U.S. patent number RE33,403 [Application Number 07/296,969] was granted by the patent office on 1990-10-23 for method for treating disorders of the vascular and pulmonary systems.
This patent grant is currently assigned to Stolle Research & Development Corporation. Invention is credited to Lee R. Beck, Ralph J. Stolle.
United States Patent |
RE33,403 |
Stolle , et al. |
October 23, 1990 |
Method for treating disorders of the vascular and pulmonary
systems
Abstract
A method for treating vascular disorders or pulmonary disorders
associated with smoking in an animal which comprises: adminstering
to the animal milk collected from a bovid being maintained in a
hyperimmune state, in an amount and for a time sufficient to
produce anti arteriosclerotic or antiaging vascular effects or
sparing effects on lung tissue.
Inventors: |
Stolle; Ralph J. (Oregonia,
OH), Beck; Lee R. (Birmingham, AL) |
Assignee: |
Stolle Research & Development
Corporation (Cincinnati, OH)
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Family
ID: |
27404470 |
Appl.
No.: |
07/296,969 |
Filed: |
January 13, 1989 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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384625 |
Jun 3, 1982 |
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Reissue of: |
546162 |
Oct 27, 1983 |
04636384 |
Jan 13, 1987 |
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Current U.S.
Class: |
424/157.1;
424/163.1; 424/165.1; 424/169.1; 424/234.1; 424/244.1;
424/257.1 |
Current CPC
Class: |
A61K
35/20 (20130101); A61K 35/54 (20130101); C07K
2317/11 (20130101) |
Current International
Class: |
A61K
35/48 (20060101); A61K 35/20 (20060101); A61K
35/54 (20060101); A61K 039/40 (); A61K 039/42 ();
A61K 039/395 (); A61K 035/20 () |
Field of
Search: |
;424/87,92,85.8,86 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Lascelles, A., Dairy Science, vol. 25, pp. 359-364, 1963..
|
Primary Examiner: Hazel; Blondel
Attorney, Agent or Firm: Saidman, Sterne, Kessler &
Goldstein
Parent Case Text
This application is a continuation-in-part of application Ser. No.
384,625, filed June 3, 1982, now abandoned.
Claims
What is new and claimed and intended to be covered by Letters
Patent of the United States is:
1. A method of lowering lipid blood stream levels in an animal
which comprises:
administering to said animal milk collected from a bovid being
maintained in a hyperimmune state against a mixture of antigens
comprising Staphylococcus aureus; Staphylococcus epidermidis;
Streptococcus pyogenes, A. Type 1; Streptococcus pyogenes, A. Type
3; Streptococcus pyogenes, A. Type 5; Streptococcus pyogenes, A.
Type 8; Streptococcus pyogenes, A. Type 12; Streptococcus pyogenes,
A. Type 14; Streptococcus pyogenes, A. Type 18; Streptococcus
pyogenes, A. Type 22; Aerobacter aerogenes; Escherichia coli;
Salmonella enteritidis; Pseudomonas aeruginosa; Klebsiella
pneumoniae; Salmonella typhimurium; Haemophilus influenzae;
Streptococcus viridans; Proteus vulgaris; Shigella dysenteriae;
Streptococcus, Group B; Diplococcus pneumoniae; Streptococcus
mutans; and Corynebacterium Acne, Types 1 and 2, in an amount and
for a time sufficient to produce said lowering effect.
2. The method of claim 1 wherein said milk is in powdery form.
3. The method of claim 1 wherein said milk is in fluid form.
4. The method of claim 1 wherein said milk is in concentrated
form.
5. The method of claim 1 wherein said milk is incorporated into a
food product.
6. The method of claim 4 wherein said product is yogurt.
7. The method of claim 1 wherein said bovid is a cow.
8. The method of claim 1 wherein said bovid is maintained in said
hyperimmune state by administering to said bovid booster injections
of a mixture of antigens comprising Staphylococcus aureus;
Staphylococcus epidermus; Streptococcus pyogenes, A. Type 1;
Streptococcus pyogenes, A. Type 3; Streptococcus pyogenes, A. Type
5; Streptococcus pyogens, A. Type 8; Streptococcus pyogenes, A.
Type 12; Streptococcus pyogenes, A. Type 14; Streptococcus
pyogenes, A. Type 18; Streptococcus pyogenes, A. Type 22;
Aerobacter aerogenes; Escherichia coli; Salmonella enteritidis;
Pseudomonas aeruginosa; Klebsiella pneumoniae; Salmonella
typhimurium; Haemophilus influenzae; Streptococcus viridans;
Proteus vulgaris; Shigella dysenteriae; Streptococcus, Group B;
Diplococcus pneumoniae; Streptococcus mutans; and Corynebacterium
Acne, Types 1 and 2.
9. The method of claim 8 wherein said antigen or mixture of
antigens booster is injected in a dose of 10.sup.6 cells to
10.sup.20 cells.
10. The method of claim 4 wherein said milk is prepared by a
process which comprises:
sensitizing a bovid with said mixture of antigens;
administering boosters of said mixture of antigens of a dosage
sufficient to induce and maintain a hyperimmume state in said
bovid, and thereafter collecting said milk from said bovid.
11. The method of claim 10 wherein said process further
comprises:
pasteurizing said collected milk; and
removing the fat from said milk.
12. The method of claim 1 wherein said milk is administered
daily.
13. The method of claim 1, wherein said lipids are selected from
the group consisting of triglycerides, cholesterol, low density
lipids and mixtures thereof. .Iadd.
14. A method of lowering lipid blood stream levels in an animal
which comprises administering to said animal, in an amount and for
a time sufficient to produce said lowering effect, milk collected
from a bovid being maintained in a hyperimmune state against
bacterial, viral or cellular antigens or a mixture of two or more
of said antigens. .Iaddend. .Iadd.
15. The method according to claim 14 wherein said bovid is
maintained in a hyperimmune state against a bacterial antigen or
antigens. .Iaddend. .Iadd.16. A method according to claim 15
wherein said bacterial antigens are selected from the group
consisting of Staphylococcus aureus; Staphylococcus epidermidis;
Streptococcus pyogenes, A. Type 1; Streptococcusi pyogenes, A. Type
3; Streptococcus pyogenes, A. Type 5; Streptococcus pyogenes, A.
