WO1994026114A1

WO1994026114A1 - Method of stimulating and modulating the immune system of animals with microbial compositions

Info

Publication number: WO1994026114A1
Application number: PCT/US1994/005414
Authority: WO
Inventors: William E. Marshall
Original assignee: Immunom Technologies Inc
Current assignee: Immunom Technologies Inc
Priority date: 1993-05-11
Filing date: 1994-05-11
Publication date: 1994-11-24
Anticipated expiration: 1995-11-11

Abstract

The invention comprises method and compositions for stimulating the immune system of an animal by administering an apathogenic bacteria selected from the family Lactobacilliaceae in a ratio of between about 500:1 and 1:500 bacteria to macrophage, the macrophage being present in the oropharyngeal-alveolar cavity of the animal, in an acceptable carrier effective to deliver said bacteria to said oropharyngeal-alveolar cavity of the animal. The methods and compositions are particularly useful in modulating the responses of macrophages in animals to E. coli and other pathogens to reduce the risk of septic shock upon exposure to such pathogens.

Description

METHOD OF STIMULATING AND MODULATING THE IMMUNE SYSTEM OF ANIMALS WITH MICROBIAL COMPOSITIONS

FIELD OF THE INVENTION

This invention relates to a method of stimulating the immune system of an animal or human by administering effective amounts of apathogenic bacteria effective to prevent or reduce

infections. More particularly, this invention relates to a method of stimulating oropharyngeal-alveolar macrophages in animals or humans by administering effective amounts of

apathogenic bacteria selected from the family of Lactobacillaceae to prevent or reduce infections.

BACKGROUND OF THE INVENTION

Macrophages, sometimes called "sentry cells," are large phagocytic cells of. tϋe reticuloendothelial system which engulf and digest or phagocytize, cells, microorganisms or other foreign materials in the bloodstream and tissues of humans and animals. As used hereafter "animal" refers to humans and animals, and includes mammals, birds and fish.

Macrophages reside in tissues where infections can originate such as in the oral, pharyngeal and alveolar cavities, the gastrointestinal tract, urethra, vagina, etc. It is believed that during their evolution and exposure to natural immunogens, macrophages begin producing cytokine-based signals that report the nature of the immunogen to the associated immune system, as well as certain organ tissue cells. sampling by macrophages of microbes, pathogens and other foreign materials includes

adsorption, ingestion and display of components of the foreign materials on the macrophage's surface in conjunction with the major histocompatibility complex (MHC) that identifies the macrophages to other cells.

Thus in their sentry role, macrophages survey the host's internal and external environments, and through the release of approximately 75 different molecules, are capable of both

defending the host as well as signalling to other immunocytes the nature of the detected immunogen, infectious agent or foreign material. Furthermore, they present the immunogen or foreign material to thymocytes, (T-cells) for appropriate responses.

T-cells, in turn, signal their response as an alert to additional cells of the immune system.

Circulating monocytes are found within the peripheral blood and lymph. These cells phagocytize aberrant molecules

synthesized by the host or tissue fragments resulting from wounds or tissue breakdown. Certain tissues also have associated mature macrophages exercising the same roles.

Interleukin-1 (IL-1) is a hormonal peptide released by animal macrophages as an alert signal to T-cells and other immunocytes as well as tissues of the host. The synthesis of IL-1 is induced when foreign materials are phagocytized by a

macrophage. IL-1 and other cytokines released by the macrophage (e.g. IL-6, tumor necrosis factor ("TNF") and others) are thought to present a profile regarding the nature of the invader which has been impressed over time into the memories of the immunocytes receiving the signals.

The outer membranes of Gram-negative bacteria contain lipopolysaccharide (LPS or endotoxin), a large molecule

consisting of three covalently linked parts: lipid A,

polysaccharide core and O-antigens. The structures of lipid A and the polysaccharide core are well-conserved between and among Gram-negative bacterial genera and species. The O-antigen region differs markedly between species and can be changed by mutation at a high frequency within the same species, a condition called "phase variation".

When a macrophage is exposed to LPS or just to the lipid A component of LPS, it releases cytokines including IL-1 in an as yet unidentified profile. This profile can lead to an

over-reaction by host cells leading to many pathophysiological effects, such as pulmonary hypertension, inflammation and fever. When high levels of IL-1 are injected into a host. animal, proinflammatory effects are observed followed by acute phase inflammation marked by molecular changes in many tissues. A state of shock, remarkably similar to septic shock, ensues followed by death. However, if monoclonal antibodies against IL-1 are injected with IL-1, the toxic response can be averted. The sentry-role of the macrophages requires them to possess a rapid detection and alert system. Cytokines, polypeptides synthesized and released by macrophages, are responsible for cell-to-cell communications to other member cells of the host, as well as its immune system. The macrophage is capable of sending an "acute phase" signal after detecting life-threatening levels of pathogens, LPS and other microbial components. This acute phase signal is believed to activate thymocytes and other cells through the multiplicity of interleukins which it can release, e.g. IL-1, IL-6, TNF, etc.

