SE448344B - ANTIBODY FOR TREATMENT OF REUMATOID ARTHRIT AND SETTING TO MAKE IT - Google Patents

Description

j44s 344 2 mechanism has been established (Sikes et al, 1955).

In studies of rheumatoid arthritis, however, researchers are still puzzled by certain recurring motifs, which indicate a connection between infections and joint diseases. Thus, e.g. Gonorrhea not only produces typical gonorial arthritis but sometimes also chronic arthritis, which can turn into rheumatoid arthritis. There are no statistics on this occurrence, so it is not known how much importance can be attached to the relationship. Tonsillitis or pharyngitis can be accompanied by a polyarthritis, which first appears as rheumatic fever but which develops into rheumatoid arthritis. Acute viral infections, especially rubella in young women, can be accompanied by prolonged = polyarthritis times involving both small and large joints. These arthritis usually occurs for several months, causing joint problems reminiscent of rheumatoid arthritis, after which the disease gradually returns. Although chronic infection with an unknown substance is still a common description for the etiology of rheumatoid arthritis, there are no published data to support this assumption. Some researchers who have studied the disease suspect that if infection is a factor, it can not be an infection of any specific type of microorganism but rather an infection of a large number of banal microorganisms with an altered response part of the host organism generated by the infections responsible for the disease. The present invention is based on this theory regarding the cause of rheumatoid arthritis. Although an infectious etiology has never been established, several recent findings seem to support this theory.

Some of these are: 1. Patients with rheumatoid arthritis have lower than normal levels of IgA, ie the class of immunoglobulin found in secretions from the gastrointestinal tract. Immunoglobulin A generated as a result of immunization via saliva, mucous membranes is found in serum colustrum and milk as well as 1 saliva. It is believed that IgA is transmitted to these various body fluids via the gastrointestinal tract and lymphatic system (Michelok et al, 1975). i 'i V f 448 344 3 i 3. After a bypass bowel operation due to obesity, symptoms develop in some patients; which are in fact identical to those of rheumatoid arthritis. The development of arthritis is accompanied by the appearance of circulating cryoproteins composed of IgG, IgM, IgA, complement components G3, G4, C5 and IgG antibodies to E. coli and B. Fragilis in blood serum; If one bypasses the bypass-intestinal tract, the symptoms disappear completely (Woods et al, 1976). __4: One type of Diplosgreptococcus agalaotiae belonging to the streptococcal group B has been reported as an etiologic substance in rheumatoid arthritis (Svartz; 1§72). This streptococcus occurs for the most part mark- iíiaaysffsra paetöšiseàfàd injöik but not in ii.

* S2 A predisposition for rheumatic diseases seems to be inherited via the = histocompatibility antigens = (HL-A). These antigens are likely to determine the response of the host organism to infectious agents. In view of these facts, it can be stated that renmatoid arthritis has an infectious origin, the infection occurring in the intestines, 4, that a large number of bacterial strains are involved in the infection, that the infection is probably due to a breakdown in the host organism's immune system. and that the most effective way to treat the disease is to restore immune protection against the infectious agent in the intestines.

Treatment of the infection There are essentially two methods which can be used for the treatment of infection, namely immunotherapy which involves either active or passive immunization against the infectious pathogens and the use of antibiotics such as penicillin, tetraycline, ampicillin and the like. Antibiotics have no specific effect and they kill a wide range of both beneficial and harmful bacteria. On the other hand, the year-immune treatment is very specific. Against a specific strain of bacteria produced, bactericidal antibodies react only with this strain and have no detrimental effect on other types of bacteria. In addition, unlike antibiotics, antibodies are natural body products and have no known side effects.

Since the object of the invention is to treat infections of a "specific group of bacteria without affecting" beneficial "bacteria in the intestines, the immunotherapy treatment technique is chosen. Active and passive immunization ¶ There are two different methods of achieving immune protection. Active immunization is the process in which the host organism is actively immunized with a vaccine; which stimulates the immune system to produce protective antibodies against factors contained in the vaccine. Active immunization takes place under natural conditions when the host organism is exposed to infectious pathogens. Passive immunization is a precaution. In this technique, the protective antibodies are transferred from the immune host organism to the recipient. Passive immune protection is temporary and lasts only as long as the passively recovered antibodies survive in the host. recipient systems, for example, antibodies from horses Immunosuppressed against tetanus toxin is given to people infected with tetanus provided that temporal protection against the toxin produced by tetanus bacteria must be achieved. U.S. Patent No. 3,626,057 discloses a process for the production of tetanus toxin in milk.

