U.S. patent number RE33,565 [Application Number 07/231,219] was granted by the patent office on 1991-04-02 for prevention and treatment of rheumatioid arthritis.
This patent grant is currently assigned to Stolle Research and Development Corporation. Invention is credited to Lee R. Beck, Ralph J. Stolle.
United States Patent |
RE33,565 |
Stolle , et al. |
April 2, 1991 |
Prevention and treatment of rheumatioid arthritis
Abstract
There is disclosed a novel method and product for the treatment
and prevention of rheumatoid arthritis. The method involves passive
immunization against a mixed spectrum of infectious bacteria which
reside in the human gastrointestinal tract. The passive
immunization is accomplished by oral.[.injestion.]. .Iadd.ingestion
.Iaddend.of IgG immunoglobulin obtained from the milk of cows that
have been immunized against a specific spectrum of bacterial types.
A unique combination of bacterial species is formulated into a
vaccine which is used to immunize dairy cattle. The IgG antibody
obtained from the milk of the immunized cows constitutes the
product of the invention.
Inventors: |
Stolle; Ralph J. (Lebanon,
OH), Beck; Lee R. (Birmingham, AL) |
Assignee: |
Stolle Research and Development
Corporation (Cincinnati, OH)
|
Family
ID: |
26924917 |
Appl.
No.: |
07/231,219 |
Filed: |
August 11, 1988 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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875140 |
Feb 6, 1978 |
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Reissue of: |
558909 |
Dec 9, 1983 |
04732757 |
Mar 22, 1988 |
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Current U.S.
Class: |
424/157.1;
424/163.1; 424/203.1; 424/810 |
Current CPC
Class: |
A61K
39/39508 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); A61K 38/00 (20060101); A61K
039/116 (); A61K 039/085 (); A61K 039/112 (); A61K
037/04 () |
Field of
Search: |
;424/85.1,86,87,88,89,90,91,92 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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214582 |
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Sep 1956 |
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AU |
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587849 |
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Dec 1959 |
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CA |
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0064103 |
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Oct 1982 |
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EP |
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307975 |
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Apr 1914 |
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DE2 |
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516403 |
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Dec 1930 |
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DE2 |
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1161659 |
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Jan 1964 |
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DE |
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32472 |
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Oct 1962 |
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FI |
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608596 |
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Sep 1948 |
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GB |
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837695 |
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Apr 1955 |
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GB |
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988135 |
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Apr 1965 |
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GB |
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1211876 |
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Nov 1970 |
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GB |
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1505523 |
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Mar 1978 |
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GB |
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2013691A |
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Aug 1979 |
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GB |
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Other References
McGhee et al., J. Dent. Res., Special Issue C, vol. 55 (1976),
206-214, Effective Immunity to Dental Caries: Studies of Active and
Passive Immunity to Streptococus Mutans in Malnourished Rats. .
Lascelles Dairy Science Abstracts, vol. 25, No. 9 (1963), A Review
of the Literature on Some Aspects of Immune Milk, pp. 359-364.
.
Holm, U.S. patent application Ser. No. 628,987, of Nov. 15, 1945,
Abstract Published O.G., Dec. 5, 1950, 1 page, Spec. pp. 1, 3, 4, 5
and 7. .
Karel, A Dictionary of Antibiosis, Columbia U. Press, N.Y. (1951),
pp. 203-208. .
Vaughan (Hollander, Ed.), Arthritis and Allied Conditions, Lea and
Febiger, Pa., 7th Ed. (1966), pp. 574-586. .
Bauer, W., Clark, W. S. and Dienes, L. (1961), The Practitioner
166:5. .
Lewis-Fanning, E. (1950), Ann. Rheum. Dis., Suppl. 9, Sabin, A. B.
(1939), Science 89:228. .
Sikes, D., Neher, G. M. and Doyle L. P. (1955), Am. J. Vet. Res.
16..
|
Primary Examiner: Kight, III; John
Assistant Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Frost & Jacobs
Parent Case Text
This application is a continuation of application Ser. No. 875,140,
filed Feb. 6, 1978, now abandoned.
Claims
We claim:
1. A method for the treatment of rheumatoid arthritis caused by a
mixed spectrum of infectious bacteria which reside in the
gastrointestinal tract, including:
which comprises the oral administration of a quantity of non-fat
milk solids equivalent to one quart of immunized cow's milk I.sub.g
G, .[.post-prandially.]. two times daily, wherein the cow's milk
.[.IgG.]. includes antibodies produced by the following
microorganisms from American Type Culture Collection bacterial
antigens:
2. An antibody for the treatment of rheumatoid arthritis caused by
a mixed spectrum of infectious bacteria which reside in the
gastrointestinal tract, including:
produced by first preparing a vaccine from killed bacteria from the
American Type Culture Collection bacterial antigens, including:
injecting said vaccine intramuscularly in healthy cows once weekly
for four consecutive weeks, and twice monthly thereafter, each
injection involving 20.times.10.sup.8 bacterial cells; collecting
the milk from the immunized cows beginning the fourth week; and
testing .[.fro.]. .Iadd.for .Iaddend.titer to insure that the
minimum titer against each of the bacteria is 1-500, as determined
by the tube agglutination method for testing antibody titer.
3. The preparation of claim 2, wherein the antibody product type
IgG is pure immunoglobulin obtained from milk.
