WO1997011667A2 - Therapeutic applications using horse serum - Google Patents

Abstract

Novel uses of animal sera including horse sera in the treatment, diagnosis and clinical management of AIDS, cancer and other viral and bacterial diseases.

Description

THERAPEUTIC APPLICATIONS OF ANIMAL SERA INCLUDING

HORSE SERUM IN THE TREATMENT OF AIDS, CANCER AND OTHER

VIRAL AND BACTERIAL DISEASES

BACKGROUND OF THE INVENTION

The present invention is directed to novel uses of horse serum and other animal sera in the treatment, diagnosis and clinical management of viral and bacterial diseases including AIDS, cancer, and autoimmune diseases. Prior art in the use of horse/animal serum in the treatment of cancer has some use as in the case of the Glover antitoxin where antiserum raised against a microorganism associated with cancer was used to treat cancer with minimal success.

Prior to the advent of antibiotics, horse serum was used in the treatment of bacterial and viral diseases, achieving somewhat moderate efficacy. Allergic reactions, however, were relatively common and often debilitating as compared to the less severe and relatively infrequent side effects later achieved with antibiotics. Although bacterial infections have been dramatically reduced with the use of antibiotics, curiously, the incidence of cancer rose just as dramatically during this same period. One hundred years ago, cancer killed 1 in every 273 people. Toda> . cancer kills 1 in every 3 people. Yet despite modern medical advances, the etiological cause of cancer has continued to elude even the most knowledgeable in the medical field. Further, new and even more virulent and resistant diseases such as AIDS have taken center stage It is generally accepted that the human immunodeficiency virus (HIV) is the etiological cause of AIDS. Current therapies directed at the chemical, physical and biological aspects of this disease have failed for a variety of reasons. First. HIV has a high mutation rate. Secondly, normal human immunological responses to HIV are currently ineffective, if not counterproductive. Further stimulation without some alteration in mechanism or function only promotes inappropriate responses. As a result, major failures are all too common with all current broad spectrum immunological stimulation. For example, use of lymphokines in current vaccine therapy is aimed at the use of HIV or viral particles of the same. However, there is a risk of promoting or even initiating harmful responses. Lastly, effective antiviral therapies are lacking in current treatments as well as an ineffectiveness with existing antibiotics in the absence ol an effective immune response

SUMMARY OF THE INVENTION The present invention is directed to the use of immune sera including horse serum and other animal sera in the treatment of AIDS, cancer and other opportunistic infections Although cancer and AIDS are used repeatedly throughout this disclosure as the targeted diseases or conditions This is not for illustration purposes onl> and is not intended to limit the scope of this invention

The immune sera may be unmodified as it is the inventor^'s contention that anti-disease activity may be inherent or may be elicited in response to a challenge In either case, the serum may be further purified electrophoretically Electrophoresis, and any other system of selective separation, are used to carry out this invention in novel ways to isolate active components from virgin/unchallenged organisms

In severely lmmunoefficient states chances of allergic responses to even crude horse serum may be reduced Patch and other testing techniques could precede administration

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Preparation of Serum Several serum sources may be used in accordance with the present invention

Mature organisms tend to have superior development of their immunological parameters However, this is often accompanied with a greater possibility of incompatible antigens and allergic or rejection phenomenon when applied to other host organisms Another potential serum source is immature fetal and embryonic sera These sources possess inherent resistance or other useful anti-disease activity, but are not likely to be as specific or as developed as those of the mature organisms Benefits may include a decreased risk of allergic or rejection phenomenon when applied to other organisms and may confer non-specific rejuvenation effects Although the present invention relates to cellular, intracellular extracellular phenomenon as applied to immunologic and other systems, where a beneficial or useful property may be conferred directly or indirectly from in vivo or in vitro source animal or other, the following discussion is limited to the use of horse serum It should be noted that this is done for convenience purposes only, and is not intended to limit the scope of the present invention

The present invention is based in part on the idea that antibodies as well as other factors present in horse serum may be of benefit in the diagnosis and therapy of a number of viral and bacteπal diseases Although current treatment with human serum extract is restricted to IgG, acute infections are initially addressed by other immunoglobulins Immune memory is usually achieved with the IgG immunoglobulin It is possible, however, that IgG activity is optimal as a memory response after other modifications have taken place in the body following the initial infection, initial immunoglobulin cellular and other responses Immunoglobulins have been shown to carr> more than a simple action of attachment and neutralization of the offending antigens Communication and modification may be among the other functions Restricting the therapeutic field to the IgG is at best narrow-minded Other immunoglobulins may be superior at certain immunological responses It may be that a shark^'s resistance to cancer may be related to its specific serological immunity It is a further objective of the present invention to use other human serological fractions separately or in combination as opposed to simply using IgG Use of such fractions as well as even current use of IgG can be improved by selective purification, dialysis and electrophoresis to minimize risk of side effects and maximize efficacy Serum Purification A number of technologies have been developed which allow for specific isolation and purification of specific extracts These include centrifugation, dialysis, electrophoresis. as well as selective filtration procedures These and other techniques are incorporated by reference in the present invention in accordance with the use of purified serum extracts in the clinical management of various disease conditions Purification techniques refer both to the testing of serum sources and content for allergens, pathogens and pyrogens by standard laboratorv procedure as well as the further purification of product to minimize anti-human component (or component against whatever species to be treated) The present invention does not solely relate to humans and human disease but applies wherever the host of a particular disease is unable to repel a disease as well as an outside source of the same or other specie when the disease is presented in the same or altered format The present invention is further related to the idea of tagging and targeting where disease appearance may be altered so that the tagging component is then attacked by antiserum (see section on transfer factor and antiserum to primary, secondary and latter manifestations of disease as well as regression anticipation in therapy) Purification procedures to minimize allergic or other undesirable potentials range from the primitive precipitation of certain fractions of serum by simple storage in refrigeration over 6 months at 3 degrees celsius as was practiced at the turn of the century to the more sophisticated techniques today of washing the animal product against human blood to remove the antihuman fraction Further safet\ may be achieved by washing against blood of a similar type as the patient or even against patient's own blood particularh if the latter is not involved in the disease process or carrying disease antigens to significantly deplete therapeutic serum efficac> Other methods include the raising of anti-antihuman antiserum and its use in the purification of therapeutic serum or the induction of tolerance to human/subject antigens in the animal to yield therapeutic serum Technique for purifying generalized and specific sera STEP 1-

Saline A - add to 4 litres of distilled water 8g chlorocresol heated slightly to facilitate dissolution, 3ml 2% Na OH, 0 2g Mg₂C0₃, 0 4g NaHCO,. 0 8g Na₂SIO, After dissolving mixture, bubble CO₂ until clarifies, usually 20 minutes Stand overnight, at room temperature, covered

Day 2 - heat to a boil check solution every 5 minutes Checking as follows 5ml aliquot in test tube add a few drops of 2% NaOH Place in a waterbath at 70-80 degrees After 5 more minutes, flocculate should be evident Place a lew drops of flocculate onto microscope slide and X450 Crystals of a cubic shape should be seen As boiling continues, cubes replaced by cigars or stars When cigars predominate discontinue heating Saline A is completed, 5 2 litres is required per batch DO NOT BOIL MORE THAN ONE HOUR FROM THE FIRST APPEARANCE OF CIGARS Saline B - boiling for an additional 30 minutes transforms saline A to saline B, 800 mis saline B required STEP 2-

Serum solution from any animal is vaccinated against any single or mixture of diseases, for example, cancer, AIDS, heφes, leukemia, etc can pool multiple diseases or animal sources 20-30cc of serum solution added to 800-900 ml of saline B Heat at 55°C Add 15ml of 2% NaOH and heat an additional 30 minutes Add 10ml of 5% HCL and filter through number 1 filter paper, use gravity or negative pressure. 0 45 micron will probably do Add 1200 ml of saline A Add 30ml of 10% ammonium carbonate and boil on a hot plate Boil the serum solution once daily for three days The serum should be sealed to avoid evaporation and microbiological contamination 450ml of serum solution needed for process STEP 3-

1 5g chlorocresol, heat slightly to facilitate dissolution 4 Og NaCL 10 g Beef extract-can substitute patient's blood, colostrum, or other animal sources of broth Boil, then let cool Filter through a number 4 glass fiber filter Add 0 l g ammonium carbonate for each 100ml of solution (add 0 8g to 800ml) Boil the solution and let it cool Filter through a number glass fiber filter Check PH. if less that 6 add 2% NaOH to bring the PH to at least 6 (this step is hardly ever necessary) For cancer specific antibodies, PH 5 5 to 6 5 may be precipitated Filter through a number 1 Whatman or glass fiber filter Autoclave at 15psι for 15 minutes STEP 4-

Final assembly - 450mls of serum solution is added to I 50mls of broth and heat in water bath at 60-62 degrees for 10-15 minutes At the same time, heat four litres of saline A in the same bath Mix together and keep at 57 degrees for 30 minutes Add the following to the mixture 90ml of 2% NaOH in divided doses. 5mls of saturated ammonium chloride Keep mixture at 50°C for one hour then leave overnight al room temperature, covered. The following day there is a clearly defined flocculate in the mixture. (If no flocculate, add 1ml of saturated ammonium chloride. Boil until cloudy and leave at room temperature overnight again.) Siphon off the supernatant gently, the flocculate is very easily disturbed. An amount of supernatant will be left behind Add the flocculate to four litres of saline with 0.01 % chlorocresol (saline C). Filter the mixture through number one filter paper. Wash the flocculate off the filter paper (or whatever filter) into 1000ml glass graduated cylinder Use saline C in a wash bottle for wash procedure. Filter again through number one filter paper. Wash flocculate into 500ml glass graduated cylinder. Add saline C to 300ml cover and autoclave at 15psi for 15 minutes. Add saline C in a wash bottle for the wash procedure. When prepared, the therapeutic dosage is about 0.05 to about l Occ intramuscular approximately every 2 days.