According to the patent, a cow is actively immunized against the tetanus toxin and the antibodies formed by the cow against the toxin are obtained from the cow's milk and these antibodies can be used to treat animals infected with tetanus bacteria in such a way that the antibodies neutralize the toxin. It was determined that the passively administered antibodies neutralize the life-grafted torin generated by the bacteria, thereby achieving temporary immunity to the toxin. Passive immunization differs from active immunization by an immune protection is temporary it only lasts as long as the protective antibodies are present. present. Active immunization is more permanent, as the host organism's immune system continues to produce protective antibodies in the presence of the stimulating antigen. * _ D d s d d Recent studies in the field of thermal immunology have shown the presence of a laconic immune mechanism in the intestinal system. This immune system pree produces a special type of antibody, which counteracts bacterial degeneration in the lumen of the intestinal system. The antibodies, termed secretory immunoelobulin or IqA, are produced by the antigen as a result of local active immunization of the term mucosa. The secretory immune system of the intestinal system works to protect the influx and growth of harmful bacterial strains in this environment. 443 544- i It is believed that a breakdown in the local immune system in the gut makes it possible for foreign harmful bacteria to establish themselves and that these bacteria cause rheumatoid arthritis. According to this theory, rheumatoid arthritis results from a breakdown in the intestinal system's local immune system, which is supposed to produce and secrete protective antibodies against harmful bacteria. The inability of the host organism to respond to active immunization precludes the possibility of using this method to treat rheumatoid arthritis.

The invention provides an antibody for treating rheumatoid arthritis caused by a mixed spectrum of infectious bacteria, which resides in the gastrointestinal tract and has the characteristics of claim 1. Administration of the antigen provides a temporal immune protection which is very specifically for the bacterial strains used to generate the antibodies and do not adversely affect the normal beneficial bacteria present in the intestinal tract.

Cow's milk is the preferred source of the antibody product of the invention. It is very specific in that it defines a unique population of antibodies in milk (IgG type), which react with a known spectrum of bacteria and this reaction is utilized for the treatment and prevention of rheumatoid arthritis.

The type of immunoglobulin is important in the context of the invention, as there are five known classes of immunoglobulins termed IgG, IgM, IgA, IgD and IgE (Nisonoff et al, 1971). These types of immunoglobulins differ structurally from each other (Waldman et al, 1970 + and have different biological functions in the body (Waldman et al., 1971, and Franklin, 1964). In addition, there are significant variations in the localization of immunoglobulins. Thus, there are clear distributional differences between the immunoglobulin classes IgA and IgG; The most prominent feature of IgA is the high concentration in body fluids such as gastrointestinal fluid. It has been clearly shown that the immune system, which contributes IgA to the stomach -intestinal- fluid, is a different system than the one that produces IgG.

In humans, IgG occurs primarily in vascular and intracellular areas of the body (Waldman et al, 1970) and a very small amount of IgG ends up in the gastrointestinal fluids. Another important difference between the classes of immunoglobulins concerns their metabolic growth. The breakdown of each class of immunoglobulin, regardless of its location in the body, appears to occur under separate control. Functional catabolic growth ranges from as low as 6.5% for IgG to as high as 90% for IgE while other immunoglobulin classes fall in between (Waldman et al, 1970). The different immunoglobulin-4 classes also differ in their propensity to bind to antigens and in their ability to combine with complement, which is one of the props for killing live bacterial cells (Heremans, 1960). It is important to emphasize these differences in the types of antibodies, as immune effects may vary depending on the type of antibody involved.

The most commonly claimed theory is that the different classes of immunoglobulin have been developed to function in different environments in the body. It is known, for example, that a special immune system exists with the task of producing antibodies, which are active in the intestinal system. Furthermore, it is generally believed that the immune functions in the intestinal system are specifically regulated by IgA antibodies and not IgG. Under normal conditions, therefore, IgA is the class of immunoglobulins that regulate the immune control of bacterial infections, which occur in the gastrointestinal tract of humans. Since IgG, IgM, IgD and IgE do not normally occur in intestinal juice, it is not logical to expect any of these types of antibodies to be effective in treating gastrointestinal infections. In The main immunoglobulin in cow's milk is IgG and not IgA (Sul- 'livan et al, 1969). Therefore, cow's milk is not a close source of antibodies for the treatment of bacterial infections in the human intestinal tract due to its high concentrations of IgG and low concentrations of IgA. "The immunization method is another important parameter when considering the different classes of immunoglobulin. It is well known to those skilled in the art that different immunization methods result in preferred production of different types of antibodies. For example, a by exposing secretory tissue to antigens achieved local immunization a preferred production and secretion of IgA-type immunoglobulins The technique of intramammoperfusion as described in U.S. Pat. No. 3,376,198 is an example of local immunization. This method stimulates the production and secretion of IgA. The invention also relates to a method for producing antibodies, which method is further stated in claim 3.