Type 8; Streptococcus pyogenes, A. Type 12; Streptococcus pyogenes,
A. Type 14; Streptococcus pyogenes, A. Type 18; Streptococcus
pyogenes, A. Type 22; Aerobacter aerogenes; Salmonella enteritidis;
Klebsiella pneumoniae; Salmonella typhimurium; Haemophilus
influenzae; Streptococcus viridans; Proteus vulgaris; Shigella
dysenteriae; Streptococcus, Group B; Diplococcus pneumoniae;
Corynebacteriumn, Acne, Types 1 and 2; Pseudomonas aeruginosa;
Pseudomonas maltophiia; Streptococcus equisimili; Streptococcus
dysgalactiae; Streptococcus uberis; Streptococcus bovis;
Pasteurella multocida; Pasteurella haemolytica; Pasteurella
multocida; Moraxella bovis; Actinobacillus lignieresi;
Corynebacterium renale; Fusobacterium necrophorum; Bacillus cereus;
Salmonella dublin; Salmonella heidleberg; Salmonella paratyphi;
Yersinia enterocolitica; Streptococcus mutans; and Escherichia
coli. .Iaddend. .Iadd.17. The method of claim 15 wherein said
bacterial antigens are selected from the group consisting of
Pseudomonas aeruginosa; Pseudomonas maltophiia; Streptococcus
equisimili; Streptococcus dysgalactiae; Streptococcus uberis;
Streptococcus bovis; Streptococcus bovis; Pasteurella multocida;
Pasteurella multocida; Pasteurella haemolytica; Pasteurella
multocida; Moraxella bovis; Actinobacillus lignieresi;
Corynebacterium renale; Fusobacterium necrophorum; Bacillus cereus;
Salmonella dublin; Salmonella heidleberg; Salmonella paratyphi; and
Yersinia enterocolitica. .Iaddend. .Iadd.18. The method of claim 15
wherein said bacterial antigen comprises Streptococcus mutans.
.Iaddend. .Iadd.19. The method of claim 18 wherein said antigen is
selected from the group consisting of Streptococcus mutans AHT,
Streptococcus mutans BHT, Streptococcus mutans 10499, and
Streptococcus mutans 6715. .Iaddend. .Iadd.20. The method of claim
18 wherein said antigen comprises Streptococcus mutans AHT,
Streptococcus mutans BHT, Streptococcus mutans 10499, and
Streptococcus mutans 6715. .Iaddend.
.Iadd.1. The method of claim 15 wherein said bacterial antigen
comprises Escherichia coli. .Iaddend. .Iadd.22. The method of claim
14 or 15 wherein said milk is in a powdery form, a fluid form, a
concentrated form, incorporated into a food product, or in yogurt.
.Iaddend. .Iadd.23. The method of claim 14 or 15 wherein said bovid
is maintained in said hyperimmune state by administering to said
bovid booster injections of an antigen or antigens. .Iaddend.
.Iadd.24. The method claim 14 or 15 wherein said milk is prepared
by a process which comprises
sensitizing a bovid with an antigen or antigens;
administering boosters of said antigen or antigens of a dosage
sufficient to induce and maintain a hyperimmune state in said bovid
and thereafter collecting said milk from said bovid. .Iaddend.
.Iadd.25. The method of claim 24 wherein said process further
comprises:
pasteurizing said collected milk; and
removing the fat from said milk. .Iaddend. .Iadd.26. The method of
claim 14 wherein said milk is administered daily. .Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for the treatment of
disorders of the vascular and pulmonary systems, such as vascular
aging and arteriosclerosis and disorders of the lungs associated
with smoking.
2. Description of the Prior Art
The normal vascular system of mammals, especially humans, includes
all of the organs, such as the heart and the arteries, involved in
blood transport and circulation. Two major disorders affect the
vascular system in animals: arteriosclerosis and aging.
Arteriosclerosis, a generic term for the thickening and hardening
of the arterial wall, is responsible for the majority of deaths in
the United States and most Westernized societies. There are various
types of arteriosclerosis such as atherosclerosis, focal
calcification, and arteriolosclerosis. The changes associated with
arteriosclerosis (of the various types) and aging are partly
overlapping. (See for example Harrison's "Principles of Internal
Medicine," 9th Edition, pp. 156-1166.)
The normal artery wall consists of three reasonably well defined
layers: the intima, the media and the adventitia. The intima is a
layer of endothelial cells lining the lumen of all arteries. The
endothelial cells are attached to each other by a series of
junctional complexes and also are attached to an underlying
meshwork of loose connective tissue, the basal lamina. The lining
endothelial cells form a barrier that controls the entry of
substances from the blood into the arterial wall. The media
consists of smooth muscle cells arranged in either single layers or
multiple layers. The outermost layer of the artery is the
advenritia which is delimited by the external elastic lumina. This
external coat consists of a loose interwoven mixture of thick
bundles of collagen, elastic fibers of varying size and a mixture
of smooth muscle cells and fibroblasts.
Maintenance of the endothelial cell lining is critical. Endothelial
cell turnover occurs at a slow rate but may accelerate in focal
areas by changing patterns of flow along the vessel wall. Intact
endothelial cells function to prevent clotting partly by
elaboration of prostacyclin that inhibits platelet function,
thereby enhancing unimpeded flow of blood. When the lining is
damaged, however, platelets adhere to it in part as the result of
production of a different class of prostaglandins, the
thromboxanes, and form a clot. The ability of the arterial wall to
maintain the integrity of its endothelium, prevent platelet
aggregation and insure the nutrition of its middle portion may be
the critical determinants of the arteriosclerotic process.
The major change that occurs with normal aging in the arterial wall
is a slow symmetrical increase in the thickness of the intima. This
results from an accumulation of small muscle cells. In the
nondiseased artery wall the lipid content, mainly cholesterol ester
and phospholipid, also progressively increases with age. While most
of the phospholipid in the normal artery wall appears to be derived
from in situ synthesis, the cholesterol ester that accumulates with
aging appears to be derived from plasma, since it contains
principally linoleic acid, the major plasma cholesterol ester of
fatty acid. As the normal artery ages, smooth muscle cells and
connective tissue accumulate in the intima, leading to progressive
thickening of the layer coupled with progressive accumulation of
fatty acid, resulting in a gradual increase in the rigidity of the
vessels. The larger arteries may become dilated, elongated and
porous and aneurysms may form in areas of encroaching degenerating
arteriosclerotic plaque.