For purposes of discussion and understanding the present invention, the following definitions are provide: DEFINITIONS

Therapeutic Serum - Serum extracted from usually extraneous in vitro or in vitro sources. Serum may also be extracted from patient's own cells in an in vitro system or from the patient^'s blood by an anticipation process. Anticipation is a technique designed to overcome threshold inhibition; it is a phenomenon where disease is perceived to be above a certain immunological load and will be protected from immune attack. The process of anticipation aims to predict changes to befall the disease condition and to develop strategies to neutralize the new forms as they arise Anticipating changes allows for the vaccination of the body against the changes before they arise, or for the raising of anti serum/transfer factor/transformation factor/other therapeutic agent against future disease manifestation

Isolation of a Specific Fragment

To minimize the chance of an allergic response while maximizing efficacy, the present invention provides some basic new methods and all modifications An animal such as a horse can be injected with disease- causing and/or synergistic and/or promoting organisms and factors Cells, serum and other physical, chemical and biological factors can be observed for changes which assist in the organism's rejection of the offending agent, for example, temperature immunological pH changes, etc Such changes can then be used or duplicated in whole part, in an extract derivative or product thereof This includes all physical, chemical and biological techniques of duplicating these directly or by genetic engineering or other techniques and such factors which can be beneficial directly or as this invention covers, by imitation, duplication or induction Of these agents which can include complex cellular or sera extracts such as transfer factor, DNA. RNA, protein, enzymes, carbohydrates, lipids, organelles, chemical, immunoglobulin changes or changes in the cell tissues or structures which produce them, our examples will be confined for discussion purposes only to animal sera, particularly that of the horse Testing of pooled serum is used to identify the serum fractions with specific anti-disease activ This can be done in several ways including gradient and other separation techniques One way is to expose a test sample of pooled or other serum to the disease or antigen that is to be targeted After exposure, the serum electrophoretic or other spectrum is compared to the unexposed sample The fractions that are missing or diminished are the serum disease specific fractions (SDSFs) Isolating the SDSFs and then treating the patient with SDSF allows for increased efficacy and higher dosage of the specific fraction Treatment with SDSF minimizes the risk of allergy, contamination or other side effects Further purification and safety techniques involve exposing pooled serum to normal cell, tissue, etc antigens from patient and then removing the fraction which attaches to these components from the treatment fraction Similar isolation and purification techniques can be applied to virtuall} any therapeutic technology in its precursor intermediate or endpoint Immunologically virgin versus immunized versus dedicated animals

While an immunologically virgin horse may carry non-specific anti-disease activit) it should also be pointed out thai specific anti-disease activity can be isolated from animals if some or all such properties are inherent Selective filtration technique-, can be performed by allowing adsorption then separation from the target antigen or b_\ knowledge of physical, chemical or biological properties which allow characterization and specific isolation A simple technique which is effective involves taking a basic electrophoresis pattern prior to and subsequent to immunization with target agents A comparison between the two at various times following immunization will indicate appropriate bands amplified by the challenge (vaccination with targeted condition) These fractions can then be isolated from organisms which have never been exposed to disease factors Isolation of inherent resistance minimizes the risk of contagion For example if upon injection of a horse with HIV, the horse shows a rise in a particular part of the IgD or IgG spectrum, isolation of similar band areas from immunologically unchallenged horses can be used therapeutically since these band areas indicate another organism s potential for disease resistance An immunized animal is likely to have a more specific response with a higher qualitative yield The risk of using compounds extracted from animals injected with live virus is that some \ iral component may lead to contagion or mutation that may be passed onto the recipient of the prophylactic or therapeutic application despite stringent purification procedures There are at least three possible preventative procedures for such an event 1 ) use of killed/inactivated viral fragment to minimize risk of contagion or promotion of mutation, 2) use of all possible variant mutants to raise polyvalent antisera, 3) use of dedicated animals, 1 e . vaccine made for each particular patient would be made from his/her specific virus isolated and/or cultured from their blood A virus could be made to mutate in vitro or in vivo through a large array of mutations against which antisera may be raised

It is the object of current therapy that in developing a response or a therapy against a factor or organism with the capacity to alter or mutate to escape the response therapy or to somehow alter the efficacv of the response The inventor believes and has demonstrated that inducing or anticipating changes to therapy, then developing or allowing treatment or antagonistic protocols, leads to greater efficacy In summary, if the animal is exposed to many if not all of the possible mutations of the targeted factor so that the immune response, such as immune serum anticipates possible changes even before they arise in the patient^'s body, then effective elimination may be accomplished Alternatively, the first animal dedicated to the patient's HIV may be used or another animal dedicated to the same patient's virus upon its mutation Any number of animals can be dedicated to deal with the virus or its mutations from the outset (multivalent systems) or as they arise

Although horse sera have been used as example in this invention, the present invention applies to many organisms whole, part, extract, product or derivative thereof immunological or other system whole, part, extract, product or derivative thereof

Horses and their sera are used to indicate that there are animals and sera with more efficient resistance to certain diseases which may be inherent or cultivated in animals by techniques that could not be done in humans for ethical and other consideration Horses and many other animals and organisms appear to have inherent and/or inducible anti-disease activity which may be different (and thus beneficial) if not superior to human responses, or with abilities which may be augmented by in vivo or in vitro manipulation or altered by such means to develop beneficial diagnostic, clinical or therapeutic applications including compositions and methods/or producing same Such products may be used to educate or modify human (or other) immune responses, e g . by transfer of genetic information, transfer factor, across interspecies or by other pathways of modification of other diseases or responses to disease Alternatively or simultaneously, direct anti-disease activity may be utilized

The essence of this invention is to use an exogenous system superior to endogenous responses either solely or in combination with the endogenous system, directly or by education of the endogenous system

An embodiment of this invention involves the use of horse serum in the treatment of AIDS Animal sera have been used unsuccessfully in the past in the treatment of cancer, however, horse sera have been demonstrated to be effective in the treatment of various infections prior to the advent of antibiotics This invention also covers the use of such sera and their application against any disease-related ιnfectιon(s), direct, induced or modified, with or without interplay with other exogenous or endogenous system, direct anti-disease activity-direct, included, modified with or without interplay with other exogenous or endogenous systems against disease in normal or altered presentation The latter point refers to a tagging concept which is covered later Tagging essentially distinguishes between the use of effective responses against disease or disease-related organisms or conditions and the use of immune or other responses aimed not against the disease but against factors which have been made to tag to it This embodiment relates to any condition but it will be broadly exemplified in the treatment of AIDS as explained below

Prior art in the treatment of AIDS involves physical, chemical and biological mechanisms of viral inhibition and treatments of the sequelae of the disease As the virus mutates, and the immune system weakens, such approaches become increasingly ineffective Attempts at ιmmunotherap\ with immunostimulating agents may create temporary increases in immune parameters However, such increases are often followed by rapid drop as the newly raised cells maintain their vulnerability to HIV Further, the supply of such cells may be nearh exhausted by the disease process

The present invention aims to increase the resistance of the cells targeted by the disease against the disease The invention further provides for effective passive and active immunological responses The concept of anticipation enables the therapeutic program to neutralize mutations before they arise and dynamic flow allows for continuous generation of effective therapy with changes in the disease

Polyphasic therapy increases the efficacy of any one modality, and polyvalent therapy allows for the combination of effective therapeutic agents to improve desired results and minimize dosages of therapeutic agents which may have toxicity, e g , AZT Another advantage is a multiple step process for eliminating the targeted condition whereby step one is the tagging of that condition with antigenetically powerful and specific agents These may induce attach by a different immunological arm. perhaps more effective than the prior one and exhibiting more resistance Passive antisera/transfer factors and other agents may be raised against the tagging component. and/or the tagging complex with the targeted condition An antiserum administered may further add to the antigenicity of the complex, and itself be viewed as a tagging agent, allowing for the repetition of the above steps

It is a further object of the invention to use any and all components of human sera not included in current preparations as well as using all such components in combination with currently available extracted components Current technology and practice is to use only IgG in pooled sera as therapy or in prevention IgA, IgD, IgM etc are not included It is the inventor^'s contention that they would serve important functions Cancer may be seen as a restricting/restraining immunological response This means that cancer will attach to a microorganism and restrain it until the immune system immobilizes against it If you attack cancer and destroy it, it allows the organism or whatever causative factor to escape When the organism or factor escapes cancer responds and spreads throughout the bodv The cancer response may follow it and enclose it again, a theoretical basis for metastasis A plant model which demonstrated this is that of the agora bacterium tumafasciens, and the cancer-like response it elicits in plant life