U.S. Patent No. 4,975,467 teaches methods by which synthetic compounds can be used to inhibit the release of IL-1 and thereby alleviate its mediated pathophysiological conditions. U.S.

Patents Nos. 4,849,506 and 5,082,657 illustrate the importance of cytokines in communicating between cells as shown by the use of leukoregulin to regulate tumor growth. U.S. Patent No. 5,055,447 provides methods and compositions for the treatment or

prophylaxis of septic shock by using transforming growth

factor-beta. That patent illustrates the complexity of the cytokine signals for thymocyte mitogenesis and teaches the use of a complex mixture of cytokines and other pharmacologically active compounds for control of shock.

U.S. Patents Nos. 5,041,427 and 5,158,939 teach the use of naturally occurring non-toxic LPS from R. spaeroides , ATCC 17023, to protect animals from toxic LPS. U.S. Patent No. 5,157,039 supports the clinical need for controlling IL-1 release by macrophages by teaching the use of 2-quinolinyl methoxy compounds to inhibit a family of

interleukins. Separately, U.S. Patent No. 5,082,838 teaches the use of sulfur-containing fused pyrimidine derivatives to inhibit IL-1 release to aid in the prevention of inflammation resulting from bactremic infections.

In U.S. Patent No. 5,151,498, a glycoprotein from oats is taught to induce the release of IL-1. However, such plant isolates are expensive to produce and often can be the cause of an undesirable allergenic response.

The acute-phase response of a macrophage to LPS or its constituent, lipid A, is believed to represent a response to an unbalanced stimulus rather than to an entire microbe. This unbalanced stimulus. is thought to result in an unbalanced cytokine signal which ultimately results in a pathophysiological state: septic shock marked by fever, inflammation and typically death of the host. In this way, the macrophage itself can be rendered unbalanced giving enhanced toxicity to Gram-negative pathogens, such as E. coli and S. typhimurium. Therefore, it is important to maintain the condition of the macrophage and not subject it to components of microbes but rather to the entire organism.

To further illustrate the importance of the cytokine mix of signals delivered to other members of an animal immune system. the intravenous administration of IL-1 to test animals in advance of a toxic dose of a pathogen or LPS has, under certain

conditions provided for the prophylaxis of septic shock. This indicates the multifunctional role that cytokines and the activated macrophage can perform depending on the nature of the activation. The level of present technology is insufficient to sort out the various possible profiles of released cytokines and their corresponding messages. However, it would be advantageous examination of an activated macrophage may give an indication that different responses are invoked. When exposed to E coli , at ratios of 1:1, the macrophage enlarges. Such is also the response to LPS when administered at nanogram levels

approximating an E coli to macrophage ratio of 100:1.

It is theorized that balanced activation of macrophages leads to the synthesis and release of IL-1 and other cytokines in ratios proper to effect a T- and B-cell stimulus that would restore homeostasis to the immune system rather than to initiate a pathophysiologic state. As discussed above, unbalanced activation can lead to the release of cytokines in ratios that initiate a pathophysiological response. For example, it has been shown by Hack, et al., Infect. & Immun. 60: 2835 - 2842, (1992), that circulating levels of IL-8, possibly of macrophage origin, correlate with important clinical parameters associated with septic shock. It has further been demonstrated by Dibb, et al., Infect. & Immun. 60: 3052 - 3058, (1992), that the release of IL-8 from macrophages is induced by LPS. In addition, tumor necrosis factor (TNF) and IL-6 have been implicated as mediators of inflammation resulting from a C. albicans infection in mice, (Steinhaus, et al., Infect. & Immun. 60: 4003-4008, 1992).

Therefore, the impact on a host of macrophage-released cytokines must be considered in light of the activating agent. The use of isolated cell components, e.g. LPS, lipoteichoic acids, muramyl dipeptide, porins and peptidoglycan fragments, leads to an unbalanced* cytokine signal. As reported by Tuomanen, et al., Infect. Pis. 151: 859-868, (1985), meningeal inflammation has been induced by injecting only components of the pneumococcal cell wall. Later, this group showed that these cell wall components stimulated the release of certain cytokines from the macrophage, but not others, (Riesenfeld-Orn, et al., Infect. & Immun. 57: 1890-1893, 1989).

Cytokine production has been induced by exposing macrophages to cell components of a number of Gram-positive pathogens and non-pathogens as well, (Bhakdi, et al., Infec. & Immun. 59:

4614-4620, 1991 and Gold, et al., Infect. & Immun. 49: 731-741, 1985).

Indeed, even when disintegrated cell wall components from approximately 1 × 10 CFUs of L. caseii , a known non-pathogen, were injected into the peritoneal cavity of genetically

predisposed 7 week-old mice, hind limb arthritis was observed, (Archer, et al., U.K. Biochem. Soc. Trans. 19: 404S, 1991). This bacterium has been in the human food system for thousands of years without being impugned as a pathogen. The injection of sonicated cell wall particulate matter only serves to underscore the point that intact whole organisms are the preferred

embodiment of a microbial prophylaxis.