By far, the leading cause of death in the United States both above
and below age 65 is atherosclerosis, the atheromatous form of
arteriosclerosis. The lesions are commonly classified as fatty
streaks, fibrous plaques and complicated lesions. The fatty streaks
are characterized by an accumulation of lipid-filled smooth muscle
cells and fibrous tissue in focal areas of the intima, and are
stained distinctively by fat soluble dyes. The lipid is mainly
cholesterol oleate. Fibrous plaques are elevated areas of intima
thickening and will present the most characteristic lesion of
advancing arteriosclerosis. They appear in the abdominal aorta,
coronary arteries and carotid arteries in the third decade and
increase progressively with age. Complicated lesions are calcified
fibrous plaques containing various degrees of necrosis, thrombosis
and ulceration.
A number of factors called "risk factors" have been identified in
individuals who develop atherosclerosis. The risk factor concept
implies that a person with at least one risk factor is more likely
to develop a clinical atherosclerotic event and to do so earlier
than a person with no risk factors. The presence of multiple risk
factors further accelerates atherosclerosis. Among the reversible
or partially reversible risk factors are hyperlipidemia
(hypercholesterolemia and/or hypertriglyceridemia), hyperglycemia
and diabetes, low levels of high density lipoproteins,
hypertension, obesity, and cigarette smoking.
As stated in Harrison's, supra (p 1166), although the emergence of
clinical consequences of atherosclerosis can be lessened, no
convincing instance of regression or interruption of progression of
atherosclerosis by removal or reversal of any single or group of
risk factors has yet been proved in humans. The trend toward lower
smoking, lower cholesterol and fat consumption and towards
reduction of overweight and exercise programs has been helpful.
Prevention rather than treatment, however, is the goal of public
health professionals. An effective program of prophylaxis has not
yet been established, although enough is known to guide in both
identification and high risk and development of measures to reduce
the risk.
Among the risk factors referred to above that mighr be particularly
well suited to therapeutic treatment is hyperlipidemia. Although
control of factors such as obesity and cigarette consumption
depend, to a great degree, on the will and inclination of the
individual, if a reasonable method for lowering, e.g., serum
cholesterol, low density lipids (LDL) and triglyceride blood stream
levels were provided, it would be suitable for treatment of a broad
spectrum of individuals.
Because of the widespread distribution of vascular disorders such
as arteriosclerosis disorders and the naturally occurring aging of
the vascular system and its accompanying problems, a need exists
for an effective method for both preventing and possibly treating
these disorders.
If a natural food product, such as milk for example, could be
obtained having anti-arteriosclerotic and aging effects it would be
an easily administerable, readily available, safe therapeutic
composition.
Another major system of mammals, especially humans, which is
characterized by the progressive degeneration of the cells of the
organs constituting that system, is the pulmonary system, and in
particular, the lungs and associated bronchii and alveoli. Like
arteriosclerosis and vascular aging, certain pulmonary disorders,
including a general breakdown of the pulmonary system, short
windedness and a decrease in the efficiency of the respiration is
associated with and due to environmental irritants and pollutants,
such as those contained in cigarette smoke.
It is known that prolonged smoking induces the accumulation of
deposits of carbon and other substances on and in the lungs, which
destroy the respiratory function of the associated area. Similarly,
smoking, and/or the presence of these deposits substantially
increases the number of neutrophilis and lymphocytes present in
mammalian lungs, both types of cells producing substances that are
injurious to lung tissue.
As with arteriosclerosis, there has been some containment or
alleviation of the disorders associated with smoking due to a
reduction in the frequency of smoking in most age groups. However,
in certain segments of the population, most notably teen age and
young women, smoking has increased, multiplying the frequency of
occurrence of disorders associated with smoking both in the current
population and importantly in the foreseeable future.
As with cardiovascular diseases, treatment based on avoidance of
pollutants such as smoking will have varying success, depending on
the individual. But, if a method for reducing the amount of
deposited matter could be found, such as stimulation of lung
macrophage activity, a treatment of benefit to most individuals
could be developed.
As with arteriosclerotic and vascular diseases in general, if a
natural food product, such as milk for example could be obtained
having a sparing effect on lungs exposed to smoke and the
associated disorders, it would be an easily administerable, readily
available, safe therapeutic composition.
It has been known in the prior art to produce milks having a
variety of therapeutic effects. Beck, for example, has disclosed a
milk containing antibody to Streptococcus mutans which has dental
caries inhibiting effects (Beck, U.S. Pat. No. 4,324,782). The milk
is obtained by immunizing a cow with S. mutans antigen in two
stages and obraining the therapeutic milk therefrom. Beck has also
described a milk having anti-arthritic properties (copending U.S.
Ser. No. 875,140 filed Feb. 6, 1978), and has described and
patented a milk having anti-inflammatory properties (U.S. Pat. No.
4,284,623). Heinbach, U.S. Pat. No. 3,128,230, has described milk
containing globulins of .alpha.,.beta.,.gamma. components, by
innoculating a cow with antiqenic mixtures. Petersen (U.S. Pat. No.
3,376,198 and Canadian Pat. No. 587,849), Holm, U.S. application
(published) Ser. No. 628,987 and Tunnak et al. (British Pat. No.
1,211,876) have also described antibody-containing milks.
None of the aforementioned references, however, disclose or suggest
milk having antiarteriosclerotic or vascular anti aging properties
or a sparing affect on lungs exposed to smoke.
SUMMARY OF THE INVENTION
It is therefore an object of the invention to provide milk having
beneficial properties towards disorders of the vascular and
pulmonary system.
Another object of the invenrion is to provide a process for
producing such milk.
A further object of the invention is to provide a method for
treating vascular disorders in animals.
A further object of the invention is to provide a method for
testing disorders in animals associated with the exposure of lungs
to smoke.
These and other objects of the invention which will hereinafter
become more readily apparent have been attained by providing a
method of treating vascular disorders in an animal which
comprises:
administering to said animal milk collected from a bovid being
maintained in a hyperimmune state, in an amount sufficient to
produce antiarteriosclerotic, antiaging vascular, or by tissue
sparing effects.