One of the things that appears to be missing in the logic of cancer therapy is that when there is a cancer remission, the cancer appears calcified with scar tissue formation around the cancer As the calcified cancer collapses, a scar remains A scar is also a restraining mechanism In order to affect a cure for cancer, the cancer can be destroyed or the causative organism Alternatively, another restraining mechanism can be used to take the place of the cancer For example, if you inject an animal with a carcinoma, a sarcoma may develop on the outside of the carcinoma or a sarcomatous- like change, whereby the development of a fibroblast reaction occurs which restricts the carcinoma development Therefore, a sarcoma can be viewed as a response to a carcinoma and a carcinoma can be seen as a immunological response to a sarcoma This may explain why, if you inject an animal which has one with the other, you aggravate the animal's condition Although efforts have focused on extracting oul immune system cells, immune system transfer factors and antisera, attention should be given to extracting out fibroblasts that have the capacity to encapsulate cancers and extracting out transfer factor from fibroblasts. which is restraining transfer factor Extracting out a restraining immunological response is important to produce scarring a_l the site of the tumor Interspecie rejection

Human cancer injected into an animal would usually be rapidly rejected Graft rejection occurs, interracial, and even among closely matched individuals It can be used in the development of therapy The antiserum and the transfer factor the animal produces are useful, but even more important is the restraining immuno response from the fibroblasts reaction that occurs around the cancer There are also fibroblasts and other cells which surround the cancer and produce precipitation of calcium in the vicinity of the cancer These fibroblasts also, if introduced to an animal, cause calcification around its particular cancer Here we are dealing with cellular responses, genetics of cellular responses, inductive agents and transfer factors It is also important to note that a tumor will contain a certain amount of tumor infiltrating lymphocytes (TIL) Cancers such as melanomas often demonstrate a small amount of TIL TILs represent white blood cells that can penetrate the cancer and either attack it or its causative organism TILs have produced remarkable shrinkages in work by Rosenberg, but have never been maintained The problem in the prior art is that TILs exist in small numbers even if stimulated and amplified because they are nearing their extinction limit also known as the Hayflick limit There are very few of the TILs left, so you can cause an expansion and explosion resulting in shrinkage of the cancer. However, the immune response becomes exhausted, and the cancer begins to grow again. This can be bypassed in several ways. One way is with the use of phages to push beyond the Hayflick limit Another way is to use reparative enzymes to push beyond the Hayflick limit from the patients own biopsy of cancer. Another way is to use tumor infiltrating lymphocytes from animals It is very hard to induce an immuno response to cancer in a patient bearing the same cancer, however TIL in animals such as a pig can easily be transplanted into a human without harmful or allergic responses One mechanism for amplifying the amount of TILs in the human body is to extract out a biopsy of the patient^'s cancer and implant it into an animal That animal will attack the cancer with an antiserum which can be used, stored and purified The animal will attack the cancer with an antiserum which will be of special use if the antiserum also relates to the causative organism, or if the animal is also vaccined against the causative organism The animal will produce a cellular response within hours of injection of the cancer, which is largely neutrophilic This cellular response can lead to cells which can cause massive tumor destruction but may have significant side effects To minimize the side effects of that cellular response, extract out the transfer factor and control the amount of cellular response that your body will then undergo The animal will also produce the restrictive immunological response, scar tissue formation and fibroblast response These cells may be used in their entirety but then you may get the species cross-reactions Transfer factors from both can be raised which will stimulate the patients own restrictive immuno response of fibroblasts and surrounding cells to encapsulate the cancer so that scar tissue will form around the cancer and calcify it

The animal will also infiltrate the cancer with TILs TILs from the animal in the cancer can then be extracted and amplified These cells will amplify to a much greater extent than ordinary human TILs because, in the animal, these cells should be at a very young stage of clonal exhaustion In other words the TILs are very far from being clonally exhausted as compared to the human TILs Therefore, implantation of a tumor into an animal will give us the systemic immunological response which can be used in the way of cells, serum transfer factors, or various types It will provide us extracts from Peyer^'s patches in the spleen, thymus and other organs which are distant to where the tumor has been implanted, but which have strong anti-cancer activity Transplantation of tissue may yield a localized fibroblast scarring tissue formation response, which is capable of enclosing the cancer TILs and other cells will also be present These may be used whole, transfer factors taken therefrom, or amplified In the amplification with phage or levamisole. cytokines or any of the mitotic stimulants, greater yield occurs because these cells are at a much younger stage than the ones inside the tumor to begin with There are mechanisms ot purification and extraction of factors that might have anti-human activity as well as anti-cancer activity which will be discussed in greater detail below

TILs isolated in this manner should be able to demonstrate strong anti-cancer activity and should be regenerable when needed The transfer factor from these TIL's can also have strong activity in stimulating the human immune system TIL s, although near extinction, may have transfer factor overlap in activity with other lymphocytes so that different clones of cells can be recruited as TIL's The farther down the evolutionar scale, the animal implanted with the human cancer is from the human, the more varied the immune response against the cancer and the greater the generation of a resistant transfer factor and transformation factor The chances are that the cells generated in answer to this cancer will not overlap with human cells It is also possible that the primary response of the animal will deal with a cell type that is not generallv used in the fight against cancer For example, transfer factor taken in the early stages of tumor inoculation in several animals will yield transfer factor from neutrophils and this will in turn, stimulate neutrophi a against the cancer in the human

Prior art involves the isolation of TIL^'s from the tumor mass and its external in vitro amplification using interferon, interleukin and other agents Pπor art also involves implanting genetics of tumor necrosis factor, interferon. interleukin, etc into tumor cells in an attempt to stimulate the TILs The prior art also involves the raising of TIL^'s from animal cancers and injecting them back into human beings Prior art fails in that TIL's are assumed to be near the end of their clonal numbers or virtuallv clonally exhausted That is why the TILs cannot be amplified indefinitely That is why outside of the in vitro situation when re-implanted into the human TILs fail to further amplify and maintain a lasting effect Prior art in taking TIL's from animal tumor models and using them to treat human cancers fails to recognize that there is a difference between the TIL attacking an animal cancer and anticipating that they will automatically attack a human cancer The difference here in the prior art is that it is the human cancer that is implanted into an animal system to generate TIL's from the animal against the human cancer Also, the prior art differs in that the animals used are of a varied range of species so as to yield tumor infiltrating cells which may be lymphocytes or neutrophils quite unlike any cell that currently exists in the human system to get a broad spectrum or arsenal of cells that can attack as well as a broad spectrum where an arsenal of transfer factors and other factors can produce an immune response In birds, for example, a major immunological response likely to result will be B-cell related or humorally carried

Quite unlike the prior art where it is a cell to cell reaction, a tumor infiltrating B-cell or a tumor attacking B- cell from a bird will elicit antibody formation which is cancer specific Also unlike pπor art where it is simply the cancer that is being targeted and attacked by the lymphocytes, the inventor also adds the organism or the causative factor and elicits the raising of antiserum cellular responses, humoral responses, endocrine responses and transfer factor responses against the cause as well as the symptoms of the disease, the cells themselves In the present invention, following implantation of the tumor and its partial destruction by TILs and animal responses, the animal responses are subjected to an intermediate stage, whereby they are incubated in vino with the human cancer so that the surviving human cancer cells can then be re- moculated into the animal As a result, multiple generations of tumor anti-cancer specific cellular, humoral and other responses are. therefore, elicited

Upon treatment with the immune response from the animal, the human cancer will change in vivo and the dynamic format can be used and anticipated, whereby the resistance cells are repeatedly implanted into the animal to raise new clones of cells Cancer is therefore attacked immunologically by a range of specie responses which may be done simultaneously or in sequence to extend the duration of the administered therapy Cancer may be brought under fire from systems which are totally foreign in the anti-cancer defense mechanisms in the human, depending on how far removed the specie used to generate the response is from the human

Prior art will usually use transfer factors raised within weeks of inoculation of the disease into the animal In the present invention, there is recognition of an immediate anti-cancer response and that transfer factors and humoral responses can be identified by the time of the inoculation as well as by mechanism So the immune response can be separated into an early anti-cancer response, intermediate and delayed anti-cancer response with respect to hours (early), days (intermediate) weeks and months delayed) according to the particular species, and whether the challenge is primary or repeated Although isolation of the neutrophilic reaction following hours of inoculation of cancer and its use in the treatment of cancer has been attempted, use of the animal neutrophils without purification often resulted in severe systemic shock and often allergic responses

It is one object of the present invention to avoid these types of responses and to show how the anti-human factor may be removed or neutralized

It is a further object of this invention to extract transfer factor from the early neutrophils and other responses which occur within hours so as to avoid major side effects to the patient

A further object of this invention is to extract from the patient^'s blood his/her own private cells and treat them with the transfer factor raised in the animal and then re-infuse them with or without pπor amplification of the transfer factor into the host of the disease Such therapy may be produced on an autogenous basis

It is a further object of this invention to better control the extent and rate of reaction to raise animal responses, not only against the disease and the disease causing organism, but also against tagging agents which may be used to mark the disease For example, accurate elimination of the disease may be made by tagging the disease with an agent against which an antiserum, a transfer factor, and a cellular response in the animal system has been raised and humanized so that the tagged disease can be anticipated, tagged, and then attacked with the anti-tag therapy previously raised The transfer factor is administered prior to and the antiserum and cellular response can be administered post-tagging with tagging agents There are organs and organisms which are resistent to certain diseases It is known that animals, such as the horse, have a superior immunological response against several diseases, particularly bacterial and viral infections A century ago, it was common to vaccinate a horse against a particular bacteria or virus in order to induce formation of an antiserum which, with minimal purification was then injected back into the patient The antiserum raised against human tissue whether it be healthy or malignant tissue, would often result in a large bulk of anti-human antiserum formation and anti-human cellular characteristics When re-infused into the patient, the therapeutic agent would often attack many of the patient^'s healthy cells Minute dilutions were used as immune stimulants because they brought about destruction of the cells, and possibly reduced the disease load and caused reticuloendothelial system stimulation