4. The method of preparing the antibody of claim 2 which includes
preparing a vaccine from killed bacteria from the American Type
Culture Collection bacterial antigens including:
injecting said vaccine intramuscularly in healthy cows .[.and.].
weekly for four consecutive weeks, and twice monthly thereafter,
each .[.injections.]. .Iadd.injection .Iaddend.involving
20.times.10.sup.8 bacterial cells.[.,.]. .Iadd.;
.Iaddend.collecting the milk from the immunized cows beginning the
fourth week; and testing for titer to insure that the minimum titer
of antibody against each of the bacteria is 1-500, as determined by
the tube agglutination method for testing antibody titer.
Description
REFERENCE TO RELATED APPLICATIONS
This application is related to an application in the name of Lee
Randolph Beck, Ser. No. 776,249 filed Mar. 10, 1977.
BACKGROUND OF THE INVENTION
A number of years ago it was commonly believed that rheumatoid
arthritis had an infectious etiology. This view is not popular
today, although the inflammatory features and constitutional
manifestations of rheumatoid arthritis-the synovitis and
granulomatous lesions, the fever, tachycardia, leukocytosis,
lymphadenopathy and ocassional spelnomegaly, the accelerated
erythrocyte sedimentation rate and other changes in "acute phases
reactants" are all compatible with an infectious process. Competent
and repeated bacteriologic studies have failed to recover
consistently a single infectious agent from the blood, synovial
fluid, synovial tissues or subcutaneous nodules. Attempts to
transmit the disease by injecting joint fluid from patients with
rheumatoid arthritis into the joints of other human subjects have
been unsuccessful. Subcutaneous nodules have failed to survive
following homologous transplantation (Bauer, et al, 1951) the
Practitioner 166:5.
An infectious process may appear to precipitate the onset of
rheumatoid arthritis in a significant number of patients, and may
exert a deleterious influence on the course of the disease when it
has already been established. There is statistical evidence to
support this clinical impression (Lewis-Faning, 1950) Ann Rheum.
Dis., Suppl. 9.
Many attempts have been madeto produce a disease in animals similar
to rheumatoid arthritis. While a variety of bacteria can produce
arthritis in animals, they fail to reproduce the clinical and
pathologic features of rheumatoid arthritis, particularly the
self-perpetuating characters of the proliferative arthritis.
Arthritis bearing some semblance to the human disease has been
produced in mice by pleuro-pneumonia-like organisms (Sabin, 1939)
Science 89:228, and in swine by Erysipelothrix rhusiopathiae
(Sikes, et al, 1955). The concept that these organisms may initiate
a hypersensitivity mechanism has been postulated (Sikes, et al,
1955).
Students of rheumatoid arthritis nevertheless continue to be
intrigued by certain recurring themes that suggest relationships
between infections and joint disease. Gonorrhea, for instance, is
capable not only of producing typical gonorrheal arthritis but also
of occasionally introducing chronic arthritis which .[.evolved.].
.Iadd.evolves .Iaddend.into typical rheumatoid arthritis. No
statistics are available on the incidence with which this occurs,
so one cannot know how much to stress the relationship. Tonsillitis
or pharyngitis may also be followed by a polyarthritis, which at
first appears to be rheumatic fever but which evolves into
rheumatoid arthritis. Acute viral infections, especially rubella in
young women, may be followed by persistent polyarthritides
involving small joints as well as large; these arhritides generally
run a several-month course of persisting joint disease resembling
rheumatoid arthritis before gradually subsiding.
While chronic infection by an unknown agent remains a popular
assumption for the etiology of rheumatoid arthritis, no published
data exist to support the presumption. Some students of the disease
suspect that if infection is a factor it may not be infection by
any specific type of microorganism, .Iadd.but rather infections by
a wide variety of banal microorganisms .Iaddend.with an altered
host response generated by the infections .Iadd.may be
.Iaddend.responsible for the disease. The present invention is
based on this theory for the origin of rheumatoid arthritis.
Although an infectious etiology has never been established, several
recent developments appear relevant in support of this theory.
Some of these are as follows:
1. Patients with rheumatoid arthritis have lower than normal levels
of IgA, the class of immunoglobulin found in secretions of the
gastrointestinal tract.
2. Immunoglobulin A produced in response to immunization via the
salivary glands is found in serum colostrum and milk as well as in
the saliva. It is suggested that the IgA is transported to these
various fluids via the gastrointestinal tract and the lymphatic
system (Michelok, et al, 1975) Proc. Soc. Exptl. Biol. Med.
148:1114.
3. Following an intestinal bypass operation for morbid obesity,
certain patients develop symptoms that are virtually identical with
rheumatoid arthritis. The onset of arthritis is accompanied by the
appearance in blood serum of circulating cryoproteins composed of
IgG, IgM, IgA, complement components C.sub.3, C.sub.4, C.sub.5, and
IgG antibody against E. coli and B. fragilis. Removal of the
intestinal bypass results in complete remission of the symptoms
(Woods, et al, 1976) New Engl. J. Med. 294:121.
4. A type of Diplostreptococcus agalactiae belonging to the
streptococci group B has been implicated as an etiologic agent in
rheumatoid arthritis (Svartz, 1972) Acta. Med. Scand. 192:231. This
streptococcus is present in most commercially available pasteurized
milk but not in immune milk.
5. A predisposition to rheumatic disease appears to be inherited
via the histocompatibility antigens (HL-A). These antigens probably
determine host response to infective agents.
On the basis of this evidence, it is concluded that rheumatoid
arthritis has an infectious origin; the site of infection occurs in
the gut; a number of different bacterial strains are involved in
the infection; the infection probably results because of a failure
in the host's immune defense mechanism; and the most effective way
to treat the disease is to re-establish the immune protection
against the infectious agent in the gut.