BRIEF DESCRIPTION OF THE DRAWINGS
A more complete appreciation of the invention and many of the
attendant advantages thereof will be readily obtained as the same
becomes better understood by reference to the following detailed
description when considered in connection with the accompanying
drawings; wherein:
FIG. 1 shows photomicrographs of cryostat sections of rabbit aortae
stained with Oil Red O to specifically demonstrate lipids.
x300;
A. Control rabbits: Numerous, small lipid droplets (arrows) are
present;
B. Hyperimmune milk-fed rabbits: Lipid droplets are absent in the
aorta's wall. The lipid (arrow) in the tissue outside the aorta is
normal and illustrates that lipids would be visible if they were
present in the wall of the aorta.
FIG. 2 shows scanning electron micrographs of inner ventricular
surfaces of rabbit hearts. x1750;
A. Control: Two thrombi lie below the eroded epithelium of the
endocardium.
B. Hyperimmune milk: The epithelium is intact and there is no
evidence of thrombi.
FIG. 3 shows transmission electron micrographs of blood vessels in
rabbit hearts. x3420;
A. Control: Lipid vacuoles are present in the endorhelial cells
(upper arrows) and smooth muscle cells (lower arrows);
B. Hyperimmune milk: Lipid vacuoles are absent in the endorhelium
and smooth muscle cells.
FIG. 4 is an electromicrograph (SEM) of the alveolar wall of a lung
of a rat exposed to cigarette smoke for three months. x5750.
A. Control: Debris (arrows) is present on the alveolar wall and the
wall is cracked, exposing a red blood cell (RBC).
B. Hyperimmune milk: The dust cell (DC) or macrophage in the center
of the micrograph engulfs debris which helps to clear the alveolar
wall of particles carried to the lower respiratory track in the
smoke.
FIG. 5 is an electromicrograph (TEM) of a rat exposed to cigarette
smoke for three months and receiving hyperimmune milk. x5500.
Psuedopodia (P) of the dust cell (DC) have surrounded the luminal
debris (arrows) in the initial stage of phagocytosis.
FIG. 6 is an electromicrograph (TEM) of a dust cell on a type 1
alveolar cell in a rat lung exposed to cigarette smoke for three
months and receiving hyperimmune milk. x1200. The dust cell (DC)
contains phagocytosed material and is involved in the uptake of
debris (arrows) in the alveolar lumen. Red blood cells (RBC) occupy
capillary lumina in the alveolar walls.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The invention comprises a natural food product (milk) which has
beneficial properties towards animal vascular and pulmonary
systems, as well as a method for producing the same. The milk of
the presenr invention, being a natural product, can be used to
treat vascular disorders associated wirh any disease or injury,
pulmonary disorders associated with smoking or a natural condition
such as vascular aging in animals and humans without fear of side
effects.
Examples of vascular disorders which may be treated with the milk
of the present invention include aging disorders such as a decrease
in the rigidiry of vessels, and a decrease in the incidence wherein
the large arteries become dilated and elongated, as well as a
decrease in aneurisms which form in areas of encroaching
arteriosclerotic plaques. Other aging-induced vascular damage which
can be prevented or reversed with the milk of the present invention
is the increase in the thickness of the arterial intima, the
reversal of gradual accumulation of smooth muscle cells, as well as
a decrease in the accumulation of lipid content in the arterial
wall.
Among the abnormal (i.e. non-aging induced) disorders of the
vascular system which are treatable (preventable or reversable)
with the milk of the present invention is arteriosclerosis, which
includes both atheromatous forms and non-atheromatous forms. Among
the non-atheromatous forms of arteriosclerosis treatable with the
milk of the present invention is focal calcification (also called
Monckeberg's sclerosis), which is common in the lower extremities,
upper extremities, and the arterial supply of the genital tract in
both sexes. Another disorder is focal calcification, which involves
degeneration of smooth muscle cells followed by calcium deposition.
Another non-atheromatous form of arteriosclerosis is
arteriolosclerosis which involves hyaline and general changes
affecting both the intima and media of small arteries and
arterioles, particularly in the spleen, pancreas, adrenal and
kidney.
Importantly, the milk of the present invention can be utilized for
the treatment of atherosclerosis. This involves and regression of
the formation of fatty streaks, both the prevention fibrous plaques
and complicated lesions, as described previously. Athough it is
probable that irreversible risk factors for atherosclerosis, such
as male sex or genetic traits (e.g. positive family history of
premature atherosclerosis) might not be reversed with the milk of
the invention, the so-called reversible factors, however, may.
Thus, the milk of the invention is useful in reducing the
accumulation of lipids and preventing or reversing
hypercholesterolemia or hypertriglyceridemia. Various forms of
atherosclerosis can be treated.
Additionally, the milk of the invention can be utilized to reverse
some of the harmful effects of smoking on lung tissue, or at least
spare that and related tissues from the harmful effects and
disorders associated with smoking, including a reduction in the
number of macrophages present and a reduction of their activity, or
phagocytosis, increasing numbers of neutrophils and lymphocytes
present in lung tissue and the harmful secretions therefrom, and a
increase in the thickness of the blood-air barrier, and possibly
associated short windedness.
In the process of this invention, the source bovid includes any
milk-producing member of the genus Bos, preferably cows.
The invention is based on the discovery that when such bovid is
brought to a specific state of immunization by means of periodic
booster administrations of an antigen or a mixture of antigens, the
bovid will produce milk which has highly beneficial properties in
the treatment of vascular disorders. The beneficial milk properties
are not produced by all bovids that are immunized. The induction of
immune sensitivity alone is insufficient to cause the appearance of
the properties in milk, as is shown by the fact that normal cow's
milk does not contain these properties, even though cows have
become sensitized against various antigens during normal
immunization against cow diseases.
Furthermore, the properties are not always present in milk produced
by bovids maintained in the immune state by booster injections. It
is only in a specific hyperimmune state that the milk has the
desired effects. This special state is only achieved by
administering periodic boosters with sufficiently high doses of
antigens or mixtures of antigens. The preferred dose range should
be equal to or greater than 50% of the dosage necessary to cause
primary sensitization of the bovid. Thus, there is a booster dosage
threshold below which the properties are not produced in the milk
even though the cow may be in what is normally called an immune
state. In order to achieve the hyperimmune state it is essential to
test the bovid's milk after a first series of booster
administrations. If the milk does not contain the desired
properties, a second series of boosters of higher dosage has to be
administered. This process is repeated until the properties appear
in the milk.