It is an object of the present invention to use horse and other animal serum immunological response as a treatment against AIDS, the Ebola virus and modern diseases which have little to no effective therapy alternatives The horse serum can be used in its raw unpurified form or it can be purified in any number of ways so as to remove the anti-human component Resistance may be inherent or may be induced by vaccination of the animal The present invention is directed to extensive purification techniques to yield the anti-disease fraction of the antiserum One method involves the raising of an anti-human antiserum so that an animal of the same or other specie is vaccinated with normal human tissue and cellular components in order to raise a large titer of anti-human antiserum The anti-human antiserum is then introduced into another animal in order to raise anti anti-human antiserum and when the disease fraction is vaccinated into a third animal prior to its use, it is treated with the anti anti- human antiserum in order to precipitate out the fraction of the antiserum which is directed against healthy cells, resulting in an anti-disease antiserum This is a complicated method but can be done repeatedly in order to further purify the therapeutic agent whether it be antiserum or cells

A simpler method is to take the resulting therapeutic agent, the antiserum or anti-human antiserum or anti-human cellular component and wash it against normal human red blood cells including the patient's normal cells, providing that these cells are not effected by the disease situation or antigenically marked by the disease When normal human cells are used to wash the therapeutic agent, the therapeutic agent will lose the anti-serum against the normal human cells and will lose the cells against the normal human tissue and will be left only with a therapeutic agent in solution Techniques may also be done whereby the disease agent is introduced into the therapeutic solution and by repeated washings, the anti-disease fraction with high affinity for the disease is removed This anti-disease fraction is then washed against normal human cells in order to remove any fraction of it with anti-normal human cell activity The common laborator) techniques of washing against red blood cells and washing against the patient s own blood components and pyrogen assa> s were also combined with horse serum extraction to produce a novel method of therapy

There are several ways of preparing horse serum so that it is pyrogen and allergen free The horse serum is vaccined against the particular diseases including AIDS and Ebola virus The horse, or any animal, once vaccinated against the AIDS virus, for example will yield a rise in the electrophoresis pattern relating to the area affected Peaks will appear that correlate to a possible pre-existing inherent resistent factor In which case, considering electrophoretic and molecular weight parameters, animals which have never been exposed to the disease can still have that fragment ot their serum purified and raised to produce anti-disease activity with such a method there is virtually no risk of contaminating the animal serum pπor to treatment Another way of assuring that the patient will not be contaminated with another persons organism or disease is to devote one animal per patient

Use of horse and other animal serum/immunological response in the treatment of AIDS does not simply refer to anti-HIV antiserum It also includes transfer factors raised from the horse/other animal, and it also includes antiserum/immune response raised against the other opportunistic infections of AIDS such as pneumocystis etc It also includes transfer factor raised from the horse and other immunological cellular factors Anticipation and dynamic flow mechanisms are also included

Dynamic flow mechanism can easily be realized when a living system is donating the therapeutic agent One of the major problems with producing an AIDS vaccine is that the AIDS virus repeatedly and rapidly mutates A living system donating the therapeutic agent can then be vaccined with the mutated agents so that a new anti-serum can be developed so that the line of therapy is never ending So long as the horse is alive, you can repeatedly vaccinate it with a new virus that is emerging This is dynamic flow mechanism of ensuring therapy is maintain for the particular patient

Even more effective than dynamic flow mechanism is anticipating the changes that are going to happen, vaccinating the horse in anticipation of the changes, and producing antiserum and transfer factors in which you anticipate the changes that will occur in the virus These changes can be anticipated in one dimension in one phase of therapy or in multiple dimensions with multiple phases of therapy In other words, an antiserum produced against the first manifestation of the disease will act against the first manifestation of the disease An assay may be made in vitro Surviving or mutating disease fractions can be identified, amplified and vaccinated against such that the horse/other animal serum immunological response may be prepared in multiple phases with each phase anticipating the new resistent mutant about to arise In the case of AIDS, a polyvalent vaccine made against multiple AIDS viruses as they currentlv exist from multiple sources may actually anticipate and neutralize many if not all of the mutant forms that may arise from the patient Similarly, we can anticipate changes with cancer/HIV (any targeted condition) that can occur in vitro or in v vo models and vaccinate against them with transfer factor and antiserum against disease and the organism or factor of cause/synergy or promotion

It is important to note that the purification process may be to the advantage of the patient for the treating cells and serum, etc However, repeated "purification" presents some problems For example, if an antiserum from a horse tags a diseased human cell which has been tagged, it provides a secondary tagging from the horses antigens themselves which are in proximity to the diseased cell It can induce a graft rejection phenomenon . One can anticipate how the horse/animal serum immune response will attack the disease, and produce a second horse/ animal serum/immune response which will neutralize the new presentation to arise prior to its doing so "Horse serum" refers to any animal, any antiserum. cellular responses and transfer factor responses One can anticipate by repeatedly exposing the agent to the antiserum, culturing them to find the survivors and raising an anti-serum to them in anticipation mode or dynamic flow mode In anticipation, the transfer factor can be administered prior to any of the antiserum being given so that the bodv ^'s old immune system can be "warned" and prepared for everything that will arise The transfer factor may be a curative transfer factor, transformation transfer factor, transfer factor of resistance, etc against the new factors to arise The antiserum raised from the horse and the transfer factor and the transformation factor raised from the disease condition with prior exposure to other agents creates a multivalent, multi-phasic therapy For example, primary horse serum may be raised against the disease, secondary horse serum may be raised against the appearance of the disease once the disease is treated with 2-MEA or an other agent, or AZT or whatever the agent You anticipate the changes caused either by the horse serum therapy itself and/or by other agents which may be used in parallel or in series with the horse serum Seeing the changes as the> happen and providing a dynamic flow ol therapy or anticipating the changes before they occur provides an anticipatory therap} As a result, both the passive form in the bodv is being supported by the passive immuno response from the horse (or whatever animal) and in the active form in the body is being notified of changes as they happen or prior to their πse by use of transfer factors Further, the bodv is being activated b> both normal cellular response and other cellular responses can take part as well Further, multiphasic, multivalent therapy involves the use of viruses/phages and other physical, chemical and biological agents

In Krebiozen, a short-lived cancer in the earlier half of this century, horse serum stimulated with Actinomycetes was used in the treatment of cancer with almost no success Animal sera also includes human sera and one aspect of the present therapy that requires mentioning is that both in its purified and direct forms and in its form where the patient has been vaccined to anticipate changes to cancer, and its form where a patient has been vaccined and the serum made to attack both the vaccined form and the prior to vaccine form by reverse tagging Bodily fluids such as ascites plural effusions and other bodily fluids whether they be cancer origin, cardiac origin or inflammatory origin may be used These fluids seem to have very powerful anti-cancer activity, particularly where sarcoma fluid is used in the treatment of sarcoma and carcinoma fluid is used in the treatment ot carcinoma Ascites fluid has been assumed to carry some factor or agent in antiserum to the cancer and it has been assumed to be effective as an anti-cancer antiserum In ascitic fluids, cells tend to exist floating freely rather than in a mass and that allows the body a better chance to attack them with an antiserum and to generate and effective antiserum because there is no mass effect when cell surfaces are fully exposed It has been assumed that the ascites anti-cancer effect is one of antiserum versus the cancer However, there must be something more than that because antiserum would then be expected to have the best responses against cancers of the same type from which the ascites fluid was removed In other words. ascites from a breast cancer patient would be expected to have the best effects against breast cancer However, the inventor found that there is much cross reactivity between ascites fluid The inventor further observed an optimal anti-breast cancer effect with prostate cancer fluid and vice versa Furthermore, the ascites fluid appears to have more activity if it was bearing cancer cells at the time of implantation into animals Furthermore, ascites fluids seem to have even more activity when it was obtained from a separate species It appeared that the effect was more than an immunologic effect Cancer masses from prostate cancer disappeared, masses of grapefruit size melted away within 6 to 12 hours without significant side effects This remission is too fast for an immunologic reaction, however, if the cancers were varied and in type but belonged to broad categories such as carcinoma or sarcoma, and provided that they did not cross this category, because when the category was crossed and carcinoma was treated with sarcoma or vice versa occasionally improvement occurred but that was usually short lived and deterioration was usually rapid following So long as we stayed within the main headings it seemed that the more vaπed the cancers, the more rapid the breakdown It is very unlikely that a woman will ever develop prostate cancer and yet fluid from a prostate cancer caused marked reduction in the breast cancer Similarly, breast cancer is rather uncommon in men and yet fluid from a breast cancer caused devastating effects against prostate cancer which the female immune system is very unlikely to ever meet. Therefore, there seems to be another mechanism active Effusions and/or fractions thereof may be used in combination with other types and phases of therapy as well as being able to carry and deliver certain physical, chemical or biological agents to the targeted condition

One object of the invention is to identify and define human anti-serum extracted from ascites plural effusions and other bodily effusions which can best match and destroy cancer cells of different types It should be noted that ascites effusions and plural effusions carry some factor which is strongly regenerative both in the immune stimulation and as an anti-cachectic agent Anti-cachexia factors may be isolated from ascitic fluid and both fresh and old preparations of lysed cancer cells Many of these anti-cachexia factors are filterable through a 0 2 micron filter, and may coincide with or also carry the function of pain-relieving and anti-inflammatory factors isolated from the same sources Anti-cachexia factors prevented disastrous effects of weight loss and starvation in cancer patients This also ties in with the theory that the cancer response is an anti-disease response in that dead cancer cell extracts injected into the patient from which they were removed, or into other patients, often resulted in weight gain, disappearance of pain, and shrinkage of tumor So it appears that cancer cells themselves possess an anti-disease activit It can be shown that cancer cells have an encapsulating effect on various diseases including tetanus