Treatment of Infection
There are basically two methods which may be used for the treatment
of infection: the immune approach which involves either active or
passive immunization against the infectious pathogens, and the use
of antibiotics such as penicillin, tetracycline, ampicillin and the
like. Antibiotics are not specific in their activity, and they kill
a wide spectrum of beneficial as well as harmful bacteria. On the
other hand, the immune approach is highly specific. Bactericidal
antibodies produced against a specific strain of bacteria react
only with that strain and have no harmful effects on other types of
bacteria. Moreover, antibodies, unlike antibiotics, are natural
body products and have no known side effects. Since the objective
of the invention is to control infection by a specific group of
bacteria, without harming beneficial bacteria in the gut, the
immune approach is the method of choice.
Active and Passive Immunization
There are two different methods to achieve immune protection.
Active immunization is a process whereby the host is actively
immunized with a vaccine which stimulates the immune system of the
host to produce protective antibodies against factors contained in
the vaccine. Active immunization occurs under natural conditions
when the host is exposed to infectious pathogens. Passive
immunization is a process whereby antibodies obtained from one
individual who has been actively immunized are given to a second
individual. By this process, the protective antibodies are
transferred from the immune host to the recipient. Passive immune
protection is temporary and lasts only as long as the passively
acquired antibodies persist in the system of the recipient. For
example, antibodies collected from horses immunized against tetanus
toxin can be given to humans infected with tetanus in order to
obtain temporary immune protection against the toxin produced by
tetanus bacteria. In a previous patent (U.S. Pat. No. 3,626,057)
there is described a process for producing tetanus antitoxin in
milk. This patent teaches that the cow can be actively immunized
against tetanus toxin; that antibodies produced by the cow against
the toxin can be obtained from the cow's milk; and that these
antibodies can be used to treat animals infected with the tetanus
bacteria in such a manner that the antibodies neutralize the toxin.
The patent teaches that the passively administered antibodies
neutralize the life-threatening toxin produced by the bacteria,
thereby, providing temporary immunity against the toxin.
Passive immunization differs from active immunization in that the
immune protection is temporary and lasts only as long as the
protective antibodies are present. Active immunization is more
permanent because the immune system of the host continues to
produce protective antibodies in the presence of the stimulating
antigen.
Recent studies in the field of gut immunology have shown the
existence of a local immune mechanism in the gut. This immune
system of the gut produces a special type of antibody which
functions to control bacterial infestations in the lumen of the
gut. The antibody called secretory immunoglobulin or IgA is
produced in response to the local active immunization of the gut
mucosa by the antigen. The secretory immune system of the gut
functions to prevent the colonization and proliferation of harmful
bacterial species in this environment. It is believed failure of
the local immune system of the gut allows unknown harmful bacteria
to become established and that .[.this.]. .Iadd.these
.Iaddend.bacteria .[.causes.]. .Iadd.cause .Iaddend.rheumatoid
arthritis. According to this theory, rheumatoid arthritis results
from a failure of the local immune system of the gut to produce and
secrete protective antibodies against harmful bacteria. Thus, the
inability of the host to respond to active immunization precludes
this method as an approach to the treatment of rheumatoid
arthritis.
The present invention describes a method for controlling the growth
and proliferation of harmful bacterial pathogens-specifically, in
the environment of the gastrointestinal tract of man; the method
being that of passive immunization by oral ingestion of protective
antibodies produced in the cow. The method provides temporary
immune protection which is highly specific for those species of
bacteria used to produce the antibodies and does no harm to the
normal beneficial bacteria that reside in the gut. The antibodies
used in the method of this invention constitute the unique and
useful product of this invention.
Cow's milk provides 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) that react with a known
spectrum of bacteria and this reaction results in the beneficial
effect, which is treatment and prevention of rheumatoid
arthritis.
The type of immunoglobulin is an important consideration with
regard to patentability of this invention because there are five
known classes .[.if.]. .Iadd.of .Iaddend.immunoglobulin which are
designated IgG, IgM, IgA, IgD, and IgE (Nisonoff, et al, 1971)
Molecules of Immunity In Immunobiology. Eds., Good, R. A. and
Fisher, W. Sinauer Ass., Stanford, Conn. (1971). Each type of
immunoglobulin differs structurally (Waldman, et al, (1970) Plasma
Protein Metabolism, Academic Press, p. 269 (1970), and has a
different biological function within the body Waldman et al.,
Immune Mechanisms on Secretory Surfaces, Postgrad. Med. 50:78
(1971); and Franklin, Prog. Allerg., 8 p. 57 (1964). Moreover,
there are striking variations in the locations of immunoglobulins
within the body. For example, distribution clearly distinguishes
immunoglobulin classes IgA and IgG. The most striking feature of
IgA is its high concentration in external secretions of the body
including the gastrointestinal fluid. .[.It has been clearly shown
that the immune system which contributes IgA to gastrointestinal
fluid..]. It has been clearly shown that the immune system which
contributes IgA to gastointestinal fluid is a separate and distinct
system from that which produces IgG.
In the human, IgG occurs primarily in the vascular and
intracellular spaces of the body (Waldman, et al, .[.(1970).].
Plasma Protein Metabolism, Academic Press, p. 269
(1970).Iadd.).Iaddend., and very little IgG fluids its way into the
gastrointestinal fluids. Another important difference between the
classes of immunoglobulin is related to their metabolic rate. The
degradation of each class of immunoglobulin, regardless of its
location within the body, appears to be under separate control. The
functional catabolic rate varies from as low as 6.5% for IgG to as
high as 90% for IgE with other classes of immunoglobulin falling in
between (Waldman, et al, .[.(1970).]. Plasma Protein Metabolism,
Academic Press, p. 269 (1970). Further, the different
immunoglobulin classes also differ in their avidity with which
.[.the.]. .Iadd.they .Iaddend.bind to antigens, and in their
ability to combine with complement, which is one of the requisites
for killing living bacterial cells (Heremans, .[.(1960).].