In summary the process comprises the following steps:
1. Antigen selection.
2. Sensitization of the bovid by primary immunization.
3. Testing the serum of the bovid to confirm sensitivity
induction.
4. Administering boosters of appropriate dosage to induce and
maintain a hyperimmune state.
5. Testing anti aging or anti arteriosclerotic properties of
milk.
6. Collecting milk from the bovid during the hyperimmune state.
Step 1--Any antigens or combination of antigens may be employed.
The antigens can be bacterial, viral, cellular, or any other
substances to which the immune system of a bovid will respond. The
critical point in Step 1 is that the antigen must be capable of
inducing a state of immune sensitivity in the cow. The antigen can
be administered by any method which causes sensitization.
Preferably polyvalent antigens, such as bacteria, are used.
Step 2--The preferred method of immunization is by intramuscular
injection. However, other methods such as intravenous injection,
intraperineal injection, oral administration, rectal suppository,
etc., can be used, providing the dose is sufficient to induce
sensitivity. The dosage is normally 1.times.10.sup.6 cells to
1.times.10.sup.20 cells, preferably 10.sup.8 cells to 10.sup.10
cells, most preferably 2.times.10.sup.8 cells.
Step 3 is to determine whether or not the cow has become sensitive
to the antigen. There are a number of methods known to those
skilled in the art of immunology to test for sensitivity, (Methods
in Immunology and ImmunoChemistry, William, C. A., Chase, W. M.
Academic Press, N.Y., London (vols 1-5)(1977)). Examples of these
include skin sensitivity tests, serum tests for the presence of
antibodies to the stimulating antigens, and tests designed to
evaluate the ability of immune cells from the host to respond to
the antigen. The type of test employed will depend to a large
extent on the nature of the antigen used. The preferred method is
to use a polyvalent vaccine consisting of multiple bacterial
species as the antigen and to test for the presence of
agglutinating antibodies in the serum of the cow before and after
challenge with the vaccine. The appearance of milk antibodies after
immunization with the vaccine is indicative of sensitivity, and at
this point, it is possible to proceed to Step 4. The minimum dose
of antigen necessary to induce sensitivity depends on the type of
antigen used.
Step 4 involves the induction and maintenance of the hyperimmune
state. Once a bovid has been shown to be sensitized, this state is
induced by repeated booster administrations of an appropriate
dosage at fixed time intervals. The spacing of the administration
depends on the nature of the antigen. A two-week booster interval
is optimal for polyvalent bacterial antigens. Moreover, the booster
administrations must not induce a state of immune tolerance. This
will cause the animal to pass from a hyperimmune state to a state
of immune tolerance to the antigen in which case the animal will
cease to produce milk with the beneficial properties.
It might also be possible, for example, to use a combination of
different immunization procedures, i.e., intramuscular injection
for primary immunization and intravenous injection for booster
injections, etc. Many different combinations of immunization
methods might be employed by those skilled in the art to: (1)
sensitize and (2) induce the hyperimmune state.
Step 5 is to test the vascular and/or pulmonary disorder treatment
properties of the milk. A battery of research techniques can used
to test the effects of the hyperimmune milk on the vascular and
pulmonary systems of animals. Preferably, rabbit or rat tissue can
be used as the test tissue. These tests include in all cases
feeding a rabbit or rat a diet which comprises hyperimmune milk
(with a control comprising rabbits in a diet with normal milk.)
After a predetermined period of time, the animals are sacrificed
and their vascular or pulmonary system can be examined by any of
the following techniques: Scanning electron microscopy of the
endocardial surface of the heart or lungs bronchii and alocoli
searching for endothelial damage or the presence of debris,
respectively; transmission electron microscopy of vessels searching
for lipid droplets, endothelial degeneration, lipid presence in
foam cells, or tendency of fibrin or platelets to adhere to the
luminal surface of endothelial cells; histological analyses of
hearts searching for lipids, e.g. demonstration of lipids with
oil-soluble dyes such as Oil Red or Sudan Black in sections of
frozen tissue, or presence of enzymes, especially cytochrome
oxidase or of lung tissue for evidence of phagocytosis; as well as
histological examination of lung tissue for neutraphil and
leukocyte infiltration.
Step 6 involves collection and processing of the milk. The milk can
be collected by conventional methods; however, special processing
is necessary to protect the beneficial properties of the milk. The
beneficial properties of the milk are heat sensitive. Accordingly,
low temperature pasteurization is preferred. The pasteurization
temperature should not exceed 160.degree. F. for 15 seconds.
Following pasteurization, the fat is removed by standard procedures
and the milk is spray dried. Conventional spray-drying procedures
are used, with the exception that the milk is concentrated under
vacuum at low temperature so as not to destroy the beneficial
properties. (See e.g. Kosikowski, F., "Cheese and Fermented Milk
Products," 2nd Ed, 1977). The final product is a milk powder which
has beneficial properties.
Fluid milk can also be used, as well as concentrated milk products
or a fraction of the milk containing the biologically active factor
or factors such as the acid whey fraction.
The invention is based in part on the discovery that the milk from
a hyperimmune bovid has beneficial properties on the cardiovascular
system. For example, it has been discovered that in hearts of
female rabbits which have been fed a steady diet of hyperimmune
milk, the endothelial cells of the heart were protected against
extensive endothelial damage of varying extent and severity
observed in rabbits fed normal milk. In the latter rabbits, craters
or holes were present where one or more cells had degenerated and
detached, whereas in hyperimmune milk fed rabbits these were not
present. Transmission electron microscopy of both populations of
rabbits showed major differences in the blood vessels. Significant
pathological features of blood vessels in control hearts included
large lipid droplets, endothelial degeneration, multiple small
lipid vacuoles, single or multiple large lipid droplets filling the
cytoplasm of endothelial cells, foam cells latent with lipid, and a
strong tendency of fibrin platelets to adhere to the luminal
surface of endothelial cells. All of the aforementioned
derangements accompany the pathogenesis of atherosclerosis. These
derangements were not found in blood vessels from representative
areas of rabbit populations which were on a steady diet of
hyperimmune milk. Histological sections of hearts from the rabbits
fed the hyperimmune milk and of rabbits fed control milk showed
that lipid was present in the lumina of some blood vessels of
control hearts and cardiac muscle fibers of control hearts were
filled with lipid. Coronary blood vessels from rabbits on
hyperimmune milk lacked the atherosclerotic lipid deposits which
are observed in control vessels.