Cytokines are multifunctional with at least dual roles in infections. Certain levels are correlated with the fatal outcome of septicemia in humans, while others indicate their role as mediators of septic shock.. In addition the macrophage response can differ among a number of biochemical pathways, (Kantengwa, et al.. Infect. & Immun. 61: 1281-1287, 1993). Selective profiles of cytokine production has been shown in human urinary tracts infected with E. coli, (Agace, et al., Infect. & Immun. 61:

602-609, 1993).

Therefore, it would be advantageous to provide methods of proper activation of macrophages by using intact, whole

apathogenic microorganisms at optimum levels, administered orally for the purposes of encountering antigen-sampling macrophages in the oropharyngeal cavity. Such methods would optimally enable the macrophage to remain functional in its response to pathogens.

Methods therefore useful in the balanced activation of an impaired immune system and or in the modulation of the macrophage response to pathogens would be of utility, not only in the maintenance of human and animal health, but also in decreasing salmonellosis in commercial poultry production, reducing reliance on sub-therapeutic doses of antibiotics in animal feeds, and maintaining human and animal cell-line cultures, as well as other uses.

U.S. Pat. No. 4,347,240 teaches the use of selected strains of L. caseii , YIT 9018, for treating and/or preventing tumor growth. However, the daily effective doses were ideally 1 to 2 g/kg for oral administration. This amounts to 1.5 × 10 CFUs per day for a 180 lb. person. Besides being uneconomical, this is not a physiological dose and is toxic to the oropharyngeal macrophages as well as to tumor cells. An accompanying

publication by the inventors indicated that the results were based entirely on injected L . caseii , rather than ingested. As such, its function would be difficult to control, (Kato, et al., Microbiol. Immunol. 27: 611-618, 1983).

U.S. Pat. No. 3,953,609 teaches the use of a strain of L. lactis , NRRL B-5628, to restrict the growth of undesirable bacteria in the mouth or crops of animals and fowl. The amounts claimed are more than 1 × 10¹⁰ CFUs per day per kg of body weight and for an extensive period of time. Again, these levels are unaffordable and, in addition, are believed to be dysfunctional to the host's macrophage population.

U.S. Pat. No. 4,314,995 discloses the local treatment of patients suffering from a bacterial infection such as

appendicitis, sinusitis or hemorrhoids with a special strain of antibiotic-producing Lactobacillus distinguished by its ability to grow in a culture that does not sustain the growth of ordinary

Lactobacilli . The levels of recommended use were 20 g of dried

Lactobacillus as a drip twice daily for 21 consecutive days.

This calculates to 5 × 10¹² CFUs per day or 1 × 10¹⁴ over the

course of treatment. Cost and the risk of dysfunctionality would be believed to greatly reduce the utility of this treatment.

To reduce noxious odors and substances as well as to control intestinal microflora, U.S. Pat. No. 4,579,734 teaches the use of pure cultures of L. clearans, L. sulfurica and L . nitrosus in

yogurt at a level of 4 × 10¹¹ twice daily per person. Changing

the bacterial composition of a baby pig's gut is the object of

U.S. Pat. No. 3,984,575 which teaches the feeding of L. lactis,

NRRL-B-5628, at levels of 7.5 × 10¹¹ CFUs per day for up to 6

months. Again, these levels are believed to be too high to

provide normal functionality of the oropharyngeal macrophages.

Also, neither of these references teach the modulation of

macrophages of the oropharyngeal-alveolar cavity.

U.S. Pat. No. 3,320,130 teaches the economic value of

feeding 10 CFUs of a mixed culture containing L . acidophilus or

Bifidobacterium bifidus and antibiotic resistant, non-pathogenic

E. coli grown on purified pig mucin for the control of colitis

and associated gastro-intestinal diseases. As stated above,

these levels are believed to be economically unsound for a

livestock raiser, and such levels are apt to impair the animals

antigen-sampling-cells of the mucosa-associated-lymphatic-tissue (MALT). It is also known in the prior art to use bacteria belonging to species and genera of the family Lactobacillaceae in the treatment of gastrointestinal disorders or as topical

therapeutics to control infections. However, the prior art is based on the theory of competitive exclusion wherein one

bacterium (an apathogen) occupies an environmental niche at the expense of another (a pathogen), and further teaches the use of much higher levels of bacteria than the present invention. In addition, the prior art does not address the use of special product formulations to deliver specific bacterial amounts directly to macrophage. Also, the prior art does not teach the use of these bacteria to activate and modulate the macrophages of the oropharyngeal cavity in a product form targeted to cells of that region.

Mitsuoka, J. Indus. Micro. 6: 263-268, 1990, has published a review of Bifidobacterium and their role in human health stating that these bacteria aid in the stimulation of the immune system when incorporated in food. Although no optimal ranges were presented, the referenced papers all recommend high levels (i.e. in excess of 1 × 10 CFUs daily) taken as foods. These levels and mode of administration are markedly different from those of my invention.