.[.Lesglobulenes.]. .Iadd.Les Globulenes .Iaddend.Seriques du
Systeme Gamma Leur Nature Et Leur Pathologie, Arscia Brusseles and
Masson, Paris (1960). It is important to emphasize these
differences in the types of antibodies because immune effects may
vary depending on the type of antibody involved.
The most commonly held theory is that the different classes of
immunoglobulin have evolved to function in different environments
within the body. It is .[.know.]. .Iadd.known.Iaddend., for
example, that a special and distinct immune system exists for the
production of antibodies which function in the environment of the
gut. Moreover, there is general agreement that the immune functions
of the gut are controlled specifically by IgA antibodies and not
IgG. Therefore, under natural conditions, IgA is the class of
immunoglobulin which regulates immune control over bacterial
infections which occur in the gastrointestinal cavity of man. Since
IgG, IgM, IgD, and IgE are not normally found in the intestinal
secretions, it is not logical to expect that any of these types of
antibodies would be effective in treating infections in the
environment of the gut.
The principal immunoglobulin in the milk of cows in IgG, not IgA
(Sullivan, et al. .[.1969).]. T. B. Jour of Immunol, 2, p. 103
(196.Iadd.9.Iaddend.). Therefore, bovine milk is not an obvious
source of antibody for treating bacterial infections of the gut in
man because of its high concentrations of IgG and low
concentrations of IgA.
The method of immunization is another important parameter when
considering the different classes of immunoglobulin. It is
well-known to those skilled in the art that different methods of
immunization result in the preferential production of different
types of antibodies. For example, local immunization of secretory
tissues achieved by exposing the tissue to antigens stimulates the
preferential production and secretion of IgA type immunoglobulins.
The technique of .[.intramammory.]. .Iadd.intramammary
.Iaddend.perfusion as described in the Petersen patent (U.S. Pat.
No. 3,376,198) is an example of local immunization. This method
stimulates production and secretion of IgA antibodies and is not an
effective method for producing IgG.
According to the present invention, intramuscular injection is used
to produce the product of the invention because IgG is the
principal immunoglobulin in cow's milk, not IgA, and in the cow,
systemic immunization is the preferred method for generating IgG
type antibodies in milk. The distinction between the IgG and IgA
type immunoglobulin is important because it teaches that systemic
immunization and not local immunization is the preferred method for
obtaining milk antibodies of high titer. Moreover, this distinction
teaches that the immune products produced by mammary perfusion of a
vaccine are distinctly different from the immune product produced
by intramuscular injection of the identical vaccine. Thus, the
product of this invention (IgG antibodies) is distinctly different
from the product obtained by the Petersen process.
The immune product of this invention is an improvement over the
product of Petersen's invention because the concentration of
antibodies of the IgG type is significantly higher than the
concentration of antibodies of the IgA type. There is no evidence
in the literature to support the claim that IgG antibodies can be
produced by intramammary perfusion of antigens. .[.Mover .].
.Iadd.Moreover.Iaddend.since the levels of IgA immunoglobulins are
either non-extant or extremely low in cow's milk, it is
unreasonable to suggest that the teaching of Petersen's patent has
any relevance to the claim of this invention. On the contrary, the
teaching of the Petersen patent leads away from the discovery of
this invention since it implies that IgA is a biologically active
factor in cow's milk, which has potential therapeutic
application.
BRIEF SUMMARY OF THE INVENTION
According to the present invention, a unique combination of
bacterial species is formulated into a vaccine, which is
administered to healthy dairy cows. The IgG antibodies obtained
from the milk of the immunized cows constitute the products of the
invention. The method of the invention involves the passive
immunization of the patient by oral .[.injestion.]. .Iadd.ingestion
.Iaddend.of the IgG immunoglobulin, which passively immunizes
against a mixed spectrum of infectious bacteria which reside in the
gastrointestinal tract. This treatment eliminates conditions in the
gastrointestinal tract which cause rheumatoid arthritis.
BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWINGS
FIG. 1 is a specimen of a questionnaire referred to in the
specification.
FIG. 1a is a continuation of the .[.questionnarire.].
.Iadd.questionnaire .Iaddend.of FIG. 1.
FIG. 2 is a graph plotting results of .[.test.]. .Iadd.tests
.Iaddend.in terms of RF titer against time, over a 12 month period,
6 months on immune milk and 6 months on placebo.
DETAILED DESCRIPTION
The product of this invention is a low-fat .[.powered.].
.Iadd.powdered .Iaddend.milk which optimally contains a population
of natural IgG type antibodies that react with the bacterial
species listed in Table 1.
TABLE 1 ______________________________________ Bacterial Antigens
ORGANISM *ATCC NO. ______________________________________
Staphylococcus aureus 11631 Staphylococcus epidermidis 155
Streptococcus pyogenes, A. Type 1 8671 Streptococcus pyogenes, A.
Type 3 10389 Streptococcus pyogenes, A. Type 5 12347 Streptococcus
pyogenes, A. Type 8 12349 Streptococcus pyogenes, A. Type 12 11434
Streptococcus pyogenes, A. Type 14 12972 Streptococcus pyogenes, A.