An experiment was conducted where animals were injected with tetanus and cancer, and remained healthy However, when the animals were injected with cancer first and two weeks later injected with tetanus they developed cancer but not tetanus When the animals develop cancer, they may resists a tetanus challenge subsequently This indicates that cancer, for over a two week period, seems to have an effect in synergy with the immune system The two week period is important because it may represent the time peπod between cellular activity and antibody activity where something may be needed to contain the disease so that it does not become lethal

Although ascitic fluid is repeatedly used as an example, the present invention also includes pleural effusions, inflammatory effusions, as well as other bodily fluids and sera Ascites fluid was used from patients who had long term disease and appeared to be fighting it well, and was used largely in patients of similar disease In the present invention, ascites fluid is superconcentrated and lyophilized so that the dosages of hundreds of mis can be obtained within lOcc and much higher doses can, therefore, be used Ascites fluid is also not only raised in humans, but also in animals against not only the cancer but also the causative organism and also against tagged forms of the cancer Ascites fluid from humans has the capacity of being able to attack and destroy cancer cells without causing any anti-human reaction It is an agent which is freely available and disposed of in thousands of liters every day in hospitals around the world One of the novel points in the present invention is to lyophihze the fluid crude and/or purify and then lyophihze the ascites fluid Also a feature of the present invention is modern testing of fluid for dangerous contaminants such as HIV, hepatitis C and other physical, chemical and biological factors including other infectious organisms

Another important factor about ascites fluid is that the cells existing in the acidic fluid are unlike other cancer cells in the body It is either that, or one of the basic theories may be flawed A few years ago constant drainage was said to be so damaging on the heart and system in general that doctors developed what is called the ascites shunt, whereby a catheter was passed from the peritoneum into the right atnum of the heart Ascites fluid was pumped through the heart and out into the circulatory system so that the tension was minimized and so that the protein, instead of escaping to the peritoneum, stayed in the circulation to stop swelling effects and heart failure, circulatory failure, and respiratory failure What would be expected according to the theories of cancers spread, I e , cancer spreads as single cells freely circulating in the bloodstream settling down in areas and growing would have been cancer metastases Yet with the shunt technique, it appears that no further metastases resulted in several cases

Another therapy used in the present invention is extraction of the single cells from ascites fluid, centrifugation of the cells, which are also washed and purified These single cells have been attacked by the body s antibodv response inherent to the ascites fluid These cells form an excellent basis for the formation of vaccines both against that type of cancer and other types Cells extracted from ascites fluid can be processed so that they do not seed in other animals Cells extracted from ascites fluid and injected into animals bearing tumors will not seed However, these cells can cause a localized response which can cause rapid and dramatic shrinkage of cancer Prior art included injection of one person^'s cancer into someone else who was bearing cancer or grafting of one person's cancer into someone else who is bearing cancer An immunological response follows which destroys the transplanted cancer It was believed that the immune system would be so angered that some of this would have a cross over effect, and destroy some of the patient^'s primary cancer as well, so that one could use cancer against cancer This often occurred to some extent With ascites fluid injection and ascitic cellular extract injections, it was found that the cancer shrinkage occurred within hours whether or not the ascites fluid was injected into the cancer It was more dramatic though if the ascites fluid was injected in the vicinity of the cancer Softening of the cancer tissue also occurred within hours Why then should that have happened over such a short time period⁰ Why should n be more dramatic when the cancers are unrelated than when they are related⁰ Reasons for this may be that the prostate cancer is easiest recognized by a breast cancers patient's system as being foreign and would be the most rapid to be destroyed particularly because it is a foreign tissue from the opposite sex This would then yield what the inventor describes as death sequence (ds) fragments such as dsRNA and other fragments As these ds fragments are released, they are specific for cancer and thev cause destruction of the other cancer DsRNA, ds fragments, and ds enzymes are compounds which appear to behave as small filterable compounds filterable through about 0 2 microns They appear to be agents of nucleic acid consistencv although thev have not been defined as such They may be capable of short range effects Evidence for this is from what is known as the "sympathetic effect," where multiple tumors in the brain, for example, may exist One tumor may be injected with the herpes virus and destroyed by that virus while another tumor within the vicinity which has not been affected by the v irus will also show signs of death Sympathetic death also occurs around normal tissues not simpl} cancerous When somebody suffers a heart attack there is often a zone of death surrounding the dead tissue although that zone of death is not tissue which is perfused by blood. Therefore, there appears to be a factor released from dead and dying cells which causes death to neighboring cells or in related cells. Death sequence complexes also seem to have something in common with phages and plasmids from bacteria which are dying as they too carry out the same effect. Death sequence complexes in dying bacteria and other micro organisms can simply be defined as phages and plasmids carrying the death sequence. When the bacteria dies, these death sequences are released and cause the death of similar bacteria in the vicinity. These are the mammalian counterparts of the death sequence.

In the present invention, ascites fluid is purified. Ascites fluid can also be used as a very broad spectrum, anti-inflammatory agent. Ascites fluids and extracts of ascites cells added to normal human cell cause extension of the Hayflick limit and culture. Extracts of cytoplasm of cancer cells of ascites fluid and of ascites fluid cells cause significant extension of cell survival in some normal cell lines dependant on the continued supply of those factors. In other words, when this factor is removed from a healthy cell, the healthy cell returns to normal, lives a normal life span and then dies. But ascites fluid in cancer cytoplasmic extract can be used to extend the Hayflick limit. Apparently the death sequence released by the Hayflick limit of normal cells can be immunized against or neutralized by cancer cell extracts in a passive way, whereby a constant supply of cancer cell extract can maintain a healthy system for a very long period of time (other mechanisms of neutralizing the death sequence accomplish the same.

In tissue regeneration and life extension, there is reproduction of high fidelity in the prevention of cellular catastrophe. One cannot simply keep multiplying cells and maintain cell health unless your enzyme factors and DNA reproduction factors so that cellular catastrophies such as cancer or mutation do not result. This ascites fluid removed from a different source, therefore, can directly cause death by release of the death sequence, can directly cause death by an anti-serum anti-cancer effect, and can directly destroy by attacking the causative organism if vaccinated against it. and under certain conditions may even be used to culture the causative organism, and can have a secondary tagging effect by carrying its foreign antigens from the host, animal, specie. or other patient to attach to the patient s own cancer and mark it with foreign antigens and exert regenerative capacity

The use of ascites fluid in the treatment of cancer in accordance with the present invention is not only amplified to produce more concentrated sera, but multivalent sera from various types of cancer is produced to account for various antigen antibody responses Ascites fluid is purified One can also quantitate the particular fraction of the ascites fluid which has the anti-cancer activity More importantlv . cancer cells as they exist in the acidic fluid may form excellent vaccines because ascites fluid is usually one of the terminal stages of cancer Therefore if someone has lung cancer in its initial stages and you vaccinate him with cells from lung cancer and ascites fluid, he is essentially vaccinated against his own terminal stage Pπor art differs from the present invention in that now we can purify components farther, check farther for contaminates, concentrate further, and perform a much broader spectrum ascites fluid preparation An ascites fluid screen can be performed, wherebv in the same way as with antibiograms, cancer cells are cultured in a petri dish or placed in culture bottles and different types of ascites fluid and combinations arc added from different diseases and different sources including different species to see which has the greater cancer cell destruction Ascites fluid can also be combined with ascites fluid raised against the organism in animals, ascites fluid raised against the organism with the tagged cells in animals or in people, antibiotics against the organisms, anti-serum against the organisms, phages against the organisms in a multivalent. multiphasic type treatment In patients who have ascites fluid, cells can be removed and tagged, and then treated much the same way as before Once all of the cells are tagged, the body can then be re-vaccinated with the original cells so as to accomplish a reverse evolution vaccine Cancer cells exist in a form and quantity that causes threshold inhibition f the immune system If tagging can sufficiently alter their appearance en masse, the system will not be inhibited against their preliminary appearance Vaccination against cancer cells in their original presentation may now raise an effective immune response, particularly when combined with transfer factors and other agents previously discussed herein Ascitic fluid may also be a source of many of these agents including transfer factors As the tagging agents are assimilated by the cancer and/or eliminated from the system, and as the cancer begins to revert to its original form, the immunological response will be waiting

Rats bearing Morris TC-hepatitis showed survival after application of lOcc of human breast ascitic fluid intrapeπoneally every second day Having the animal model enables us to raise ascitic fluid as tagging complexes and the cancer or targeted disease as it would appear after tagging, this yields ascitic fluid of greater efficacy in destroying a targeted condition. Ascitic fluid extracted from humans treated with vaccines which tag cancers with microorganism extracts will be rich in antiserum against the tagged manifestation of the targeted condition