According to the invention, intramuscular injection is used to produce a product useful in the invention, since IgG is the major immunoglobulin in cow's milk and not IgA and since in cow system immunization the preferred method is to produce IgG-type antibodies in milk. This difference between IgG-type and IgA-type immunoglobulin is important, since it can be learned from this that system immunization and non-local immunization are the preferred method for obtaining high titer milk antibodies. From this distinction one can also learn that the immune products generated by mammary infusion of a vaccine are clearly different from the immune product formed by intramuscular injection of the same vaccine.

The product prepared according to the invention (IgG antibodies) - thus clearly differs from the product prepared according to U.S. Pat. No. 3,376,198. The immune product prepared according to the present invention is an improvement over the product prepared according to U.S. Pat. No. 3,376,198 in that the concentration of IgG-type antibodies is considerably higher than the concentration of IgA-type antibodies; There is no support in the literature for the notion that IgG antibodies can be formed by intramammary administration of antigens. Furthermore, since the amount of IgG immunoglobulins in cow's milk is either zero or extremely low, said patent has nothing to do with the present invention. On the contrary, the contents of said patent lead away from the present invention, since the patent claims that IgA is a biologically active factor in cow's milk, which has potential therapeutic application.

According to the present invention, a unique combination of bacteria is prepared into a vaccine which is administered to healthy dairy cows. The IgG antibodies obtained from the milk of immunized cows constitute the product produced according to the invention. The method of the invention involves a passive immunization of the patents by oral injection of IgG immunoglobulin, which passively immunizes against a mixed spectrum of infectious bacteria occurring in the gastrointestinal tract. This treatment eliminates conditions in the gastrointestinal tract. who are responsible for rheumatoid arthritis.

Fig. 1 shows an example of a questionnaire, to which reference is made in the following description. Fig. 1a is a continuation of the questionnaire in Fig. 1. Fig. 2 graphically shows the results of experiments performed during a 12-month period of 6 months on immune milk and 6 months on a control sample, the RF titer being listed as a function of the time specified in months.

The product prepared according to the invention is a low fat dry milk containing a population of natural IgG-type antibodies which react with the bacteria listed in Table 1 below.

TabeIl ~ 1 §ateriel1a.antigener Qäeafriåfß 1 3 * è fl lëgg fl r- i Staphylococcus aureus' 3 7 I 7 11631 Staphylococcus epidermidis _ 7 155 Streptococcus pyogenes, A, typ 1 I 8671 1 "1" 3 _3 10339 "23 _ _ _. 2 2 12347 '* V1 2 "s 12349" 2 1 "12 1 11434 P'" 14 12972 'f - 2 1 _ *' 18 1 ._ 12357 F '1 3 1 "22 of 10403 Aerobacter aerogenes -' 884 Escherichia coli 3 "3 26 Salmonella enteritidis *, * 'p 7 7 3 13076 Pseudomonas aeruginosa 7 7 7700 Klebsiella pneumoniae 2 3 d 9590 Salmonella typhimurium' 1 3 13311 Haemophilus influenzae 3 _f 9333 Streptococcus viridansp I de 1 6249 3 Proteus vulte dysenteriae * W 3 11635 Streptococcus, _group B_ 1 448 544 * Table 1, cont.

Diplococcus pneumoniae D Streptococcus mutans eCorvnebacterium, Acne) type 1 & 2 * American Type Cultnre Collection 12301 Parklawn Dr .; R0ckVille, 'Md. 20852 The antibacterial milk contains all the substances that are normally found in low-fat dry milk. The main constituents found in antibacterial milk are listed in Table 2 below.

Table 2 Quantitative and qualitative analysis of antibacterial milk Proteinerr _ 'k k'. V '35.6% Fat ¿-D - 1, o% Carbohydrates DI _ 4 52% Minerals "7 J 7 Ü 7,8% - Moisture ~,' 'i - 3,54% Each in liquid form return liters of 85 -113 g fat-free dry milk contains about 1200 mg calcium '_ e * l 157% 935 mg phosphorus VI - 125% 0.3 mg thiamine 7 32% 1.78 mg riboflavin ~ .- _ 140% _1.04 mg niacin " _ 10% 324 calories Antibacterial milk and normal xem milk contain approximately the same percentage by weight of concentrations of ingredients. In addition, the concentration of IgG immunoglobulin in antibacterial milk and normal milk is the same. It is therefore only in the case of the specific composition of antibodies that the antibacterial milk differs from normal milk. By specific composition is meant here the spectrum of bacterial species with which the antibodies react, D d44sís44id Antibacterial milk does not contain additives of medicines or other components, which do not constitute natural products from the cow.