Type 18 12357 Streptococcus pyogenes, A. Type 22 10403 Aerobacter
aerogenes 884 Escherichia coli 26 Salmonella enteritidis 13076
Pseudomonas aeruginosa 7700 Klebsiella pneumoniae 9590 Salmonella
typhimurium 13311 Haemophilus influenzae 9333 Streptococcus
viridans 6249 Proteus vulgaris 13315 Shigella dysenteriae 11835
Streptococcus, Group B Diplococcus pneumoniae Streptococcus mutans
Corynebacterium, Acne, Types 1 & 2
______________________________________ *American Type Culture
Collection, 12301 Parklawn Dr., Rockville, Md. 20852
The antibacterial milk contains all of the substances normally
found in low-fat powdered milk. The principal constituents
comprising antibacterial milk are shown in Table 2.
TABLE 2 ______________________________________ Quantitative and
Qualitative Analysis of Antibacterial Milk Proteins 35.6% Fat 1.0%
Carbohydrates 52% Minerals 7.8% Moisture 3.5% Each reliquified
quart of 3-4 ounces of non-fat dry milk contains approximately:
1200 mg calcium 157% 935 mg phosphorous 125% 0.3 mg thiamine 32%
1.78 mg riboflavin 140% 1.04 mg niacin 10% 324 Calories
______________________________________
Antibacterial milk and normal cow's milk contain the same
approximate percent by weight concentration of ingredients.
Moreover, the concentration of type IgG immunoglobulin in
antibacterial milk and normal milk is identical. Therefore, it is
only the specificity of antibodies comprising the antibacterial
milk which distinguishes it from normal milk. By specificity of the
immunoglobulin is meant the spectrum of bacterial species .[.that
.Iadd.with which .Iaddend.the antibodies react .[.with.]..
Antibacterial milk contains no drug additives or any other
components which are not natural food products of the cow.
The immune milk of the present invention is also useful in the
control of auto-immune diseases, e.g. lupus erythematosus and the
like, which are caused or aggravated by bacterial infectious in the
gastrointestinal tract.
The polyvalent antigen used for the induction of antibacterial milk
is prepared as follows:
Preparation of the Vaccine
The bacterial strains listed in Table 1 were obtained from the
American Type Culture Collection, which ensures authenticity of
bacterial strains and the highest standard of purity that is
available. Upon receipt, each individual bacterial strain was grown
on a blood agar plate to test the viability of the culture and to
determine if growth pattern is typical or atypical of the bacteria
in question. A single colony from each of the test cultures was
taken for histological examination to further ensure authenticity
and purity of the culture. A single colony of each culture was used
to inoculate 500 ml of standard culture broth. The standard broths
recommended by the American Type Culture Collection were used to
grow each of the specific bacteria listed in Table 1.
All organisms were incubated as static cultures with the exception
of 12, 13, 14 and 16, which were incubated in the shaker to provide
agitation. Identification of bacterial strains and the American
Type Culture Collection catalog numbers are shown in Table 1. Each
culture was cultivated for 48 hours at 37.degree. C. Following
incubation, the cultures were killed by heating at 60.degree. C.
for two hours. Samples of the killed bacteria were used to
inoculate fresh broth which was then incubated for 24 hours at
37.degree. C. to determine if the .[.filling.]. .Iadd.killing
.Iaddend.process was complete. Only cultures proven sterile by this
procedure were used for further processing. Sterile cultures were
then washed five times in distilled water and the cells were
recovered by centrifugation. The bacterial cells were frozen by
immersion in liquid nitrogen and freeze-dried by the process of
lyophilization. The lyophilized cells were stored in sterile vials
until used for production of the polyvalent vaccine. The polyvalent
vaccine was prepared by weighting out one gram quantities of each
of the bacterial strains. The dry cells were mixed together and
this mixture was suspended in sterile physiological saline (20
grams of bacteria per 500 ml saline).
A sample of the concentrated solution was diluted in serial fashion
with saline to determine .Iadd.the .Iaddend.dilution which gives a
concentration of 4.times.10.sup.8 .[.ml.]. .Iadd.CL .Iaddend.per
cc. The stock 55 concentrated polvalent vaccine was
.[.diskpersed.]. .Iadd.dispersed .Iaddend.into multiple containers
and stored frozen. A sufficient amount of concentrated antigen was
included in each individual container to immunize 50 cows. The
final dilution of concentrate was made just prior to immunization
The preferred procedure is to remove a sufficient number of vials
to immunize the number of cows to be treated. For example, the
vials are removed 24 hours prior to the planned time of
immunization; a sample of the concentrate is then diluted in a
sterile container to a final concentration of 4.times.10.sup.8
cells per ml. The maximum response in cows is obtained by injecting
20.times.10.sup.8 bacterial cells or 5 cc of the sterile
preparation which is 4.times.10.sup.8 cells per ml according to the
method of immunization described below.
Preferred Process for Immunization of Cows
The antibody product of the invention is produced by immunizing
cows with the polyvalent antigen prepared as described above. The
cows are injected with 5 cc of polyvalent antigen containing
20.times.10.sup.8 bacterial cells. The injection is made
intramuscularly in the gluteus maximus muscle of the hind leg. This
procedure is repeated at one week intervals for four consecutive
weeks beginning 2-3 weeks prior to the predicted day of
parturition. Following the primary immunization, booster injections
using the same concentration of the antigen, are given every 14
days. This method of immunization gives the maximum antibody
titer.
Collection, Handling and Processing of Milk
The milk is collected from immunized cows in a modern dairy parlor.
