toxic effects on the animals and that it just “activates” the “virus”
• It was found that when an inactive domestic rabbit wart suspension was mixed with an equal part of a suspension prepared from wild rabbit warts of known infectivity, the resulting mixture was either completely non-infectious or the incubation period was prolonged and the resulting warts few in number as compared with control animals (control animals got warts…?)
• Shope made no systematic attempt to render experimental domestic rabbit warts infectious by removal of a hypothetical inhibitory substance
• He tried to infect rabbits with inactive experimental domestic rabbit wart suspensions that had been heated to 60°C. for 30 minutes yet all three of these procedures yielded completely negative results
• Shopes criteria for the successful “isolation” of an invisible “virus” was:
  1. The absence of significant visible bacterial forms
  2. The supposed filtrability of the
  3. etiological agent and the inability to cultivate, on lifeless media, any visible microbial form
  4. The invisible agent’s ability to transmit in series through wild rabbits (but not domestic)
  5. Its glycerol resistance
  6. Its ability to induce in its hosts an immunity which is constant although of variable degree
  7. Its apparent tropism for one type of tissue place
• The non-transrnissibility of the agent in series through one of its demonstrably susceptible hosts, the domestic rabbit, is not a characteristic of most of the known “virus” diseases
• Another property of the wart-producing agent that is unusual among “viruses” causing diseases in animals is its resistance to heat
• “Virus” heated to 67°C. for 30 minutes, while still active, produced, in his limited number of experiments, warts which either developed scantily or retrogressed after a few days’ growth
• He was aware of no other animal “virus” which could withstand so high a temperature in the moist state; most are completely inactivated at much lower temperatures
• Shope claimed that plant “viruses” like TMV are resistant to heat and for that reason, it did not seem necessary to consider seriously the possibility that the unusual heat resistance of the wart-producing agent eliminated it from classification as a “virus”
• The not infrequent shortening of the incubation period in animals inoculated either with “virus” heated to from 45-65°C. or with “virus” that had been filtered through Berkefeld V or N candles could not be explained
• It had not been previously observed in studies of mammalian warts of this kind that an epithelial neoplastic process of identical gross and histological appearance can be induced in two animal species, in one of which the condition is not only transmissible in series, but transmissible by cell-free filtrates, and in the other of which it is not transmissible at all
• In other words, they had never seen a case where a “virus” could be transferred from one species of animal (in this case wild and domestic rabbits…not different species) to another but not vice versa
• The papillomata induced in domestic rabbits, while initiated by the same “virus,” resisted transmission either to domestic or wild rabbits
• A study of this epithelial new growth in domestic rabbits without knowledge of its causation would probably lead an investigator to classify it as one of that large group of so called “spontaneous” mammalian tumors that are non-transmissible
• It would not even be suspected that the papillomata had been caused by a filtrable “virus” of wild rabbit origin
• Shope states that the question which was naturally brought to mind by the experiments of des Ligneris and Andrewes with fowl tumors and his own with rabbit warts is whether certain “spontaneous” non-transmissible  or not readily transmissible tumors may not originally have been caused by “viruses” which produce transmissible tumors in some other species
• According to Shope, the activities and properties of the papilloma-producing agent warrant its classification as a filtrable “virus” even though the condition was not transmissible in series through domestic rabbits

There you have it. To “prove” a “virus” caused warts in rabbits, Shope and co. minced and ground up glycerolated rabbit warts with sterile sand and added heaping amounts of physiological saline solution along with culture broth of B. Prodigiosus. They then tried every which way to infect the rabbits. They injected these rabbits directly in their veins, in their stomachs, in the fat layers of their skin, in their testicles, and in their brains and all attempts ended in negative results. They could only achieve results about 50% of the time when they inoculated the rabbits directly under their skin yet reactions only occured at the site of injection. They could not transmit infection from domestic rabbits to other domestic rabbits or to wild rabbits. They only succeeded when using wart material from wild rabbits. The only way they ever had success with this material was through scarification by using needles and sandpaper directly on the shaved skin of the rabbits. If they wanted a small amount of warts, the needle was used. If they wanted many warts, sandpaper was used. Never once did they consider the method of scarification as a potential cause of the warts, even in the face of numerous negative results attempting to inject an invisible “virus.”

It is truly amazing the lengths these “scientists” will go to in order to attempt to “prove” their assumptions correct. Because of Shope’s work with rabbits, we got the Shope “papillomavirus.” This paved the way for researchers to do the same ridiculous method of grinding up human wart tissue and comparing it under EM to plant “viruses” and rabbit warts in order to create the human “papillomavirus” (HPV). Shope’s work led to the disastrous HPV and flu vaccines that have ruined countless lives. We have all of this suffering thanks to the shoddy pseudoscientific work of one man.