Members of the family Lactobacillaceae have been used in several human and animal studies indicating that feeding

protected the hosts from infections and enhanced their immune systems. L . bulgaricus and S . thermophilus at 2.4 × 10¹¹ CFUs were fed daily to mice for 29 days before challenge with S .

typhimurium. The resistance to infection was interpreted as evidence for the successful competitive exclusion of

S. typhimurium from niches in the gut, (DeSimone, et al . ,

Immunophar. & Immunotox. 10: 399-415, 1988, ibid, 14: 331-340,

1992).

A series of experiments by Perdigon yielded similar results. Mice fed 2.4 × 10⁹ CFUs per day of a mixture of L. acidophilus and caseii for 8 days demonstrated increased resistance to infection by S. typhimurium. The lack of colonization by S.

typhimurium was attributed to the precolonization of these sites by the Lactobacilli . However, mice fed the individual strains showed an increased susceptibility to infection, (Perdigon, et al., J. Food Protection 57: 255-264. 1990, ibid, 53: 404-410, 1990, Immunology 63: 17-23, 1988, J. Food Protection 49: 986-989, 1986). Peritoneal macrophages were shown to be activated by feeding 1 × 10⁹ CFUs. of L. caseii and L. bulgaricus daily for 8 days to young mice as determined by increases in their enzymic activity and their ability to phagocytize colloidal carbon, (Perdigon, et al., Infect. & Immun. 53: 404-410, 1986). In these experiments, no attempt was made to target the macrophage population of the oropharyngeal cavity with lower levels of bacteria, based on ratios to macrophages, and in the absence of food.

It is therefore an object of this invention to provide an effective method for maintaining the homeostasis of the immune system of animals by modulating the response of antigen-sampling macrophages of the oropharyngeal-alveolar cavity to pathogens. It is a further object of this invention to employ these methods for the activation or modulation of an immune system impaired by stress and disease. It is a further object to use levels of apathogens which interact with macrophages at physiological ratios typically found in the host's environment.

SUMMARY OF THE INVENTION

The invention comprises methods and compositions for stimulating an animal immune system by administering an

apathogenic bacteria selected from the family Lactobacilliaceae in a ratio of between about 500:1 and 1:500 bacteria to

macrophage, the macrophage being present in the oropharyngeal-alveolar cavity, in an acceptable carrier effective to deliver said bacteria to said oropharyngeal-alveolar cavity of the animal. In a preferred embodiment, the ratio of apathogenic bacteria delivered to the macrophage in the oropharyngeal-alveolar cavity is between about 100:1 and about 1:100. In a most preferred embodiment, the ratio of bacteria to macrophage is between about 10:1 and about 1:10. By "acceptable carrier," it is meant that the vehicle which delivers the apathogenic bacteria to the oropharyngeal-alveolar cavity will do so in a form effective to enable a macrophage-bacteria interaction. Such interaction optimally induces a proper cytokine release. Such acceptable carrier forms may comprise sprays, lozenges, gums, gels, and pastes. The invention is especially useful for modulation of the macrophages response to activation by a pathogen and reduces the risk of septic shock in an animal upon exposure to a pathogen.

DETAILED DESCRIPTION OF THE INVENTION

Macrophages from the lungs of mice (alveolar macrophages), from human peripheral blood (monocytes), and a cell line of human tumor origin (MonoMac 6), were used to study the interaction of apathogenic bacteria and macrophages.

First, these bacteria activate the quiescent macrophage as seen by microscopic observation (irregular shaping, clumping, granule formation), and the production of cytokines as measured by thymocyte mitogenesis. The optimum ratios for activation lie between about 500 bacterial cells to 1 macrophage cell to 1 bacterial cell to 500 macrophage cells.

Second, in the presence of E. coli or LPS, the type of activation was the same as when exposure was to the apathogens in the absence of E. coli or LPS. -The interaction of the

macrophage to E. coli or to LPS alone is marked by an enlargement of the macrophage typically referred to as an "angry" state. The presence of apathogens modulates the macrophage response to typical clumping, but not an angry state of enlargement.

Individual apathogenic bacteria modulate this response when present in the range of about 10 bacterial cells to about 1 macrophage cell to about 1 to about lb. The ratio of E. coli to macrophage was held at about 1:1.

Third, since monocultures seldom if ever exist in a host's environment, the interaction of mixed bacterial cultures and the macrophage was studied in the presence and absence of E. coli or LPS. Again, the apathogenic bacteria activated the macrophage and also modulated its response to E. coli or LPS as stated above. Optimum ratios of the mixed cultures to each other and to the macrophage were in the above stated ranges.

The method of the present invention comprises using certain apathogenic bacteria commensal to the host in proper levels to cause (1) . activation of the macrophages from a quiescent or impaired immune state and, (2) in the presence of these selected apathogens, modulation to normalcy the macrophage's response to a pathogen or components thereby ensuring that the macrophage ' s response will not lead to a pathological state of septic shock. The macrophages can be of animal, fowl or fish origin.