A fully automated milking system collects and stores the milk under
complete sanitary conditions. The milking system consists of
automated machines connected directly to refrigerated storage tanks
by a closed system of pipes. The complete system is cleaned and
sterilized following each milking to ensure maximum sanitary
conditions. It is important to take careful steps to prevent the
growth of bacteria .[.to.]. .Iadd.in .Iaddend.immune milk during
processing, since such bacteria can lower the titer of antibodies
in the milk.
Milk is transported daily from the refrigerated holding tanks to a
dairy processing plant by milk transport trucks. At the dairy plant
a high temperature short-time system is used to pasteurize the
antibacterial milk. Specialized dairy machinery provides the flash
heating of a continuous flow of milk to 155.degree. F. for a period
of not more than 15 seconds. Temperature and time is critical since
antibody is susceptible to degradation by heat. Milk antibody is
destroyed at temperatures above 165.degree. F., if held for periods
longer than one minute. Following pasteurization, the whole milk is
immediately cooled and the fat is removed by centrifugation, and
the skimmed whole antibacterial milk is powdered by a spray
process. The spray process consists of a large drying chamber into
which hot air (350.degree. F.) is blown at high velocity. The
skimmed milk is atomized into the chamber where the finely divided
milk particles are instantly dried as they fall to the bottom of
the tank. The dried milk is removed automatically by means of
mechanical devices and the milk powder is packaged under sanitary
conditions. Prior to atomizing, the skimmed milk is condensed by
boiling in a chamber under vacuum (100.degree.-110.degree. F.). At
each step it is critical to keep the bacteria from contaminating
the milk since this reduces the titer of the antibody.
Testing Procedures
Immune milk was prepared in inbred Holstein cows. The cows were
immunized by the intramuscular injection of a mixture of bacterial
antigens identified in Table 1. The vaccine was prepared by the
process described above. The immunologic response of the cows was
boosted by bi-weekly injections of the vaccine. The milk from these
cows was pooled, the fat removed, and the non-fat milk was
pasteurized by exposure to 162.degree. F. for 16 seconds followed
by a spray-drying process in which the temperature of the milk did
not exceed 85.degree. F. The milk was packaged in one quart
polyethylene containers. Control milk (placebo) was non-fat
.[.powedered.]. .Iadd.powdered .Iaddend.milk purchased from a local
producer.
Erythrocyte sedimentation rates were determined on freshly
collected blood by the method of Westergren and corrected for
hematocrit according to the method of Wintrobe & Landsberg
(1935). Rheumatoid factor titers were determined by the
Singer-Plotz (1966) macroscopic tube test.
Patients were accepted for the study on the basis of an elevated
erythrocyte sedimentation rate and a positive rheumatoid factor
titer. Nine patients were studied for 12 months and 11 patients
were studied 18 months. The patient group was composed of thirteen
caucasian females ranging in age from 32 to 69 years with an
average of 50.4 years, and seven caucasian males ranging in age
from 43 to 70 years with an average age of 58.1 years. The mean
duration of arthritis was 10.8 years for the females and 11.0 for
the males. Patients were randomly placed either on immune milk or
on non-immune milk (a commercial product purchased in the Dayton
area that served as a placebo). Both milk products were packaged in
identical containers and were identified as being immune milk or
placebo, respectively, by a blue or red pressure-sensitive label
that was attached to each container at the time it was filled. The
labels were removed just prior to dispensing the milk to the
patients. Thus, at no time did the patients know whether they were
receiving immune milk or placebo. Patients were randomly (as
determined by the flip of a coin) selected to receive either immune
milk or the placebo during the first six-month period. At the end
of this time, those that were receiving immune milk were placed on
the placebo and those that were receiving placebo were placed on
immune milk for the second six-month period.
At the end of the second six-month period, 11 patients volunteered
to remain on the study for an additional six months. The type of
milk (immune or placebo) was again changed at this time and
observations were continued. Thus, the study was comprised of three
six-month periods, 11 of the patients participating for three
periods and nine participating for two periods. Patients were seen
at monthly intervals at which time a one month supply of milk was
dispensed, an evaluation questionnaire were filled out and a blood
sample was collected for rheumatoid factor titer, erythrocyte
sedimentation rate and hematocrit determination.
Patients were instructed to take a quantity of non-fat milk solids
equivalent to one quart of milk post prandially two times daily.
The milk solids were freshly dissolved in one pint of cool tap
water immediately before ingestion shortly after awakening in the
morning and again just prior to retiring at night. They were told
to see their physician as usual and to follow the treatment
regimens prescribed by him. Medication was to be taken ad libitum
or as prescribed by their regular doctor. We requested only that
they report the quantity of medicines taken.
A questionnaire was completed by each patient at monthly intervals.
It was divided into six sections that deal with:
(1) duration of morning stiffness,
(2) severity of pain experienced in each of eight joints,
(3) type and quantity of drugs with short-term actions that were
taken,
(4) type and quantity of drugs with long-lasting actions that were
taken,
(5) ability of patient to conduct his normal activities, and
(6) severity of symptoms of rheumatoid arthritis. The numbers shown
in the spaces next to each answer indicate the score assigned to
that answer in the course of evaluating the questionnaires. In
scoring the sections dealing with medications, an effort was made
to reflect the relative anti-inflammatory and analgesic activities
of the various drugs used. A five-grain aspirin tablet .[.as.].