These results show conclusively that hyperimmune milk slows and/or
represses the pathogeneses of arteriosclerosis and aging of the
heart.
The same tests of rabbit populations, as well as clinical trials
with human subjects, has conclusively demonstrated that diets
incorporating the hyperimmune milk of this invention results in a
reduction of serum cholesterol levels, as well as marked reductions
in triglyceride levels and low density lipid levels, all of which
are key risk factors associated with cardiovascular disease.
The invention is also based, in part, on the discovery that the
milk from a hyperimmune bovid has beneficial properties on the
pulmonary system of animals exposed to smoke, and particularly a
sparing effect on the lung tissue thereof. For example, it has been
discovered that in the lungs of rats which have been fed a steady
diet of hyperimmune milk and exposed to cigarette smoke, the number
of neutrophils and lymphocytes infiltrating the lungs upon exposure
to smoke are substantially reduced as compared with control groups.
Also, the surface of the alveoli is "cleaner" in rats receiving the
hyperimmune milk and exposed to smoke then rats exposed to smoke
only or smoke plus control or normal milk. The hyperimmune milk
appeared to increase the activity of the dust cells or lung
macrophages. The dust cells in the bronchioles and alveoli of rats
fed a diet of hyperimmune milk were more active in the phagocytosis
of smoke-associated debris in lungs of rats exposed to smoke than
of those rats exposed to smoke only or smoke plus a diet of
non-hyperimmune milk. The blood-air barrier was also observed to be
thinner in rats exposed to smoke and fed a diet of hyperimmune milk
than rats exposed to smoke only or rats exposed to smoke and fed
normal or controlled milk.
In addition to cardiovascular tissue, 16 other tissues representing
one or more components of each major organ system have also been
studied from the same animals. Hyperimmune milk has no adverse
effects on any of these tissues.
The milk of the invention can be provided in any amount which
effects or maintains the reversal of vascular disorders or smoking
associated pulmonary disorders in warm-blooded animals. Daily
amounts of 1 ml to 10 liters based on fluid milk can be provided,
depending on the particular circumstance of the disorder and the
animal species.
The same amounts can be utilized in normal subjects when operating
in a preventive mode.
In a preferred mode the milk is administered periodically (e.g.
daily in dosages of approximately 40 g) for a prolonged period of
time such as at least 15-30 days and more, up to several years. As
will be recognized, the treatment duration can be shortened by
increasing daily dosages. Additionally, smaller dosages spread out
throughout the day may be more effective than a single dosage taken
once a day.
The fat-free milk can be incorporated into any food product, as
long as the food product is not treated at a temperature which is
too elevated and would inactivate the vascular treatment properties
of the product. A temperature lower than 150.degree. C. is
preferred. For example puddings or yogurt may be prepared with
hyperimmune milk.
Further, it has been found that the whey fraction contains the
agent or agents responsible for the beneficial properties observed
and referred to above. This acid whey fraction may also be added to
syrups, ice-cream mixes, candy, beverages, cattle feeds or the
like. (See Kosikowski, Supra, p 446). Those of ordinary skill in
the art, knowing that the whey fraction contains the factors of
importance, would clearly recognize that further separation can be
made to obtain more potent fractions.
Further, where alleviating disorders associated with smoking, the
milk may be incorporated in the substance being smoked, such as by
spraying tobacco with an aerosol form of the milk.
Having now generally described this invention, the same will be
further described by reference to certain specific examples which
are provided herein for purposes of illustration only and are not
intended to be limiting unless otherwise specified.
PREPARATION OF MILKS
Example 1
Five Holstein cows were immunized against Escherichia coli
(American Type Culture Strain No. 13076). The primary immunization
was accomplished by intramuscular injection of a vaccine containing
heat-killed E. coli cells suspended in physiological saline. The
concentration of bacterial cells was 4.times.10.sup.8 cc. A dose of
5 cc (20.times.10.sup.8) cells was injected i.m. once weekly for
four consecutive weeks. Milk collected during the fifth week was
tested for the presence of antibodies against E. coli. The presence
of antibody against E. coli was determined using a
micro-agglutination procedure. This procedure involves reacting
different dilutions of milk whey with a fixed concentration of E.
coli bacterial cells suspended in buffer. The presence of
antibodies in the milk causes agglutination of the bacterial cells.
The milk is diluted in a serial fashion and there comes a point
when the concentration of antibodies is too low to cause the
agglutination reaction. The maximum dilution which causes
agglutination is the antibody titer. The presence of high antibody
concentration in milk is an indication that the immunization
procedure causes sensitization of the cow's immune system against
the antigen. Table 1 compares the antibody titer against E. coli in
the five cows before and after primary immunization.
TABLE 1 ______________________________________ Milk Anitbody Titer
in 5 Cows Before and After Immunization Against E. coli Cow No.
Before Immunization After Immunization
______________________________________ 1 0 640 2 0 1,280 3 0 5,000
4 0 1,280 5 0 10,000 ______________________________________
In each case there was a significant increase in the milk titer
against E. coli following immunization. From this it was concluded
that the immunization caused sensitization of the cow against the
E. coli. Having induced a state of sensitivity, the cows were given
booster injections of the same dose of antigen every 14 days, thus
establishing and maintaining a period of hyperimmune state during
which time the milk was collected daily and processed to obtain
skimmed powdered milk.
The skimmed powdered milk induced by the method outlined above was
treated for beneficial properties using a number of tests,
infra.
Example 2
The identical experiment as described in Example 1 was undertaken
with the exception that a polyvalent vaccine comprised of the
bacterial strains listed below in Table 2 was used as the selected
antigen. The different bacterial strains were combined by mixing
equal weights of the lyophilized bacterial cells and diluting the
mixture in saline to obtain a concentration identical to that used
in Example 1. Results of the tests on milk produced using these
selected antigens were positive, infra.
TABLE 2 ______________________________________ Bacterial Antigens
Organism ATTC No. ______________________________________
Staphylococcus aureus 11.631 Staphylococcus epidermidis 155 5
Streptococcus pyrogenes. A. Type 1 8,671 Streptococcus pyrogenes.