The criteria for the selection of the modulating/activating bacteria are as follows: known apathogenicity, possessing a stable genome, and containing an A4alpha interpeptide bridge. An A4alpha bridge describes the structure and linkage in the interpeptide bridge linking the two strands of peptidoglycan in the cell-wall or murein of Gram-positive bacteria. The A4alpha consists of L-lysine covalently bonded to D-aspartic acid and in turn, covalently bound to L-alanine of the next peptidoglycan chain. L-lysine occurs in position 3 of the peptide stem descending from the muramyl residue and D-aspartic or

D-isoasparagine bridges it to L-alanine of the next peptide stem of the adjacent peptidoglycan polymer (Fig. 9 in Schleifer, et al., Bacter. Rev. 36: 407, 1972). A review of the scientific literature (Bergey ' s Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, MD, 1986) indicates that these criteria are best met in the

Lactobacillaceae family of bacteria. A number of these bacteria are indigenous to the oropharyngeal-alveolar cavity of animal and fowl, including humans and poultry. In addition, some have been used as ingredients in the preservation and product formulations of milk-based foods, and as such are considered

generally-regarded-as-safe (GRAS) by the United States Food and Drug Administration. Microbial ecologists consider them to be commensals, i.e. microbes associated with healthy humans.

The Lactobacillaceae family consists of 7 genera:

Bifidobacterium, Diplococcus, Lactobacillus, Leuconostoc,

Pediococcus, Peptostreptococcus, Streptococcus and four

sub-genera of the genus Lactobacillus . Only the following four genera and sub-genera of this family fulfill the above criteria (i.e. known apathogenicity, possessing a stable genome, and containing an A4alpha interpeptide bridge) and are the specific subject matter of this invention:

Lactobacillaceae Bifidobacterium: indicum, coryne forme and eriksonii.

Lactobacillaceae Lactobacillus Thermobacterium: acidophilus, bulgaricus, helveticus, jugurti, lactis, lactis, salivarius, delbruckii, leichmannii, and jensenii . Lactobacillaceae Lactobacillus Streptobacterium: caseii and sub-species, sake, coryneformis, curvatus, xylosus, and zeae .

Lactobacillaceae Lactobacillus Betabacterium: fermentum.

Pediococcus: cerevisiae, acidolactici, and pentosaceus .

According to this invention the range of usefulness of these bacteria lies in their ratio to the macrophage. Very high doses are toxic, while very low doses do not provide for a modulated response or activation. Beneficial results are obtained within the ratio of bacteria to macrophage of about 500:1 to 1:500 and preferably 100:1 to 1:100. The optimum range is approximately from 50:1 to 1:50.

Ratios immediately outside of this range give uneven results. Delivering these ranges to receptive macrophages requires the formulation of products that can be held in the oropharyngeal-alveolar cavity rather than swallowed into the stomach and lower intestines. This necessitates the use of a medicament developed to insure that the active ingredients are held in the oropharyngeal cavity long enough to allow a

macrophage-bacteria interaction. Such formulations can be sprays, lozenges, gums, gels, pastes, etc. Of further

importance is to present these ratios and absolute numbers of CFUs to the host as compounded without dilution by food, beverage or saliva. They should not be taken as part of, or with or before meals. The ingestion of foods would alter the bacteria to macrophage ratios. The approximate population of antigen-sampling-macrophages in the oropharyngeal cavity of animal, human and fowl requires a formulated dose in the range of 1 × 10⁷ colony-forming-units (CFUs) and 10³ CFUs of bacteria per host.

If more than one species or strain of commensal bacteria is employed in the formulation, of further importance is to compound the bacteria in ratios to each other ranging from 10:1 to 1:10.

Although the bacteria can be administered as dead cells, the modulation is not as effective as when viable cells are used. If non-viable cells are employed, it is beneficial to maintain their surface antigens intact. This necessitates a "soft kill" of the bacteria by radiation, U.V. radiation techniques employing psoralens, mild heat, repeated freezing, etc. PREPARATION OF BACTERIA

Representative strains of the above-listed bacteria were obtained from national culture collections in lyophilized pure form and stored as directed on the labels.

Strains of the above listed bacteria were grown at 37°C overnight in a suitable medium - MRS broth. The cells were quickly chilled to 4°C and collected by centrifugation at 5000 g for 5 minutes and washed twice with cold isotonic saline (0.9% sodium chloride) and centrifugation. The pellet of bacteria was resuspended in cold saline and the Absorbance at 550 millimicrons adjusted to 0.75, 0.90 and 1.0 with saline. These A₅₅₀ approximate CFUs of 10⁶, 10⁷ and 10⁸ per ml. The exact bacterial numbers were determined by plating the diluted suspensions on MRS agar and counting the colonies after 48 to 72 hrs. at 37°C.

These preparations were stable for 72 hours at 4°C without detectable loss in CFU per ml. After 1 week at 4°C,

approximately 90% of the cells were no longer viable. However, the remaining viable strains were still active in modulating macrophages. Longer term stability could be obtained by freezing or lyophilization incorporating commercially available

protectants.