.Iadd.was .Iaddend.assigned a value of one. All other drugs (with
the .[.except.]. .Iadd.exception .Iaddend.of gold, plaquenil and
cortisone shots which were considered separately) were arbitrarily
assigned values relative to aspirin. Thus, all salicylate
preparations, Tylenol, Darvon, Motrin, etc. were considered
equivalent to a five-grain aspirin tablet and were also assigned a
value of one. The number of mg of Prednisone was multiplied times
four, the number of Indocin capsules taken was multiplied times
2.5. The number of grains of codeine was multiplied times two, and
the number of Butazoladin tablets taken was multiplied times
seven.
The mean scores in each category were calculated for each six-month
period. The differences of the means where then calculated by
subtracting the mean values scored during administration of immune
milk from those scored during administration of placebo. When the
results were calculated in this manner, improvement in the
patient's condition during the period he received immune milk was
indicated by negative values for questions one and six, and by
positive values for all other questions. Mean corrected erythrocyte
sedimentation rates (ESR) and rheumatoid factor titers (RF) were
respectively shown in a similar manner. There were calculated in
such a way that positive values reflect a lower erythrocyte
sedimentation rate or .[.rehumatoid.]. .Iadd.rheumatoid
.Iaddend.factor titer during administration of immune milk. The
data were statistically evaluated using the Statistical Analysis
System of Goodnight et al. (Computer Program Used for the
Statistical Analyses, Statistical Systems Institute, Raleigh,
N.C.). Calculations were performed with the aid of an IBM model
370/155 computer.
Results
The immune milk was well tolerated by all patients with the
exception of one who had pernicious anemia. This patient complained
of diarrhea and was terminated from the study. Some patients
reported a weight gain during the course of the study. This may
have been due to the increased caloric intake from the milk or
possibly reflects a generalized improvement in their physical
condition.
TABLE 3
__________________________________________________________________________
Periods of Treatment Regimen Observation Placebo Immune Mean
Control Immune Mean C.V.* Mean C.V.* Difference P
__________________________________________________________________________
A.M. Stiffness 27 24 0.322 95.7 0.679 35.6 -0.347 0.0001 Joint Pain
a. Shoulder 27 24 0.954 67.1 0.716 60.2 +0.238 0.0420 b. Elbow 27
24 0.752 83.7 0.613 65.9 +0.139 0.0511 c. Wrist 27 24 0.824 73.6
0.539 74.8 +0.285 0.0010 d. Hand 27 24 1.073 54.7 0.828 56.5 +0.245
0.0011 e. Hip 27 24 0.533 90.3 0.227 135.0 +0.306 0.0005 f. Knee 27
24 0.904 74.1 0.683 59.0 +0.221 0.0015 g. Ankle 26 22 0.7811 66.9
0.659 65.7 +0.1221 0.0127 h. Feet 26 22 0.948 63.7 0.729 50.9
+0.219 0.0010 Pills 27 24 20.663 104.1 16.515 101.3 +4.148 0.0405
Other 27 24 0.325 140.7 0.244 175.6 +0.081 0.0276 Medication ADL 27
24 2.224 36.1 1.874 29.1 +0.350 0.0023 Monthly change a. Pain 27 24
1.903 21.8 2.247 14.1 -0.344 0.0042 b. Stiffness 27 24 1.985 18.8
2.254 12.4 -0.269 0.0024 c. Swelling 27 24 1.924 17.9 2.117 13.3
-0.193 0.00153 ESR 25 23 36.293 29.7 35.922 38.2 +0.371 0.7376 RF
27 24 6.698 45.5 6.834 41.7 -0.136 0.9635
__________________________________________________________________________
*Coefficient of variation.
As shown in Table 3, patients were observed during a total of 27
control periods (six-month periods during which they received
placebo) and 24 test periods (six-month periods during which they
received immune milk). One patient had sustained a physical injury
to one of his ankles and feet. The pain in these joints was not
evaluated, which accounts for there being a smaller number of
periods of evaluation for these joints. The erythrocyte
sedimentation rates for one patient were so extremely abnormal
(more than two standard deviations removed from the mean of the
values for the other patients) that they were not included. This
omission accounts for the smaller number of observations reported
for that variable.
The mean values and coefficients of variation (C.V.) are listed in
the table for each variable. Differences between the means were
calculated by subtracting the mean value obtained during the
periods the patients received immune milk from that obtained during
the periods they received the placebo. A favorable response to
immune milk is indicated by negative values for AM stiffness
(question 1) and Monthly change (questions 6a, b, and c) and by
positive values for all other variables. An effective response to
immune milk was obtained for all data obtained from the
questionnaires. Probabilities (P) indicate a high degree of
statistical significance in every instance. The small mean
differences obtained for erythrocyte sedimentation rate and
rheumatoid factor titer were not significant. When erythrocyte
sedimentation rates were considered on an individual basis,
however, four of the twenty patients studied had statistically
significant decreases while receiving immune milk.
Although immune milk had no significant effect on the mean values
for rheumatoid factor titer, examination of individual patients
revealed some interesting responses. Seven of the twenty patients
studied had negative rheumatoid factor titers on at least one
occasion during the period they were receiving immune milk. Four of
them became negative during the period that they received immune
milk and their titers failed to become positive during the
following six-month period when they received the control (placebo)
milk as shown in FIG. 2. Continuation of the study past this
reporting period reveals that 13 of 25 patients lost the rheumatoid
factor from their blood.
Discussion
The scientist in charge of this study personally interviewed each
patient at monthly intervals.[., .]. and recorded their answers to
the questions. Every effort was made not to influence the patient's
answers. The patients were initially informed and were frequently
reminded that, during certain periods of the study, they would
receive a placebo. It was anticipated that this knowledge would
serve as an inducement for the patients to answer the questions
objectively and without bias. At no time were the patients informed
whether they were receiving immune milk or the placebo.