A. Type 3 10,389 Streptococcus pyrogenes. A. Type 5 12,347
Streptococcus pyrogenes. A. Type 8 12,389 Streptococcus pyrogenes.
A. Type 12 11,434 Streptococcus pyrogenes. A. Type 14 12,972 10
Streptococcus pyrogenes. A. Type 18 12,357 Streptococcus pyrogenes.
A. Type 22 10,403 Aerobacter aerogenes 884 Escherichia coli 26
Salmonella enteritidis 13,076 Pseudomonas aeruginosa 7,700 15
Klesbiella pneumoniae 9,590 Salmonella typhimurium 13,311
Haemophilus influenzae 9,333 Streptococcus viridans 6,249 Proteus
vulgaris 13,315 Shigella dysenteriae 11,835 20 Streptococcus Group
B Diplococcus pneumoniae Streptococcus mutans Corynebacterium Acne,
Types 1 and 2 ______________________________________ *American Type
Culture Collection 12301 Parklawn Drive Rockville, Marylan
20652
It should be noted here that the milk produced in this Example is
identical to that produced in a preferred embodiment of the
invention disclosed in copending U.S. application Ser. No. 875,140,
filed Feb. 6, 1978 by Beck and in U.S. Pat. No. 4,284,623. The
application discloses that the milk of the present Example also has
highly beneficial anti-arthritic properties, and the patent
discloses that the milk has anti-inflammatory properties.
Example 3
A strain of Streptococcus mutans was cultured in accordance with
established techniques. Cultures of S. mutans AHT (serological a),
BHT (group b), 10449 (group c) and 6715 (group d) were grown in
dialyzed tryptose medium. The cells were harvested by
centrifugation at 4000.times.G and washed five times with 0.1 M
phosphate buffered saline, pH 7.0. The cells were inactivated by
heating at 60.degree. C. for 30 minutes and resuspended to a final
concentration of S. mutans AHT, BHT, 10449 and 6715 at
5.times.10.sup.8 cells/ml. This preparation was used to immunize
two cows. Each cow was immunized on two separate occasions with
fresh antigen from all four groups of S. mutans (groups a, b, c and
d). A cow was then immunized in accordance with the established
techniques of this invention to generate a milk product. Following
immunization, blood samples of the cow were taken until the serum
anribody titer reached its highest level, then the milk was
collected. The milk itself was then dried and powdered, again in
accordance with established techniques, to produce a powdered milk
having positive beneficial effects.
It should be noted here that the milk produced in this Example is
identical to the caries-inhibiting milk disclosed by Beck in B.P.
No. 1,505,513.
Example 4
The polyvalent vaccine shown below in Table 3 was utilized as in
Example 2 to immunize a cow and obtain hyperimmune milk with
cardiovascular or pulmonary effects.
TABLE 3 ______________________________________ Bacteria ATTC No.
______________________________________ 1. Pseudomonas aeruginosa
14212 2. Pseudomonas maltophiia 17666 3. Streptococcus equismili
9542 4. Streptococcus dysgalactiae 27957 5. Streptococcus uberis
27958 6. Streptococcus bovis 15351 7. Streptococcus bovis 27960 8.
Pasteurella multocida 9659 9. Pasteurella multocida 6533 10.
Pasteurella haemolytica 14003 11. Pasteurella multocida 15743 12.
Moraxella bovis 10900 13. Actinobacillus ligineresi 13372 14.
Corynebacterium renale 10848 15. Fusobacterium necrophorum 25286
16. Bacillus cereus 25621 17. Salmonella dublin 15480 18.
Salmonella heidleberg 8326 19. Salmonella paratyphi 11511 20.
Yersinia enterocolitica 9610
______________________________________
BIOLOGICAL STUDIES
A. Cardiovascular Effects
Materials and Methods
1. Animals
Nine, young adult female rabbits of the New Zealand White strain
were used for this study. Rabbits were housed in the College of
Medicine's Vivarium at the University of South Alabama in Mobile.
The room in which the rabbits were kept was maintained at
72.degree. F. with 12 hours of light followed by 12 hours of
darkness during each 24 hr. period. Only one rabbit was housed in
each cage.
The rabbits were fed the diets (below) and sacrificed during the
second and third weeks after end of the diets.
2. Diets
The rabbits were divided into groups. Both groups received a
balanced laboratory chow ad libitum. Control rabbits in Group I
(N=4) received Jerrell's nonfat dry milk dissolved in tap water (15
g dry milk/500 ml water). Experimental rabbits in Group II (N=5)
received the same concentration of dry hyperimmune milk from either
Examples 1, 2, 3 or 4 which was also dissolved in tap water. The
water-milk mixture was changed daily. The fresh mixture was placed
in sterilized glass or plastic bottles. Rabbits received no liquid
other than water used to dissolve the dry milk.
3. Methods of Study
After 3 months of drinking the dissolved, non-fat dry milk (Group
I) or the hyperimmune milk rabbits were weighed and anaesthetized
by injecting sodium pentabarbital into an ear vein. The body weight
of each rabbit at the time of sacrifice was compared to its body
weight on the first day of the diet. There was no significant
difference between the body weights of rabbits receiving
hyperimmune milk and those receiving the non-fat dry milk. Hearts
and the great vessels at the base of the heart were removed and
prepared for study by several different procedures. Specimens were
coded so that the investigator was unaware whether the material was
control or experimental.
Results and Comments
1. Scanning Electron Microscopy
For SEM, the inner (endocardial) surface of the heart was scanned
using variable magnifications (X10-40,000). Areas of specific
interest (minimum of 5 randomly selected areas/heart) were then
studied and photographed.
Control hearts, in contrast to experimental hearts, showed
extensive endothelial damage that varied in extent and severity.
Craters or holes were present where one or more cells had
degenerated and detached. Small holes were present in many cells.
In some instances, a fibrin-like substance mixed with blood cells
and platelets was around or within the craters. Such sites were
interpreted as stages in the formation of atheromatous thrombi.
Loss of endothelial cells as evident in the control hearts would
expose blood in the heart chambers to endocardial connective tissue
and lead to the formation of thrombi. SEM showed that hyperimmune
milk protected rabbit endothelial cells and prevented
thrombogenesis.