Organisms may be seen in many disease conditions including cardiovascular as well as acute and chronic degenerative diseases Whether the organisms isolated from AIDS or other diseases play a role in cause or simply synergize with the disease. Raising vaccines and antisera against them appear beneficial, even in absence of anti- HIV antisera in therapy Blood from AIDS patients can be lysed by physical/chemical/biological means as previously described Plating on blood agar and or inoculation into TSB as well as many other media may be used 500cc of TSB are inoculated and cultured for three days Bacterial growth is centrifuged and resuspended in 30cc of normal saline Suspension is boiled for 15 minutes every three days then used to immunize an animal (horse) by injections intradermally of l cc every three days Animals can also be injected with living organisms or those prepared in vaccine format to anticipate a physical, chemical or biological therapy which the patient will undergo For example, targeted condition and/or associated organisms or factors may be heated so as to reveal antigens and properties which will be brought out by hyperthermia. Targeted condition and/or associated organisms or factors may be irradiated and administered to the patient prior to the patient receiving radiotherapy By preparing the vaccines in a manner which mimics whatever therapy which the patient is about to undergo and by allowing the vaccine protocol appropriate dosage and time to elicit effective immunological response, the vaccine response which may be further aided by various transfer factors will augment the other therapy by joining the therapy in its attack against the targeted condition and by providing an immunological modality for elimination of cells which are resistant to the other physical, chemical and biological therapies

Vaccines referred to above may be active (inducing host immunological response) or passive raisin antisera and other useful immunological fractions from outside the host Attenuation Factor

Cancers in culture and in living systems have the capacity to attenuate many forms of microbial infections Although this capacity for production of attenuation factors is not unique to cancer cells, the attenuation factor in this invention may be isolated from other cell types However cancer cells appear to have an incredible propensity for attenuation of infectious agents in vivo and in vitro For example, leukemia cells infected with feline panleukopenia virus will be destroyed and the animal may appear in remission for a short period of time, usually measurable in weeks When the leukemia recurs, vacuolation will still be seen in a significant percent of cells, suggesting that whereas the v irus initially cause vacuolation and extensive destruction, the virus is now incapable of destruction of the leukemia as previously The virus appears to exist in synergy or a non-threatening manner in the cells The virus appeared to be no longer as infective as initially This is evidenced b> a lower percentage of affected cells, suggesting a) higher cellular resistance, and b) viral attenuation Cells with higher resistance which do not exhibit vacuolation may be used to yield transfer factor of resistance against the virus

Attenuation factor, however, is evident as successive generations of infected cancer cells yield viruses of diminished ineffective capacity Attenuation factor exists m many cancer cell types and cancer-related fluids, and may be of assistance in placing viruses in a proviral state Ascitic fluid extracted from a patient with hepatoma who had been treated by tagging with various microbial factors and extracts showed greater than 99% inhibition of HIV cultured in vitro in human T-cell lives Other ascitic extracts have shown strong attenuation features in that, despite a rise in HIV yield, the T-cells remained largely unaffected In every animal species, there is an immunological response with great specificit and affinity In a diseased host, that response may be depleted TILs. for example, exist in many cancer patients, but are of too low a number and/or activity to affect a cure TILs isolated from animals beaπng similar cancers and/or from animals inoculated or transplanted with the human cancer may be used to yield an effective cellular immune response which can be tolerated by the patient by any means of administration depending on the cancer and/or transfer factors isolated from the TILs against the disease and as it exists or as it may be anticipated or as it may appear tagged maj be of use A transfer factor of resistance which is isolated from surviving animal or human cells exposed to certain conditions such as radiotherapy and chemotherapy, wherein the cells are normal cells of the diseased host being treated, surviving members can be used to yield transfer factors of resistance to augment and support similar cells in the host Cells which are damaged or killed by certain conditions such as the therapeutic modalities named above can be used to yield transfer factors of sensitivity These would be of use in specifically programming cancer cells to be more sensitive to therapy

There exists a restrictive immunological response This can be seen in the scarring that occurs around old tuberculosis lesions and around shrinking tumors Cells involved in this process including fibroblasts and cells which cause local deposition of calcium can be used whole or m part of (including transfer factor) to induce restriction responses in the host Multiphasic. Multimodal Therapy Multiphasic therapy may be used in synergistic multimodal approach That is to say that various modes may be used to augment each other's function Phages raised against or with activity against penicillin resistant organisms will augment the function of said antibiotic Function is even augmented against penicillin sensitive organisms An example of this is the plating on blood agar of streptococcal culture with penicillin sensitivity Streptococcus was isolated from skin sample and possessed penicillin sensitivity (test was repeated on several occasions using penicillin as well as other types of antibiotics and streptococcal, staphylococcal and other host organisms as well as their phages as supplied by ATCC and/or raised independently, with similar results) after plating and culturing for 3 days at room temperature, the plate was covered with many purely streptococcal colonies On the third day, a disk saturated with procaine penicillin was placed in the center of the agar plate Within 24 hours a zone of clearance was evident around the disk as a halo measuring 2mm in depth The following day the zone measured 3mm, there was no further enlargement of zone with further incubation Streptococcal phage was added to the penicillin-saturated disk Phage was either one obtained from ATCC which had demonstrated efficacy in lysis of this particular streptococcus or one raised from the bacteria by techniques previously noted Solution of phage estimated at 10 to the ninth power of plaque forming units 0 5cc of phage TSB suspension was added to disc saturated with procaine penicillin Zone of clearance formed as in previous test, zone of clearance was marginallv larger 0 5mm, more importantly, within 24 hours plate contained no viable colonies within 48 hours In other test systems where total colonv clearance did not occur multiphased (usually no more than 3) were required to achieve total bacterial lysis

This invitro data does not easi translate in invivo data as phage of single phase is usually unable to eradicate infection from a living body, not only is a living open system conducive to the bacterial target s development of resistance, but there is also the problem of therapy reaching target, much of the phage load may be intercepted by the host's immune response and eliminated pπor to reaching the bacteria Prior mechanisms and examples described show the ability of multiphased phages and immune responses to augment each others ability as well as other pathways of therapv such as antibiotic therapy

Multiphasic therapy in multiple modes can enhance each others function as well as offer prevention against the development of resistance Phages can be raised specifically against antibiotic resistant strains of target organism Immune response such as antiserum, may be raised against organisms Other techniques including selective filtration, electrophoresis and other technologies including isolation and genetic amplification technology including monoclonal antibody production and other mechanisms involving hybrid and other cell lines The patent covers the use of anti sera/white blood cell extract/immunostimulation as adjunct to antibiotic therapy both directly and by raising response against target previously treated with antibiotic so that there is a passive as well as a latter active immune response against therapy resistant organisms It is possible to incubate patient cells with transfer factor from immunized cells of other source, to induce their activity against target organism, to recognize, isolate and amplify using interferon, interleukin and/or other modalities patient cells with activity against target

Following several months of cold storage precipitates form in the serum which are filtered out Serum is then filtered through 0 2 microns 0 3cc of serum is injected intradermally Doses up to 5cc hav e been used lntradermally/subcutaneously/ intramuscularly without harmful results The low dose is used as in theory, the target resistant population exists in either low dose or hav e not yet been generated

Furthermore we can use the antisera to inhibit donation of plasmids or other resistance- donating factors Even where resistant strains exist in significant amounts, it is unlikely that they will survive both antibiotic and phage attack, again leaving relatively little for the antisera to overcome THERAPY

One million units of procaine penicillin. 0 6cc of phage lysate ( 10 to the ninety power plaque forming units), 0 3cc horse serum (anecdotal cases have suggested that human an/or animal immunoglobulins including the commonly available forms such as pooled immunoglobulins even if not specifically raised against target may be of benefit but tend to not be as effective, current risk of hepatitis and AIDS contamination of human stock may make animal sera more appealing )

TECHNIQUE- 1 million units of procaine penicillin were injected intramuscularly 0 6cc of phage lysate was injected intradermally at the same time 0 3cc of antiserum was injected intradermally 6 hours later The reason for the delay is the theoretical possibility that antiserum may bind and neutralize phage lysate prior to its circulation and reaching targets

The phage injection in all three patients gave red marks 1 -4 inches in diameter and was itchy for several minutes One reported a mild headache and temperature of 100 degrees celsius that lasted for 6 hours Tonsillitis resolved in all patients within 48 hours of treatment Follow up for 1 year revealed no recurrence of tonsillitis in any of the three This was quite unusual in view of previous history of at least two yearly bouts amongst them

There exist other invitro and therapeutic data suggesting the efficacy of multimodal therapy combined with current standard therapy as well as multiphased multimodal therapy alone and/or in combination with standard therapy

Table A below represents activity of animal sera, ascitic fluid, phages. attenuation factors (not restricted to those derived from cancer cells), and other agents as prepared in accordance with the methods outlined in the present invention

TABLE A

Neutralizing P24 Antigen Percent Sample (ng/ml) Neutralization Triplicates (Mean)

Virus + HIV -negative 198/210/210(206) N/A serum

Virus alone 172/1 88/1 76( 179) N/A

Virus + HIV -positive 74/76/130(93) >99 serum n\ 0 5/0 60 3(0 8) >99

#2 0 1 /0 2/0 1 (0 1 ) >99

#3 <0 l /<0 1 /0 1 (<0 1 ) >99

#4A <0 l /<0 1/0 1 (<0 1 ) >99

#4B 0 1/0 1 /0 1 (0 1 ) >99

#5 0 3/0 3/0 3(0 3) >99

#6 0 6/0 4/0 4(0 4) >99 #7-1 0.7/0.8/0.7(0.7) >99

#7-2 0.2/0.5/0.2(0.3) >99

#8 0.2/0.3/0.1(0.2) >99

#9B 0.1/0.1/0.1(0.1) >99

#10 76/62/56(65) 69

#1 IC 106/55/54(55) 65

#12 0.4/0.4/0.9(0.6) >99

#13 0.2/0.2/0.2(0.2) >99

#14 0.1/<0.1/<0.1(<0.1) >99

#15 0.3/0.3/0.6(0.4) >99

#16 0.7/0.6/0.2(0.5) >99

#I8A 0.1/<0.1/<0.1(<0.1) >99

#18B-1 0.1/<0.1/<0.1(<0.1) >99

#18B-2 0.1/<0.1/<0.1(<0.1) >99

#18C 0.1/<0.1/0.1(<0.1) >99

Results indicate strong efficacy in the inhibition of viral growth. Analyses also showed the capacity of transfer factors of resistance and synergy to protect the T-cells from viral destruction even where the virus was not fully inhibited.