The question regarding medication taken "yesterday" (question #3)
and the question regarding gold shots, Plaquenil and cortisone
shots (question #4) are objective and are of primary importance in
considering answers given to the other questions. These questions
are important for two reasons:
(1) if the immune milk is effective in relieving symptoms of the
disease, the patient would be expected to take fewer medicines that
were allowed ad libitum. On an average, patients reported that they
took four less aspirins or their equivalent per day during the
periods that they received immune milk. They also reported that
they received fewer gold shots. Plaquenil and cortisone shots
during these periods; and
(2) if patients took smaller quantities of analgesics and other
medicines useful in the treatment of rheumatoid arthritis, one
would expect them to report increased discomfort unless the immune
milk was influencing the disease favorably.
As noted in Table .[.2.]. .Iadd.3.Iaddend.significantly less joint
involvement was reported during periods that the patients received
immune milk even though they were taking less medicines for their
arthritis.
Patients started on the study at monthly intervals over a one-year
period, and the type of milk product (immune milk or placebo) that
they initially received was randomized. The observation that
positive responses or improvement were obtained for all parameters
of the questionnaire, and that these mean responses were
statistically significant strongly indicate that 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 in erythocryte
sedimentation rate while receiving immune milk.
Results of the rheumatoid factor titers are difficult to evaluate.
This is due .[.at least in part.]. .Iadd., at least in part,
.Iaddend.to the fact that the origin and role of rheumatoid factors
in the etiology and prognosis of rheumatoid arthritis is not
understood. Rose et al.Iadd., Proc. Soc. Exp. Biol. Med. 68:1
.Iaddend.(1948) showed that sheep red blood cells that were
sensitized with rabbit antibody underwent agglutination in the
presence of blood serum from patients with rheumatoid arthritis.
The test depends on the specific reaction between normal
immunoglobulin (either rabbit or human I.sub.g G) with rheumatoid
factors. The specificities exhibited by .Iadd.the
.Iaddend.rheumatoid factor are like those that would be expected of
antibody against I.sub.g G (Epstein, et al, .[.1956).]. Proc. Soc.
Exp. Biol. Med. 91:235.Iadd.(1956)).Iaddend..
The presence of rheumatoid factors has been correlated with disease
severity in rheumatoid arthritis and can be identified in proteins
precipitated in the tissues of patients with rheumatoid arthritis.
Although a small percentage of patients with rheumatoid arthritis
do not have positive rheumatoid factor titers, it is generally
agreed by most rheumatologists that positive agglutination
reactions do not revert to negative even when the disease is in
remission. DeForest, et al .[.(1958).]. Arth. & Rheum. 1:387,
.Iadd.(1958).Iaddend., however, described a small number of
patients who had positive rheumatoid factor titers that reverted to
negative following a remission. When recrudescense of the disease
occurred, the test again became positive. Aho, et al .[.(1959).].
Ann. Exp. Fenn. 37:377 .Iadd.(1959) .Iaddend.noted, however, the
most patients whose disease had become inactive remained
serologically positive. The fact that negative titers were observed
in 60% of our patients and that in half of these, the titers
remained negative for six months, proves that immune milk is
affecting a primary etiologic factor responsible for rheumatoid
arthritis.
The effect of immune milk in alleviating the symptoms of rheumatoid
arthritis is particularly relevant when considered on the basis of
the recently described relationship between the histocompatibility
antigens (HL-A) and the susceptibility to rheumatic disease
(Brewerton, .[.1976).]. Arth. & Rheum. 19:656.
.Iadd.(1976)).Iaddend.. Histocompatibility antigens are genetically
determined antigens that are found on all human cells. The genes
controlling their inheritance are called histocompatibility genes.
There are now known to be over 40 of these genetically determined
antigens. They are responsible for rejection of tissue grafts made
between individuals other than identical twins. Superficially the
HL-A antigens resemble ABO blood groups in that they are inherited
for a lifetime. Their functions .[.is.]. .Iadd.are .Iaddend.not yet
known, except in the highly artificial situation produced by
transplantation. It is known, however, that the histocompatibility
genes are closely linked with the immune response genes on the
sixth chromosome. In this relationship, they may determine the
immune response of the individual to a foreign invader, such as a
.[.bacteria..]. .Iadd.bacterium. .Iaddend.
Persons with HLA-B27 appear to be particularly susceptible to a
variety of rheumatic diseases. It is postulated that this
histocompatibility antigen dictates a type of immune response which
in the presence of other predisposing factors leads to rheumatoid
arthritis. After an intestinal infection with Yersinia
enterocolitica, some patients develop an acute peripheral arthritis
(Ahvonen, et al, .[.1969).]. Acta. Rheum. Scand. 15:232
.Iadd.(1969)).Iaddend.. Similarly, after salmonella infection,
about 2% of patients develop acute peripheral arthritis (Warren,
Am. Rheum. Dis. 29;484 1970). HLA-B27 was found in 43 of 49
patients with yersinia arthritis and in 15 of 16 with salmonella
arthritis (Aho. .[.1974).]. Ann. Exp. Fenn. 37:377
.Iadd.(1974)).Iaddend.. It is an attractive possibility that
infective agents may thrive in the intestinal tract without giving
rise to local symptoms. In patients with HLA-B27, a host response
is established that results in arthritis. Thus, it is not necessary
for the infective agent to gain entry into the joints. Immune milk
is beneficial to patients with rheumatoid arthritis because it
contains antibodies that effectively inactivate or neutralize
offending bacteria and/or their metabolic products.
* * * * *