2. Transmission Electron Microscopy
TEM showed major differences in the blood vessels of control and
experimental hearts. Significant pathological features of blood
vessels in control hearts included: (1) large lipid droplets that
sometimes occluded vascular lumina; (2) endothelial degeneration;
(3) multiple, small lipid vacuoles in endothelial cells; (4) single
or multiple large lipid droplets that almost filled the cytoplasm
of endothelial cells; (5) foam cells laden with lipid; (6) strong
tendency of fibrin and platelets to adhere to the luminal surface
of endothelial cells.
All of the aforementioned derrangements accompany the pathogenesis
of atherosclerosis. Coronary, renal, and cerebral arteries are
known to be especially susceptible to atherosclerosis. There is
focal involvement of the blood vessels, rather than total
involvement throughout their length. The disease was not found in
blood vessels from representative areas of the hearts of rabbits
who drank immune milk.
3. Histology
Portions of hearts from all control rabbits were divided; 1 part
was embedded in paraffin, and 1 part in plastic. Sections of
plastic specimens cut at a thickness of 1 micrometer and stained
with toluidine blue or paragon clearly showed two differences
between control and experimental hearts: (1) lipid was present in
the lumina of some blood vessels of control hearts, and (2) some
cardiac muscle fibers of control hearts were filled with lipid.
Hearts from rabbits who drank immune milk did not manifest these
features.
4. Demonstration of Lipids
Lipids were localized by two standard methods, Oil Red O and Sudan
Black B. Both methods selectively demonstrate lipids in sections of
frozen tissue.
Each method showed lipid deposits in the tunica intima and tunica
media of blood vessels in control hearts. These two tunics of
control aortas were particularly heavily infiltrated with lipid.
Tunica intimas of blood vessels in control rabbits contained
thickenings associated with lipid that were typical of
atherosclerosis. Coronary blood vessels from rabbits on hyperimmune
milk, however, lacked atherosclerotic lipid deposits.
5. Enzymes
Activities of the following enzymes were studied and evaluated in
frozen sections of control and experimental rabbit hearts: (1) acid
phosphatase, (2) alkaline phosphatase, (3) isocitrate
dehydrogenase, (4) succinate dehydrogenase, and (5) cytochrome
oxidase. The activity of acid phosphatase was also determined
biochemically.
Of the several enzymes whose activities were evaluated, only
cryochrome oxidase (an enzyme associated with aging) showed
significantly greater activity in control hearts than in
experimental hearts. It is noteworthy that control hearts contained
foci of cardiac muscle fibers, measuring about 1 mm in diameter,
that stained intensely. Such areas were evidently very rich in
mitochondria and may represent focal areas of aging.
6. Blood Stream Levels
Analysis of the serum cholesterol levels in the rabbits of these
studies revealed a marked decrease in serum cholesterol levels.
Those control rabbits fed a die without hyperimmune milk
demonstrate an average serum cholesterol level of 4,450 mg/dl. In
contrast, the rabbits fed a diet of hyperimmune milk exhibited a
serum cholesterol level average less than half of that figure,
2,167 mg/dl. This is believed the mechanism whereby the
physiological improvements discussed above are affected.
7. Clinical Studies
Clinical studies have now been initiated for three human subjects,
to determine the value of the hyperimmune milk of this invention in
the reduction of plasma lipid values. Although by no means
complete, these studies clearly indicate the value of the
hyperimmune milk diet in reducing lipid values. Although not tested
against controls for obvious reasons, beginning lipid values have
been markedly reduced for all three subjects. The exact lipid
values are reflected in Table 4.
B. Pulmonary Effects
Pulmonary effects studies similar to those related above with
regard to cardiovascular effects were performed on rats to
determine the effect of a diet of hyperimmune milk on mammals
exposed to cigarette smoke. The diets and method of study were
substantially similar to those related above with regard to the
studies on cardiovascular effects, except the subject animals were
female albino rats, of the Charles Rivers, CD strain. A total of 68
rats were involved in the studies.
TABLE 4 ______________________________________ Plasma Lipid Values
For Human Subjects TRIG CHOL LDL
______________________________________ SUBJECT 1F Beginning 88 170
107 6 wk 1M 61 140 83 SUBJECT 2F Beginning 71 173 114 6 wk 1M 69
156 97 12 wk 1M 87 150 88 SUBJECT 3M Beginning 675 269 -- 6 wk 1M
190 264 181 12 wk 1M 210 265 178
______________________________________ Values = mg/dl
RESULTS AND COMMENTS
Through histological study, it was determined the the lungs of
control rats not exposed to smoke and the lungs of rats on a
hyperimmune milk diet and exposed to smoke contained far fewer
neutrophils than the lungs of rats exposed to smoke or to smoke and
fed a diet of normal or control milk. Lymphocytes were also fewer
in number in the lungs of the control animals, and even further
reduced in the bronchial-associated lymphoid tissue (BALT) in the
lungs of rats that were fed a diet of hyperimmune milk and exposed
to smoke.
Scanning electron microscopy showed the surface of the alveoli of
rats exposed to smoke and fed a diet of hyperimmune milk to be
markedly" cleaner than those of rats exposed to smoke or exposed to
smoke and fed a diet of normal or control milk. The hyperimmune
milk diet appeared to increase the activity of the dust cells, or
lung macrophages. The dust cells in bronchii, bronchioles and
alveoli were far more active in phagocytosis in the lungs of rats
exposed to the smoke and fed a diet of hyperimmune milk than after
the other experimental conditions. Of the rats exposed to smoke,
only those fed a hyperimmune diet exhibited dust cells consistently
on the surfaces of Type 1 alveolar cells in the gas exchange
passages and on the epithelial cells of the air conducting
passages. Pseudopodia of dust cells engulfed products of the
epithelial cells and other debris that was present. Thus, the
physiological "patentcy" of the air-conducting and respiratory
portions of the lung was maintained in rats fed hyperimmune milk,
even in the presence of excessive secretion and debris associated
with cigarette smoking.
The blood-air barrier was observed to be thicker in the lungs of
rats exposed to smoke with or without normal or control milk than
in rats exposed to smoke and fed a diet of hyperimmune milk. This
phenomenon is believed to be closely associated with the
short-windedness often observed in conjunction with cigarette
smoking.
Having now generally described this invention it will become
readily apparent to one skilled in the art that many changes and
modifications can be made thereto without affecting the spirit or
scope thereof.
* * * * *