The immune milk prepared according to the invention can also be used to fight autoimmune diseases; for example lupus erythematosus and the like, which are caused by or aggravated by bacterial infestations in the gastrointestinal tract.

The polyvalent antigen used for the induction of antibacterial milk is prepared as follows.

Preparation of vaccine The bacterial strains listed in Table 1 above were obtained from the American Type Culture Collection, which guarantees authentic bacterial strains and the highest quality of purity. Upon receipt, each bacterial strain was allowed to grow on blood agar plates to determine the viability of the culture and to determine whether the growth pattern was ambiguous or not typical of the bacterium in question.¿ A single colony was taken for each of the tested cultures for histological examination to further state that the culture was authentic and pure. A single colony of each culture was used to inoculate 500 ml of a standardized culture medium. The substrates recommended by the American Type Culture Collection were used for culturing each of the specific bacteria listed in Table 1. All organisms were incubated as static cultures with the exception of 12, 13, 14 and 60, which were incubated in a shaker for agitation. The identification of the bacterial strains and the catalog numbers according to the American Type Culture Collection are given in Table 1.

Each culture was grown for 48 hours at 37 ° C. After incubation, the cultures were killed by heating at -60 ° C for two hours.

Samples of the killed bacteria were used to inoculate fresh culture medium, which was then incubated for 24 hours at 37 ° C to determine if the killing process was efficient and complete. Only cultures that were found to be sterile in this study were used in the further work. Sterile cultures were then washed five times in distilled water and the cells were recovered by centrifugation. The cells were frozen by immersion in liquid nitrogen and lyophilized by lyophilization. The lyophilized cells were stored in sterile vials until used for testing. The cells were injected with 5 ml of polyvalent antigen containing 20 x 10 4 444 544 11 11 "production of the polyvalent vaccine. The polyvalent vaccine is prepared by weighing 1 g of each of the bacterial strains, the dry cells were mixed together and this mixture was suspended in sterile physiological saline (20 g of bacteria per 500 ml of saline).

A sample of the concentrated solution was diluted in series with saline to determine the dilution which was a concentration of 4 x 108 cells per cm 3. The stored concentrated polyvalent vaccine was dispersed in multiple containers and stored frozen. Concentrated antigen was contained in each individual container in an amount sufficient to immunize 50 cows. The final dilution of the concentrate was made just before immunization. A preferred approach was to take out a sufficient number of bottles to immunize the number of cows to be treated. For example, the vials were withdrawn 24 hours before the scheduled immunization and a sample of the concentrate was then diluted in a sterile container to a final concentration of 4 x 10 8 cells per ml. The maximum response of cows was achieved by injecting 20 x 10 108 bacterial cells or 5 cm 3 of the sterile preparation, which held 4 x 10 8 cells per ml according to the injection method described below; Preferred Method for Immunizing Cows The final antibody product was prepared by immunizing cows with the polyvalent antigen prepared on the bacterial cells described above. The injection was made intramuscularly into the gluteal musculature of the hind leg. This procedure was repeated at one-week intervals for four consecutive weeks beginning 2-3 weeks before an expected calving. After the primary immunization, buffer injections were given every 14 days using the same concentration of antigen. This method of immunization gave the maximum antibody titer. Collection, handling and further processing of milk In milk, a modern milk chamber was collected from immunizing cows.

A fully automated milking system collected and stored the milk under completely sanitary conditions. The milking system consisted of automatic machines directly connected to storage containers placed during cooling through a closed pipe system. The complete system was cleaned and sterilized after each milking to ensure maximum sanitary conditions. It is important to take precautionary measures to prevent the growth of bacteria in immune milk during processing, as such bacteria may lower the titer of antibodies in the milk.

The milk was transported every day from the refrigerated storage containers to a milk handling plant with the help of special 'milk transport vehicles'. In the milk handling plant, the antibacterial milk was subjected to short-term high-temperature treatment for * pasteurization. Using special dairy machines, heating was carried out during continuous flow of milk to 68 ° C for a maximum period of 15 seconds. The temperature and time were critical, as antibodies are broken down in heat. The antibodies in the milk are destroyed at temperatures above 73 ° C, if they are kept at this temperature for a longer period than 1 minute. After pasteurization, all milk was cooled immediately and the fat was removed by centrifugation, and the antibacterial skim milk thus obtained was transferred in powder form by spray drying. The spray drying took place in a large drying chamber, in which hot air (177 ° C) was blown in at high speed. The skimmed milk was atomized in the chamber, where the comminuted milk particles as they fell to the bottom of the container were dried. The dry milk was extracted automatically by means of mechanical devices and the milk powder was packaged under sanitary conditions lxw Before atomization, the skimmed milk was condensed by boiling in a chamber under vacuum (38-43oC). In each step, it was important to pour bacteria away from the milk, as these could reduce the antibody titer. In Test Methods, the immune milk was prepared using cows of the Holstein breed.