E. coli strain 1090 hsdR was grown in trytone-soy-broth overnight and collected as described above.

PREPARATION OF THE MACROPHAGES

Three sources of monocyte/macrophages were obtained: from peripheral blood of adult human volunteers, a macrophage cell line "MonoMac6" maintained at the New York Medical College by Dr. Myung Chun originally obtained from Dr. H. W. L.

Zeigler-Heitbrock arid described in Zeigler-Heitbrock, et al., Int. J. Cancer 41: 456-461, 1988, and alveolar macrophages obtained from 6-week old BALB/c female mice. The isolation of the monocyte/macrophages was as follows:

Peripheral venous blood monocytes (mononuclear leukocytes) were separated from 40 cc of heparinized freshly-drawn blood by means of gradient-density centrifugation on Ficoll-Hypaque (Sigma Chemicals, St. Louis, MO). The buffy-coat was collected and washed once with fresh Ficoll-Hypaque and the cells counted in a hemocytometer and placed in an RPMI 1640 (GIBCO Labs., Grand Island, NY) buffer supplemented with 5% heat-inactivated (56°, 30 min.) fetal calf serum (Sigma Chemicals) and distributed in 0.5 ml aliquots in 24-well tissue culture plates. After a 3-hr.

incubation at 37°C in 5% CO₂ the non-adherent cells were removed by aspiration. The adherent cells, numbering approximately 2 × 10e⁵ per well were.maintained in fresh RPMI 1640 with 2 mM L-glutamine.

The MonoMac6 macrophage cell line was maintained in RPMI buffer at 37°C in an atmosphere containing 5% CO₂ as described in Zeigler-Heitbrock, et al., Int. J. Cancer 41: 456-461, 1988, an equal quantity of fresh buffer was added every 48 hours. Cells were counted in a hemocytometer and distributed in 24-well tissue culture plates as described above.

Alveolar macrophages were collected by teasing murine lung tissue in RPMI buffer. Cells were counted in a hemocytometer and distributed in 24-well tissue culture plates as described above.

TESTING THE ACTIVATING EFFECTS

Selected macrophages were placed in a 24-well micropiate at a concentration of 1 × 10⁵ cells per well. To these were added aliquots of the specific bacteria containing from 1 × 10³ to 1 × 10⁷ CFUs per well. This cellular mixture was incubated for 24 hours in the case of fresh peripheral blood monocytes or murine alveolar macrophages and for 48 hours in the case of the MonoMac6 cell line macrophages. This period of time permitted a generation of macrophage growth permitting the induction of synthesis of cytokines. After the doubling time, the

supernatants were collected by filtering through a 0.2 micron filter and held frozen at -20°C until they were assayed for interleukin activity.

Cytokine release was quantitated in the frozen supernatants by the lymphocyte activating factor in the absence of exogenous stimulus. Each culture supernatant was tested at multiple dilutions in 24-well culture plates. The multiple dilutions were obtained by serial dilutions of one-third to a maximum of 12 dilutions or 1, 0.33, 0.11, 0.037, 0.0124, 1 × 10^-3, 1.4 × 10^-3, 4.5 × 10^-4, 1.5 × 10^-4, 5. × 10^-5, 1.7 × 10^-5, 5.5 × 10^-6. The highest dilution in which radioactivity above the background was observed was considered the endpoint of stimulus.

The uptake of tritiated thymidine (DuPont, NEN Research Products, Boston, MA) by mouse thymocytes which had been exposed to the supernatant obtained from the 24 hour exposure to bacteria and macrophages was determined. The thawed supernatants were mixed with freshly prepared murine thymocytes for 68 hrs. and then pulsed with 0.5 microCuries of [³H]thymidine for 4 hrs. in 96-well tissue culture plates. The cells were harvested in a cell harvester and the incorporated radioactivity measured in a scintillation counter. Control wells containing no stimulus, recombinant IL-1 and lipopolysaccharide (Sigma Chemicals) were included in each test series. In total, some 14 test series were run numbering approximately 450 determinations.

Results were quantitated by plotting radioactivity

reflecting the uptake of tritiated thymidine by thymocytes against the serial dilutions. The height of the radioactive peak as well as the highest reciprocal-dilution was used to express the positive degree of stimulus. Radioactive peaks reflecting thymocyte mitogenesis typically measured 3 to 25 fold higher than baseline controls. The reciprocal dilutions were 1, 3, 9, 27, 81, 240, 730, 2190, 6560, 19680, 59050, 181820.

EXAMPLES EXAMPLE 1

L. casei , subspecies caseii ATCC 393, was grown overnight in 200 ml of MRS broth to a concentration of 1.0 × 10⁹ CFU/ml, chilled rapidly, and centrifuged to a pellet. The pellet was washed three times with 200 ml of cold saline and finally suspended in 20 ml with a resultant concentration of 1.0 × 10¹¹ CFU/ml. Seven dilutions of the bacteria were made with cold saline and added to wells containing 8 × 10⁵ of human peripheral blood monocytes at ratios of bacteria to monocytes ranging from 1250:1 to 1:800. A stock solution of lipopolysaccharide (LPS) was diluted and added to 8 × 10⁵ monocytes at 5, 0.5, and 0.05 micrograms per well. A stock solution of 100 units of recombinant IL-1 was added in another well to 8 × 10⁵ monocytes.