Table B demonstrates the safety and non-toxicity against normal tissue of products of the present invention.

TABLE B

Cytotoxicity (Cells + serum) @ 7 days of culture {Rephcates-Cells x \ Q (mean)}

Sample 1 20 Dilution 1 100 Dilution 1 1000 Dilution

HIV-neg. serum 74/76 (75) 68/51 (69) 121 /1 14 ( 1 1 7)

#1 91/80 (85) 80/90 (85) 86/87 (87)

#2 89/90 (89) 142/1 14 ( 128) 99/103 ( 101 )

#3 48/41 (45) 108/121 ( 1 15) 67/79 (73)

#4A 52/42 (47) 87/83 (85) 102/106 ( 104)

#1 1 C 121/156 ( 138) 148/130 (139) 136/89 ( 1 13 )

#13 74/58 (66) 92/97 (94) 128/91 ( 109)

#15 63/69 (66) 134/106 (120) 92/105 (98)

#16 77/85 (69) 97/83 (90) 1 12/92 ( 102)

#18B- 1 74/64 (69) 83/94 (88) 93/87 (90)

Results demonstrate almost no toxicity and thus, extreme safety at various dilutions Evaluation of the products of the present invention to produce the data in Tables A & B was performed at Cedars Sinai Medical Center in Los Angeles California

Antisera raised against the heat killed organisms as described above is combined with antisera raised against the living bacteria (same preparation technique without the heat killing after resuspension in saline) vaccination is intradermal 0 5cc three times a week for three weeks, followed by antiserum preparation as previously described Antisera raised against these organisms appear capable of maintaining immune status and at least preventing deterioration in the cases studied Patient age 42 diagnosed for three years T-cell count dropping 80- 100 points per month over past four months T- cell count 31 1 1 4 8 ratio 0 2 Patient was treated with antiserum (0 6ct intradermal three times a week) raised against organisms isolated from his blood as per previous discussion

Day 3 - T-cell count 330 T4/T8 ratio 0 2

Day 10 - T-cell count 31 8 T4/T8 ratio 0 2 Day 60 - T-cell count 350 T4/T8 ratio 0 3

Day 120 - T-cell count 300 T4/T8 ratio 0 3 Fatigue and rapidly decreasing T-cell count Readings taken over the previous months had shown a monthly drop in T-cell counts by 150-200 per month At presentation T- cell count total 350, T4/T8 ratio 0 8 Specific antiserum (raised against pooled aids blood treated with antihuman antiserum) 0 6cc were administered intradermally three times a week

Day 3 - T-cell count 340 T4/T8 ratio 0 8

Day 10 - T-cell count 310 T4/T8 ratio 0 7

Day 30 - T-cell count 400 T4/T8 ratio 0 9 Day 50 - T-cell count 510 T4/T8 ratio 1 0

Day 60 - T-cell count 480 T4/T8 ratio 1 0

Day 90 - T-cell count 550 T4/T8 ratio 1 0

Day 120 - T-cell count 515 T4/T8 ratio 1 0 Use of multiphasic antisera pooled aids blood was treated with antihuman antisera and used to vaccinate horse as per above method Horse serum was allowed to stand at 3° for nine months patients (3) were injected with 0 6cc of antiserum three times a week for three weeks, their blood was then pooled and prepared as per previous protocol to revaccinate horse as well as vaccinating new animals The antiserum raised as per previous protocol was then processed Antiserum raised from previously immunized animal was labelled second challenge antiserum, animals vaccinated de-novo generate a purely second generation antiserum This process may be repeated indefinitely Original animal will be in its third year by the time the third phase is ready (as this technique requires nine months for dehumanizing) Other techniques of preparing animal sera for human use. such as plasmapheresis and/or washing against human blood etc can greatly speed multiphasic antisera generation Patients treated with multiphasic antisera are exemplified by the following case male 52, three year history since diagnosis T-cell count 220 T4/T8 ratio 0 6 T reated by 0 6cc antisera intradermal every three days

Day 3 - T-cell count 280 T4/T8 ratio 0 8 Day 10 - T-cell count 350 T4/T8 ratio 0 8

Day 30 - T-cell count 418 T4/T8 ratio 0 8

Day 60 - T -cell count 410 T4/T8 ratio 1 0

Day 120 - T-cell count 400 T4/T8 ratio 1 0 In most of the above cases patients were either asymptomatic or suffered from fatigue which readily resolved within the first 24 hours

Where patients presented with specific infections, which recurred frequently or were resistant to antibiotic therapy, animal sera (both general and specific) was useful in eliminating these diseases

Aids patient. 28 year old male, 4 year history since diagnosis Asymptomatic T-cell count 350, T4/T8 ratio 0 6 0 05cc Goat blood was injected intradermally day 1

Day 3 - T-cell count 250 T4/T8 ratio 0 8 Day 10 - T-cell count 450 T4/T8 ratio 0 9

Aids patient, 45 year old male, 2 years since diagnosis T-cell count 240. T4/T8 ratio 0 5 Horse blood 0 1 cc crude lysed injected subdermally day 1

Day 3 - T-cell count 248 T4/T8 ratio 0 5 Day 10 - T-cell count 330 T4/T8 ratio 0 6

Aids patient, 50 year old female. 6 months since diagnosis T-cell count 150 T4/T8 ratio 0 4 Goat serum - 0 lcc injected intradermally Day 3 - T-cell count 90 T4/T8 ratio 0 3 Day 10 - T-cell count 210 T4/T8 ratio 0 6

The above cases were only provided to demonstrate that the overall immunostimulant ability of foreign blood fractions could be achieved by whole blood and/or extracts from a variety of animals and is often predated by an initial drop in parameters

The initial drop may be attributable to either the antihuman effect of animal blood and/or to the lysis of infected/diseased cells There is data to suggest that the effect of decrease and subsequent augmentation of immune response may be more dramatic when the animal has been immunized against the HIV/diseased cells/blood of patient Again, the two phenomenon are thought to be at least partially responsible

Removing antihuman component techniques for this range from simple to complicated Many were covered in preliminary discussion the simplest technique will be demonstrated Serum is stored at 3° celsius for nine months Precipitation forms and is removed lt was found by previous workers in the early parts of this century that prolonged cold storage resulted in antisera which demonstrated markedly decreased side-effects and therefore was assumed to be of less antihuman activity

In this inventor's field of experience, both immunized and non-immunized blood was studied, the disadvantage of the above dehumanizing method is the long time needed to manufacture specific vaccines The idea of using a living resistant extraneous system to combat HIV is to be able to modify therapy (as in raising new antiserum for example) as the virus mutates into a form that can inhibit/elude/otherwise survive previous immune response Preparation of broad spectrum antisera against a large variety of HIV samples as collected or raised and use of that or the specific fraction of it shown to act specifically against the patent^'s viral load (as previously discussed, this would be the fraction altered electrophoretically after exposure to HIV, it can then be identified by its physical characteristics, other methods of precipitating it out with target cell or virus then reconstituting it may also apply May overcome this problem as would the use of more rapid 'dehumanizing^' techniques such as washing serum against normal blood cells etc Perhaps the greatest success was noted when the antisera was used as part of multimodal/multiphasic application Several cases were treated with antisera alone to demonstrate efficiency of this simple pathwav as sole therapy as well Use of Non-Immunized Antisera in the Treatment of AIDS The preparations used were crude by modern standards 1 year old horse was bled and l OOcc of serum were removed following centrifugation of blood Serum was stored at 3°C for nine months as were sera from sheep and goat Precipitates were marked by 3 months and all precipitate was removed by 0 2 micron filtration at the end of nine months

Use of Horse serum

23 year old female, 18 months since diagnosis T-cell count 320, T4/T8 ratio 0 8 O.όcc antiserum administered intradermally

Day 3 - T-cell count 280 T4/T8 ratio 0 6 Day 10 - T-celi count 420 T4/T8 ratio 1 0

Day 30 - T-cell count 380 T4/T8 ratio 0 9

45 year old male, 1 year since diagnosis T-cell count 140, T4/T8 ratio 0 5 0 1 Goat antiserum injected intradermally

Day 3 - T-cell count 90 T4/T8 ratio 0 3 Day 10 - T-cell count 210 T4/T8 ratio 0 8

Day 30 - T-cell count 340. T4/T8 ratio 1 0

18 year old male. 8 months since diagnosis T-cell count 200. T4/T8 ratio 0 8 0 l cc Sheep antiserum used intradermally

Day 3 - T-cell count 210 T4/T8 ratio 0.8 Day 10 - T-cell count 310 T4/T8 ratio 0.8