The cows were immunized by intramuscular injection of a mixture of the bacterial antigens listed in Table 1 above. The vaccine was prepared as described above. The immunological response of the cows was enhanced by injections twice a week of the vaccine. The milk from these cows was collected, the fat was removed and the fat-free milk was pasteurized at 71 ° C for 16 seconds, after which spray drying was carried out, during which the temperature of the milk did not exceed 2900. The milk was then packaged for about 1 hour. 448 344 1 ters packages of polyethylene. Control milk samples were performed on 'fat-free dry milk purchased from a local producer.

The rate of erythrocyte sedimentation rate was determined on fresh blood samples by Westergren's method and corrected for hematocrit according to the method described by Wintrode & Langsberg (1935). Rheumatoid factor titers were determined according to Singer-Flotz (1966) macroscopic method with test tubes.

Patients were admitted for studies based on an elevated erythrocyte sedimentation rate and a positive rheumatoid factor titer. Nine patients were studied for 12 months and 11 patients were studied for 18 months. The patient group consisted of 13 Caucasian women_year aged between 32 and 69 years with a mean age of 50.4 years and 7 Caucasian men aged between 43 and 70 years with a mean * age of 58.1 years. The mean time that patients suffered from arthritis was 10.8 years for women and 11.0 years for men. Patients were arbitrarily placed in groups for treatment with immune milk or non-immune milk (a commercial product purchased in the Dayton district and acting as a control fluid). Both dairy products were packaged in the same packaging and were identified as immune milk or control samples with a blue or red, pressure-sensitive label; which is affixed to each package at the time of refilling. ”The labels were removed just before patients were allowed to drink the milk. Thus, at no time could the patients know whether they were drinking immune milk or control milk. Patients were arbitrarily selected (by tossing coins) to drink either immune milk or control milk during the first six-month period. After one week, those who had previously received immune milk received control milk and. the; who had previously received control milk, now received immune milk during the second six-month period. 7At the end of the second six-month period, 11 patients voluntarily remained for study for another six-month period.

The type of milk (immune: or control milk) was changed again at this time and the observations continued. The study thus included three six-month periods, with 11 of the patients participating for three periods and nine participating for two periods. In Patients were allowed to come once a month, leaving powdered milk as. values in relation to "Aspirin 'include the said" Aspirin 448 544 14 would suffice one month aaiaaes out and a questionnaire was filled out 1 and blood samples were collected for determination of rheumatoid factor titer, erythema sedimentation rate and hematocrit.

Patients were instructed to consume a quantity of dry milk equivalent to T, 1 l of whole milk twice daily. The dry milk was dissolved in a fresh state in 0.55 l of cold tap water immediately before ingestion and shortly after waking up in the morning and just before bedtime in the evening; They were asked to visit their doctor as usual and to follow the treatment instructions proposed by him. The medication should be taken ad libitum or as prescribed by their regular doctor. The only requirement was that they de-report the amount of medication they were taking.

A questionnaire was completed by each patient each month. It was divided into six sections and concerned the following issues: 1) Duration of morning stiffness, l_ 2) assessment of pain observed in each of eight joints, 3) type and quantity of medicine with short-term effect taken, 4) type and quantity of medicine with long-term effect taken,) ability of the patient to perform their normal activities and 6) assessment of the severity of the disease symptoms.

The numerical values given in connection with the questions indicate the points that have been assigned to each answer for evaluating the questionnaire. In assessing the questions regarding medication, an attempt was made to reflect the relative anti-inflammatory and analgesic effect of various medications used. A 324 mg "Aspirin-E) tablet" was loaded with hydrogen 1. All other medications (with the exception of gold, "Plaguenil" and cortisone syringes, which will be assessed separately) were added. All other salicylate preparations, such as " Tylenol "," Darvon "and" Mortrin "were assessed as the equivalent" tablet "and were also assigned a value of 1.

The number of grams of cortisone was multiplied by four and the number of indocin-å (vikranhat = 0, o64s "tab capsules multiplied by 2.5. The number of grain g) codeine was multiplied by 2 and the number of" Butazoladin letter was multiplied by 7 .: I The average score for each category was calculated for each six-month period. The differences for mean values were then calculated by _ å! 448 344 subtract the mean values during administration of immune milk from the mean values during administration of the control milk. When the results were calculated in this way, an improvement in the patient's condition during the period in which he received immune milk was indicated with negative values for samples 1 and 6 and with positive values for all other questions. Mean-corrected values for erythrocyte deposition rate (ESR) and rheumatoid factor titer (RF) were displayed in the same manner. These were calculated in such a way that positive values reflect a lower erythrocyte sedimentation rate or rheumatoid factor titers during administration of immune milk. the values were obtained statistically using the statistical analysis system according to Goodnight et al. (SAS Institute, Raleigh, N.C.).