At ratios of bacteria to monocytes of 1250 and 125 to one, the monocytes were observed under microscopic examination to be clumping to the point of dissolution and dysfunctionality to the point of mortality. The macrophages appeared overwhelmed by the high numbers of monocultures.

However, the monocytes exposed to twelve bacteria and to one bacteria per monocyte cell were observed to phagocytize the bacteria and maintain their vigor during the exposure. Exposure of less than 1:1 ratios of bacteria to monocytes resulted in reduced mitogenesis of thymocytes. At a level of 1:8, only the first dilution demonstrated macrophage activation. The ratios of 1:80 and 1:800 yielded normal-appearing macrophages and only mild stimulus of the thymocytes. Peak heights of radioactivity indicating the numbers of T-cell responders were usually between 7 and 10 fold higher than baseline.

Optimum stimulus of monocytes was observed at 12:1 and 1:1 ratios of bacteria to monocytes. The results are shown in

Table 1.

TABLE 1

Ratio of Highest

Sample Bacteria to Monocytes Reciprocal Dilution

L. caseii, caseii 1250:1 dissolution of monocytes

125:1 dissolution of monocytes

12:1 6560

1:1 2190

1:8 3

1:80 little stimulus

1:800 slight stimulus

100 units of IL-1 no bacteria 730

5.0 microg LPS no bacteria 2190

.5 microg LPS no bacteria 730

.05 microg LPS no bacteria 6560

EXAMPLE 2

L . casei , subspecies caseii ATCC 393, was grown overnight as described above, and suspended in saline with adjusted

concentrations yielding 140:1 to 1:20 of bacteria to MonoMacβ macrophages. Peak heights of radioactivity were between 4 and 8 fold higher than baseline. The results are shown in Table 2.

TABLE 2

Ratio of Highest

Sample Bacteria to Macrophages Reciprocal Dilution caseii, caseii 140:1 1

125:1 27

14:1 730

1:20 730

5 microg LPS none 6560

.5 microg LPS none 6560

.05 microg LPS none 2190

EXAMPLE 3

L. casei , subspecies rhamonosus ATCC 7469, was grown overnight in 20 ml of MRS broth to a concentration of 3.6 × 10⁹ CFU/ml, washed and pelleted as described above. The pellet was suspended in saline to provide the ratios to monocytes as shown in Table 3 below.

Stock solutions of lipopolysaccharide (LPS) and IL-1 were diluted and added to wells containing the monocytes as described in Example 1. Peak heights were between 4 and 6 fold higher than baseline.

TABLE 3

Ratio of Highest

Sample Bacteria to Monocytes Reciprocal Dilution

L. caseii, rha 5:1 181820

1:2 3

5.0 microg LPS none 240

.5 microg LPS none 240

.05 microg LPS none 240

IL-1 none 240

EXAMPLE 4

L. casei , subspecies rhamnosus ATCC 7469, was grown

overnight and pelleted as described above. The pellet was suspended in saline to provide the appropriate ratios to

monocytes.

Stock solutions of lipopolysaccharide (LPS) and IL-1 were diluted and added to wells containing the macrophages as described in Example 1. Peak heights were between 3 and 8 fold higher than baseline. The results are shown in Table 4.

TABLE 4

Ratio of Highest

Sample Bacteria to Macrophages Reciprocal Dilution L. caseii, rha 5:1 3

1:2 730

1:20 730

5.0 microg LPS none 6560

.5 microg LPS none 6560

.05 microg LPS none 2190

EXAMPLE 5

Bacteria were grown as described in Examples 1 and 3 and combined as pairs of commensals. In combination, they were immediately admixed with macrophages and added to the monocytes or macrophages as shown below. Peak heights were between 1.5 and 3 fold higher than baseline. The results are shown in Table 5.

TABLE 5

Ratio of Highest

Sample Bacteria to Monocytes Reciprocal Dilution

L. caseii, rha .5:1 3

L. caseii, cas 140:1 1

Both strains half & half 1

Ratio of Highest

Sample Bacteria to Macrophages Reciprocal Dilution

L. caseii, rha 5:1 0

L. caseii, cas 140:1 1

Both strains half & half 730 L. caseii, cas 10:1 3

E. faecium 250:1 1 in combination half & half 3

EXAMPLE 6

The macrophage is protected against a pathophysiological response to a dose of E. coli or LPS by the presence of another commensal bacterium; i.e., the appearance of the macrophage exposed to E. coli or to LPS is different than when exposed to E. coli or LPS in the presence of a commensal. The presence of the commensal population does not alter the degree of activation of the macrophages, but rather alters the cytokine response as seen by morphological changes typical of a balanced response. Radioactive peak heights were 2 to 10 fold above baselines. The results are shown in Table 6.