Day 30 - T-cell count 280 T4/T8 ratio 0.7 Use of Immunized Animal Antiserum

Antihuman antiserum was raised against normal human blood Pooled blood from aids patients were treated with antihuman antiserum and incubated for three hours (l cc of antiserum to 10cc of aids blood) lcc of supernatant was used to vaccinate horse intradermally three times a week for three weeks l OOcc of animal blood were then drawn and serum separated and stored for nine months to allow for antihuman fragment separation

Preparation ot antihuman antiserum animal (horse) was injected with 0 5cc of human blood pooled from healthy donors every three days tor three weeks Serum was extracted on fourth week and labelled antihuman serum This was used to treat blood isolated from several diseases in order to remove normal components and leave disease- related organisms/factors Use of antisera raised against this disease-specific fragment raises a theoretically much more specific antiserum All the cases and examples presented here are not offered as irrefutable proof, but rather as examples for patent cover to all animal blood/whole/extract/processes, etc T he best results and those which respond best short and long-term occur when immune therapy is used in multiphasic mode in combination with multimodal approach Multiphasic would involve a scenario exampled by the following, multiphasic general, raised by repetition of the above processes of manufacture so that every week, blood drawn from aids patients under treatment three times a week with antiserum at dosage of 0 6cc intradermal. would be used to vaccinate animals which yielded previous antisera or new ones Pooled blood may be used directly and/or first prepared by treatment with antihuman antiserum Compound multiphasic serum would be raised when blood is used from patient treated with multiphasic serum hence generating a second generation of multiphasic responses

Use of modern purification of serum techniques such as those previously mentioned in patent could accelerate the production of safe patient-specific multiphasic sera

Use of animal immune response such as that of a horse also includes in patent the use of cells and cellular components including transfer factor

Efficacy is amplified when antiserum is used as part of multiphasic/multimodal therapy and new pathways/mechanisms are also generated It is possible, for example to tag diseased cells with fractions and then vaccinate animals against either the tagging agent alone or against the tagging agent-disease complex This is of particular importance in the treatment of cancer but is mentioned here to cover the use of antisera bv this mechanism in aids

Every phase of the use of exogenous animal antisera can demonstrate benefits of preparations ranging from the crudest use of whole blood to extracts cellular/non- cellular to the use of crude and purified antisera An example will be given of the use of purified antisera raised against a general pool of aids blood treated with antihuman antiserum. Patient 45 year old male, 5 year history since diagnosis T-cell count stable at 900-1000 for that time It is a further invention in this patent to use any and all components of human sera not included in current preparations as well as using all such components in combination with currently available extracted components Current technology and practice is to use only IgG in pooled sera as therapy or prevention IgA, IgD, IgM, etc are not included, it is this inventor's contention that they would serve important Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims

Claims

I claim:

1 A method for using animal serum in the treatment, diagnosis and clinical management of AIDS, comprising the steps of a removing target cells from a patient, b injecting the target cells into an animal having resistance to the target cells to produce an antiserum against the target cells, and thereafter c administering the antiserum to the patient with AIDS in therapeutic dosages and for a sufficient period of time to disable the targeted condition^'s capacity to resist and/or destroy the patient^'s immune response 2 The method according to claim 1 , wherein the animal is a horse

3 A method for using animal antisera raised against a targeted organism in the treatment of AIDS, comprising the steps of a exposing an animal to a targeted organism, HIV, or the disease condition to produce antisera against the HIV b administering the antisera to a patient having HIV for a sufficient period of time to disable the targeted condition^'s capacity to resist and/or destroy the patient^'s immune response

4 The method according to claim 3, wherein the animal is a horse 5 A method according to claim 3. wherein the antisera includes cellular responses, transfer factors in whole, part or extracts thereof

6 A method for using animal antiserum raised against related disease conditions such as opportunistic infections, comprising the steps of a exposing an animal to an opportunistic infection or other viral or bacterial associated infections associated with a targeted condition or disease to produce an antiserum against the infection, and thereafter b administering the antiserum to a patient having the infection associated with the targeted condition 7 The method according to claim 6, wherein lhe infections include herpes, cytomegalovirus (CMV), and pneumocystis

8 The method according to claim 6, wherein the animal is a horse

9 A method for using mixed antisera in the treatment of AIDS, cancer and other viral and bacterial diseases, comprising the steps of a exposing an animal to a targeted organism or disease condition to produce antiserum against the organism or condition b exposing the animal to a related condition of the disease condition or target organism to produce antiserum against the related condition, c combining each antiserum to form mixed antisera, and d administering the mixed antisera to a patient having the targeted organism or disease condition and the related condition

10 The method according to claim 9. wherein the animal is a horse 1 1 Use of polyvalent antigens in the preparation of antisera where the polvvalency is from pools of HIV and/or cytomegalovirus and/or herpes and/or other related conditions and/or from generations of autogenous culture

12 Animal antiserum raised against the anticipated form of the targeted disease or condition, wherein the antiserum comprises target cells or disease isolated from the individual or from other individuals

13 A polyvalent animal antisera. comprising a mixture of antiserum raised against the anticipated form of the targeted disease or condition, and antiserum raised by tagging the condition as it exists

14 A resistant animal serum wherein the animal is not specifically vaccinated against the targeted cells or disease condition prior to administration into a patient

15 Use of dynamic flow principles to generate novel antisera as resistance develops and/or resistant mutations arise

16 The use according to claim 15. wherein antisera includes cellular responses, whole, part or extracts thereof including transfer factors 17 Use of anticipatory principles to predict presentation of resistant forms or mutations of targeted cells or disease conditions

18 Use of an animal^'s immune responses against targeted cells or a disease condition in combination with pharmaceuticals to eliminate treated viruses and microorganisms and the targeted condition and to overcome pharmacologically resistant strains

19 The use according to claim 18, wherein the pharmaceuticals include AZT, DDL 2-MEA and a complex formulation of 2-MEA, BHT, at least one transfer factor and living organisms 20 The use according to claim 18 wherein the pharmaceuticals include antibiotics, chemotherapeutic agents and transfer factors

21 Use of crude animal antiserum in combination with immunological extracts in the treatment of persons with AIDS, cancer and other viral and bacterial diseases 22 The use according to claim 21. wherein the animal antiserum is purified

23 A purified animal antiserum suing a humanized process including washing the animal serum against red blood cells

24 A purified animal antiserum using a specific humanization process including washing the animal serum against the patient^'s red blood cells

25 Animal antiserum for use in the treatment of AIDS, cancer and other viral and bacterial diseases, the antiserum being purified, lyophilized and reconstituted for administration to a patient with a targeted disease or condition

26 Animal antiserum raised against an animal for a targeted disease or condition as it exists

27 A polyvalent animal antiserum having the characteristics of claim 26

28 Animal antiserum raised against an animal for a targeted disease or condition as it will exist following mutations 29 A polyvalent animal antiserum having the characteristics of claim 28.

30 Animal antiserum raised against an animal for a targeted disease or condition that is tagged. 31 A polyvalent animal antiserum having the characteristics of claim 30

32 A method of using resistant organs and organisms for the preparation of extracts for treatment of AIDS, cancer and other viral and bacterial diseases, comprising the steps of: a extracting out specific transfer factors from cells and/or tissues that have resisted invasion by a targeted disease or condition; and b administering the extracts to a patient having AIDS, cancer and/or other viral and bacterial diseases

33 The method according to claim 32. wherein the extracts include fibroblasts 34 The method according to claim 32. wherein the extracts are used to replace the antigens or rejection on the targeted cells or the targeted disease or condition

35 Transfer factors and antiserum produced by the method of claim 32, and raised against the expected appearance of the disease against therapy or against the resistant forms of the disease after therapy

36. A transfer factor of anticipation for the treatment of AIDS, cancer and other viral and bacterial disease, the transfer factor being raised against anticipated changes and resistance of the targeted cells or disease/ condition in response to physical, biological or chemical factors to educate and stimulate the immune response of the recipient and to prepare the recipient for manifestations of the targeted disease which may not yet exist.

37. Use of ascitic fluid and pleural effusions from animals to produce a graft rejection of cancer in other species

38 A transfer factor of resistance isolated from surviving animal or human cells exposed to certain conditions such as chemotherapy and radiotherapy, wherein the cells are normal cells of the diseased host being treated and the surviving cells after the exposure are used to produce a transfer factor of resistance

39 An attenuation factor extracted from various cell types, wherein attenuation factor is inherent or induced against a particular microorganism by challenge with the microorganism m vitro or in vivo

40 The attenuation factor according to claim 39 for use in the treatment of AIDS, wherein a crude extract of the attenuation factor is produced from ascitic fluid where cancer cells are lysed by a physical, chemical or biological agent, the lysed cells and ascitic fluid are filtered and then administered to a patient at a dosage of up to 500cc or greater

41 A generalized death sequence factor used to cause specific cell death of targeted cells or disease, wherein the death sequence factor is isolated from dead and dying cancer cells

42 A specific death sequence factor that is elicited by a specific agent, the agent including chemotherapeutic and radiotherapeutic agents

43 The specific death sequence according to claim 42 for use in promoting cancer cell death in response to a particular therapeutic agent

44 Use of cancer cell fragments broken down by physical, chemical or biological agents from autogenous and/or pooled sources and/or source other than the patient that is capable of increasing normal cell survival both in vitro and in vivo

45 The use according to claim 44, wherein the cancer cell fragments or extracts thereof carry an anti-cachexia factor which prevents, among other things, weight loss

46 Use of other cancer cells in the regeneration of cancer cells of a different type

47 Use of a multimodal. multivalent technique of therapy