The calculations were performed using a computer IBM model 370/155.

Results '7 Immune milk was well tolerated by all patients except one who suffered from pericardial anemia. This patient complained of diarrhea and the study of this patient was discontinued. Some patients reported weight gain during treatment. This may have been due to the increased caloric intake from the milk or may reflect a general improvement in their physical well-being. 16 448 344: mw.w. # Q® fi0 HHMmoMm fl owvmwHm> # ¶ Høxmw flflfi u fl mnøm W H fi mndë mm @@, o ¶fl @ m «1o | _ß. «: m.m« w @ w, w íám ßm mm .w. @ ßßß.o «ßm.o +. ~ .wm NN @ .mm_ ß.mm mm ~ .wm mm mw mmm .ß mmvoo.o mmv.ø | ¶ mm «ßvv.N m.ß« «mm.v Å vmí ßw @« uHHø> m .U @ ~ oo.o ww ~, o1:. ~ «åmN. ~ w.wv mw @.«: N ßm »mnH @ pm..p ¶m # oo» o¶ :: mo «f. #«. ß: «. ~ w.« N mo @. «àw mm Muw> .w ¶ ¶ ¶ main ¶ ¶ ¶, ¶ nw fi wnww w flfl pmnma .w mwøo.o omm, o + vfam« ßæ. «v. @ ß ¶: Nm .N .àw ßw ¶ ¶ ¶ nnw .m wßmo.ø _ «wo.o + w.mßv :: mo ¶ß, o:« mNm.o: N ßm ¶ qø fi wøx flfl wa qm fl n <.w mo # oo m # «. : + m. «o« m «mm« «.: o« mwwfom àm um nw flfifi m 1m¶ ovoo.o ¶mfm.o + m.om mNß.ø @ ¶ß.m@¶ ¶ w: @, o ímm mm nwppmw .n ß ~ «o, o« w @ «. o + ß.m @ @ m @ .o mfww. v «wß.o, Nmí ww Hw fi mqm .w mvoø, o« - »o + o.mm mw @ .o«.: ß # om.o ám ßm qmmx, w møoo.ø woß.o + o.mmv ßwm.ø mo @ mmm, o íí àw mm ¶Hw »wwn .mv« Qo.o ¶, ¶. m # m ~ o + ¶ ¶ m.wm w ~ w.o ß. @ m .mßolv dw mm Hwøqwn .w _o «oo.ø¶¶ ¶mwN.o + ¶ ¶. w.:ß mmm.ø¶ w.mw: mw.o. : m ¶ ßw_¶ Hmwm fi wqmn. °. ¶ v «mo.o_ mm« .o + ¶ m, m @ ¶ mf @ .o_ ß.mw ¶mmw «o: N ¶ ßm fl m fl mwpanm .Q o ~ # o, o wmm.o + ¶ m, o @ @« ß, o. «, ß @ ¶: m @ .o ¶¶« m_ ßw pm fi wm. ~ _ ¶. ¶ ¶. ¶ Mnw> @wm .N voøo, o ß: m.o | @ .mß @ ßw1o¶ ß, m @ ¶H¶Nßm.o: N »N pum fl mpmnownos.« m mnønwww flfl *> o _ | Mwmmm ¶ #> o n fl wwmm _ asaw ¶ H fi onpnom @ øHw> H @@ wz¶ m aw ø fi nwøo fl nmm nm fl o fl pd> nwmßo ~ ~ ......_-............._, ......._......;, ..... ._...... 448 544 _17 As shown in Table 3, patients were observed for a total of, 27 control periods (six-month periods during which they received control milk) and 24 test periods (six-month periods during which they received immune milk). A patient had been subjected to a physical injury in one of the ankles and one foot. The pain in these joints was not evaluated, which is the reason for the lower number of periods in terms of evaluation of these joints. The erythrocyte sedimentation rate for one patient was so extremely abnormal (more than two standard deviations from the mean values for other patients) that they were not recorded. This is the reason for the lower number of observations regarding this variable.

The means and coefficients of variation (C.V;) are given in the table for each variable. The differences between the mean values were calculated by subtracting the mean value obtained during the periods when the patients received immune milk from the mean values obtained during periods when they received control milk. A favorable reaction to the immune milk is indicated with negative values for morning stiffness (question 1) and monthly change (questions 6a, b and c) and with positive values for other variables. An effective response to immune milk was obtained for all data from the questionnaires. The probability (P) indicates a high degree of statistical significance in each experiment. The small difference values obtained for the erythrocyte sedimentation rate and the rheumatoid factor titer were not significant.