TABLE 6

Ratio of Highest

Sample Bacteria to Mø Reciprocal Dilution

E coli 1 : 1 27

L. caseii. cas 1 : 10 3

Together 240

E coli 1 : 1 27

E faecium 1 : 3 9

Together 240

E coli 1 : 1 27

L. caseii. rha 1 : 1 3

Together 27

LPS 0.5 microg 2200

L. caseii. cas 8 : 1 81 Together 720

LPS 0.5 microg 2200

E faecium 12 : 1 81

Together 240

LPS 0.5 microg 2200

L. caseii, rha 24 : 1 27

Together 27

EXAMPLE 7

The macrophage is protected against a pathophysiological response to a dose of E. coli or LPS by the presence of other commensal bacteria; i.e. the appearance of the macrophage exposed to E. coli or to LPS is different than when exposed to E. coli or LPS in the presence of commensals. The presence of the commensal population does not dramatically alter the activation of the macrophages, but rather alters the cytokine response as seen by morphological changes typical of a balanced response. The results are shown in Table 7. Radioactive peak heights were 2 to 9 fold above baselines.

TABLE 7

Ratio of Highest

Sample Bacteria to Mø Reciprocal Dilution

E coli 3 : 1 81

L. caseii, cas + 1 : 10

L. caseii, rha 1 : 10 81

Together 2200

E coli 3 : 1 81

L. caseii, cas + 1 : 1

L. caseii, rha l : 10 3

Together 3

Claims

WHAT IS CLAIMED IS:

1. A method for stimulating the immune system of an animal by administering an apathogenic bacteria selected from the family Lactobacilliaceae in a ratio of between about 500:1 and 1:500 bacteria to macrophage, the macrophage being present in the oropharyngeal-alveolar cavity of the animal, in an acceptable carrier effective to deliver said bacteria to said oropharyngeal-alveolar cavity of the animal.

2. The method of Claim 1 wherein the ratio of apathogenic bacteria to macrophage in the oropharyngeal-alveolar cavity is between about 100:1 and about 1:100.

3. The method of Claim 1 wherein the ratio of apathogenic bacteria to macrophage in the oropharyngeal cavity is between about 10:1 and about 1:10.

4. The method of Claim 1 wherein the carrier is selected from the group consisting of sprays, lozenges, gums, gels, and pastes.

5. The method of Claim 1 wherein the apathogenic bacteria is selected from the group consisting of: Lactobacillaceae Bifidobacterium: indicum, coryne forme and eriksonii;

Lactobacillaceae Lactobacillus Thermobacterium: acidophilus, bulgaricus, helveticus, jugurti, lactis, lactis, salivarius, delbruckii, leichmannii, and jensenii;

Lactobacillaceae Lactobacillus Streptobacterium: caseii and sub-species, sake, coryne formis, curvatus, xylosus, and zeae;

Lactobacillaceae Lactobacillus Betabacterium: fermentum;

Pediococcus: cerevisiae, acidolactici and pentosaceus; and mixtures thereof.

6. The method of Claim 1 wherein the apathogenic bacteria comprise an A4alpha interpeptide bridge structure.

7. The method of Claim 1 wherein the apathogenic bacteria have been rendered non-viable by a soft kill technique.

8. A composition for modulating the response of animal macrophages to pathogens , said composition comprising an

apathogenic bacteria selected from the family Lactobacilliaceae said composition having an amount of bacteria effective to deliver a ratio of between about 500 : 1 and 1 : 500 bacteria to macrophage, the macrophage being present in the oropharyngeal-alveolar cavity of the animal , and an acceptable carrier effective to deliver said bacteria to said oropharyngeal-alveolar cavity of the animal.

9. The composition of Claim 8 wherein said pathogen is E. coli.

10. The composition of Claim 8 wherein the ratio of apathogenic bacteria to macrophage in the oropharyngeal-alveolar cavity is between about 100:1 and about 1:100.

11. The composition of Claim 8 wherein the ratio of apathogenic bacteria to macrophage in the oropharyngeal cavity is between about 10:1 and about 1:10.

12. The composition of Claim 8 wherein the carrier is selected from the group consisting of sprays, lozenges, gums, gels, and pastes.

13. The composition of Claim 8 wherein the apathogenic bacteria is selected from the group consisting of:

Lactobacillaceae Bifidobacterium: indicum, coryne forme and eriksonii;

Lactobacillaceae Lactobacillus Thermobacterium: acidophilus, bulgaricus, helveticus, jugurti, lactis, lactis, salivarius, delbruckii, leichmannii, and jensenii; Lactobacillaceae Lactobacillus Streptobaσterium: caseii and sub-species, sake, coryne formis, curvatus, xylosus, and zeae;

Lactobacillaceae Lactobacillus Betabacterium: fermentum;

Pediococcus: cerevisiae, acidolactici and pentosaceus; and mixtures thereof.