However, when the rate of erythrocyte sedimentation rate was assessed on an individual basis, four of the twenty patients showed statistically significant reductions in treatment with immune milk. Although immune milk did not have a significant effect on the mean values for the rheumatoid factor titer, some interesting responses were obtained when examining individual patients. Seven of the twenty patients studied had negative rheumatoid factor titers on at least one occasion during the period they received immune milk. Four of them became negative during the period they received immune milk and their titer did not become positive during the subsequent six-month period when ao received control milk, as shown in Fig. 2). A further study after this reporting period showed that thirteen of twenty-five patients received lost the roumatoid factor 1 its blood. 448544 Z i 1sf Discussion d j _ j.

The scientist responsible for these studies personally interviewed each patient at monthly intervals and recorded their answers to the questions. Every effort was made not to affect the patient's response. The patients were informed from the beginning and were reminded from time to time that during certain periods of the treatment they would receive a control sample. It was assumed that the patient with this knowledge would answer the questions more objectively and without preconceived notions. At no time were the patients informed that they had received immune milk or control milk. I The question regarding medication "yesterday" (question 3) and the question regarding long-term mediation (question 4) are objective and are of primary importance when considering the answers given to other questions. These 7 questions are important for two reasons: If the immune milk is effective in relieving symptoms of the disease, the patient would be expected to take less medication than was allowed ad-libitum; Patients reported taking an average of four tablets less äspirine or equivalent tablets per day during the periods in which they received immune milk. They also reported receiving less long-acting medication during these periods and 2 ) if patients took smaller amounts of analgesics and other drugs useful for the treatment of rheumatoid arthritis, one would expect them to report a deterioration in well-being regardless of the immune milk affecting the disease favorably. "As shown in Fig. 2, significant smaller number of joint problems during periods when patients received immune milk even if they took a smaller amount of medication for their arthritis .: The study of patients started at monthly intervals over a one-year period and the allocation of type of milk product (immune milk or control milk) was random. The observation that positive answers or improvements were achieved for all parameters in the questionnaire and that these mean rden was statistically significant is a strong indication that the immune milk had a beneficial effect on the patients. This conclusion is reinforced by the observation that 20% of the patients experienced a statistically significant (p <0.05) decrease; is .h 448 544 19 'increase in the erythrocyte sedimentation rate during treatment with immune milk. f_The results of the rheumatoid factor titer values are difficult to evaluate. This is due at least in part to the fact that the origin and role of the rheumatoid factor in the etiology and prognosis of ~. rheumatoid arthritis is not known. Rose et al (1948) have shown that red blood cells' nos sheep, sam senabilniaerat maa antibodies from aakinin, underwent agglutination in the presence of blood serum from patients with rheumatoid arthritis. The result depends on the specific reaction between normal immunoglobulin (IgG from either rabbit or human) with rheumatoid factors. The behavior of the rheumatoid factor is well in line with what can be expected in antibodies to IgG (Epstein et al, 1956). The presence of rheumatoid factors has been linked to the severity of the disease, in the case of rheumatoid. arthritis and can be identified by observing precipitated proteins in tissues in patients with rheumatoid arthritis. Although a small percentage of patients with rheumatoid arthritis do not have positive rheumatoid factor titers, most rheumatologists believe that positive agglutination reactions do not turn into negative ones, even when the disease is recurring. However, De Forest et al (1958) have described a small number of patients who had positive rheumatoid factor titers and who turned negative after the recurrence of the disease. When the disease broke out again, the test became positive again. However, Aho et al (1959) have noted that most patients whose diseases became inactive remained serologically positive. The fact that negative titer values were observed in 60% of the patients examined in the present study and that in half of them titer remained negative for six months, showing that immune milk affects a primary etiological factor responsible for rheumatoid arthritis.

The effect of immune milk in eliminating the symptoms of rheumatoid arthritis is particularly relevant in view of the recently described relationship between histocompatibility antigens (HL-A) and susceptibility to rheumatic disease (Brewerton, 1976). Histo-compatibility antigens are genetically determined antigens that are found in all human cells. The genes that determine these antigens are called histocompatibility genes. Over forty of these genetically determined antigens are currently known. They are responsible for the rejection of transferred tissue between individuals other than identical twins. In general, histocompatibility antigens resemble ABO blood groups in that they are inherited for life. Their functions are not yet known, except in the very artificial situation of transplantation. However, it is known that the histocompatibility genes are closely related to the immune genes on the sixth chromosome; In this context, they can determine the immune response of an individual to a foreign invader, such as the bacterium.

People with HLA-B27 seem to be particularly susceptible to a line! rheumatic diseases. It has been established that this histocomal -pability antigen dictates a type-of immune response, which in the presence of other predisposing factors leads to renmatoid arthritis. Following an intestinal infection with Yersinia enterooolitica¿, some patients develop acute peripheral arthritis (Ahovenen et al, 1969). Similarly, after salmonella infection, about 2% of patients develop acute peripheral arthritis (Warren, 1970). HLA-B27 was found in forty-three of forty-nine patients with yersinia arthritis and in fifteen of six- * ton patients with salmonella arthritis (Aho, 1974). It is an interesting finding that infectious agents can thrive in the intestinal tract without giving rise to local symptoms. In patients with HLA-B27, a reaction is established that results in arthritis. Thus, it is not necessary for the infectious agent to gain access to the joints.

Immune milk is beneficial for patients with rheumatoid arthritis, as it contains antibodies that effectively inactivate or neutralize harmful bacteria and / or metabolic products thereof. '7 I *

Claims (4)

z 11fL 'E! Zl 448 344 Patentkrav Antibody for the treatment of rheumatoid arthritis caused by a. mixed spectrum of infectious bacteria present in the gastrointestinal tract, characterized by being obtained by first preparing a vaccine of killed bacteria from bacterial antigens from the American Type Culture Collection including: Staphylococcus Aureus 2 11531 Staphylococcus epidermidis 155 Streptococcus pyogenes, A, type 1 'e 3671 2. "" "3 1 10389" 7 "c _ 2“ 5 '12341 J “." "S 12349, f" -_ "" 12 11434 * "3“ Å - - “14 12972" - 2 "“ 18 12357 "7" “22 10403 _dAerobacter aerogehesl 1 - 1 534 '” Escherichia coli - _ 26 fsalmonella enteritidis 3 13076 Pseudomonas aeruginosa 7700 dxiebsieila pneumoniae 3 9590 _Salmonel1a typhimurium 13311 Haemophilus 924 I3 Shigella dysenteriae I 11835 Streptococcus, group B Diplococcus pneumoniae Streptococcus mutans Corynebacterium, Acne, Type 1 & 2 Inject this vaccine intramuscularly on healthy cows once a week for four consecutive weeks and twice every month thereafter, each injection comprising 20 x 108 bacterial cells; collect the milk containing the antibody from the immunized cows beginning in the fourth week and test for titer to ensure that the minimum titer against each of the bacteria is 1-500, determined by macroscopic daily glutination test tubes to control the antibody filter. The product according to claim 1, characterized in that the antibody product type IgG is pure immunoglobulin obtained from milk. A method of producing an antibody according to claim 1, k e n n e ~. t e c k n aj of preparing a vaccine of killed bacteria from bacterial antigens from the American I lype Culture Collection including: 7 _ Staphylococcus aureus. f_f a11631 Staphylococcus epidermidis 11 '155 ištreptococcus pyogenes, A, typ 1 8671 "" iaa "3 - 10389 - i"' vs 212347 -a "l - f" saay 12349 "a" 12 'a 11434 l: a "2" 14- ia 12972 - 'P18' Å 12357 I "a 'fi * - z2 a 1o4o3 Aerobactër-aerogenesl 3 d _ ÉB4 Escherichia coli _ _ a I 2 2 26" salmonella enterltldls ai 2 "13016 Pseudomonas aeruginosa _ 7700 Klebsiella pneumoniae 2 _ 'a 1 1 9590 a Salmonella typhimuríum 13311 Haemophilus influenzae. _. and 9333 Streptococcus viridans - 6249 Proteus vulgaris _ a "Z, 13315 Shigella dysenteriaeí" 11835 Streptococcus, group B Diplococous pneumoniaea Streptococcus mutans 3 Corynebacterium; Acne, type 1 & 2 _ Inject this vaccine intramuscularly on healthy cows and every I week for four consecutive weeks and twice every month thereafter; each injection comprising 20 x 108 bacterial cells, collecting the milk containing the antibody from the immunized cows starting the fourth week and testing the titer to ensure that the minimum titer of the antibody to each of the bacteria is 1 ~ 500, determined by macroscopic agglutination in test tubes for examination of antie (fr) k! 448 344 234 krøppstiter¿-- 4. A method according to crane 3 for the preparation of a vaccine for injection into cows for production. The milk system of an antibody, known as the preparation of cultures of the bacterial strains in suitable buffers, kills the bacteria "by The heater collects the killed bacteria by centrifugation, washes the bacterial strains, subjects the washed bacterial strains to lyophilization, mixes the individual bacterial types on an equal weight basis and suspends the mixed bacterial strains in a suitable carrier for injection.