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The Mechanism of Action of MicroLactin®: Scientific

experiments used to identify the mechanism of action of the hyperimmune milk factor (HIMF)—the biologic compound found in MicroLactin®

Amy Van Gels, DVM, April 28, 2015

Introduction

The protective benefits of a mother’s milk to her baby are well known. Compared to formula-fed human babies, studies have shown that breastfed babies have a decreased risk for digestive illnesses, ear infections, respiratory infections, asthma, allergies, obesity, and childhood leukemia.1 This reduction in disease may be partially due to the natural protective properties of milk. Mother’s antibodies can protect against infectious diseases. Lactoferrin and lactoperoxidase are natural antibacterial substances found in milk. Antioxidants decrease tissue damage.2

Scientists have also discovered that during times of infection a mother’s immune system creates a specialized anti-inflammatory factor (AIF). While AIF does not always appear in milk, when it does it can decrease inflammation even more than aspirin.3 Scientists have discovered a way to trigger production of AIF in cows through the use of regularly repeated vaccinations (hyperimmunization); the product of which is referred to as hyperimmune milk factor (HIMF). Other names for this biologically active product include milk anti-inflammatory product (MAIF) and milk-derived factor (MDF); for consistency’s sake, the term HIMF will be utilized throughout the document. HIMF has been available commercially for more than a decade as the nutritional supplement MicroLactin®—the active ingredient of Duralactin® brand products.

The following paper describes the process in which scientists first proved that HIMF can inhibit inflammation and then discovered how it works—i.e., its mechanism of action. The following studies showed that HIMF:

• Reduces inflammation,4,5,6 • Prevents inflammation prior to an inflammatory trigger or after inflammation has already

started,7,8 • Decreases the number of neutrophils present at an inflammatory site,9,10,11,12,13,14 • Inhibits and reverses neutrophil attachment to the blood vessel wall via its effects on the cell

receptor protein CD18,15,16,17 • Inhibits neutrophils from being able to squeeze through tight junctions between the cells in

blood vessel walls (a process called diapedesis or transmigration),18 • Affects the function of lymphocytes (type of inflammatory/immune cell),19 • Improves efficacy and action of macrophages (a type of inflammatory cell),20,21 • Increases the ability of macrophages to engulf (phagocytize) bacteria,22,23 • Reduces the “rebound effect” of the increased tissue damage seen in bacterial infections

treated with typical anti-inflammatory medications,24,25,26 • Improves the function of inflamed tissues,27,28 • Provides a protective effect on animals with bacterial infections.29,30

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Carrageenan-Induced Inflammation

Scientists typically test the efficacy anti-inflammatory drugs by using the Rat Paw Edema Test. This simple and direct laboratory test plays a vital role in the development of new anti-inflammatory drugs.31,32 In this test, researchers inject an inflammatory substance called carrageenan into one of the paws of laboratory rats. The injection triggers an inflammatory reaction that causes swelling (edema), redness, and pain. The amount of swelling is then measured to quantify the amount of inflammation that has occurred. This measurement is then compared to the opposite (control) paw that did not receive a carrageenan injection. In rats that receive a test drug, an anti-inflammatory effect is demonstrated if there is no significant difference between fluid volumes of the two paws. That is, the lack of fluid accumulation means that the test drug prevents the inflammation caused by carrageenan.

Scientists usually include a control group of rats to prove that the carrageenan is effective. The control group is given a known anti-inflammatory drug (such as aspirin), a medication that does not contain the anti-inflammatory drug (ie, saline), and/or no medication at all. The control group is then compared to the group of rats that receives the drug that is being studied. If a significant difference occurs between the two groups, the medication is said to have anti-inflammatory effects.

The anti-inflammatory action of hyperimmune milk factor (HIMF) has been repeatedly verified by numerous rat paw edema tests. In these experiments, HIMF was administered into the vein (intravenous or IV), abdomen (intraperitoneal or IP), muscle (intramuscular or IM), under the skin (subcutaneous or SQ), or mouth (oral or PO) of treatment rats. Other control rats were administered either aspirin, water, or normal milk. The paws of rats treated with HIMF showed significantly less swelling than the paws of the rats in the control groups (see Figure 1). In fact, rats injected with intravenous (IV) HIMF showed less swelling than rats given aspirin (see Figure 2). Also, the carrageenan-injected paws of rats treated with HIMF were statistically no different from the paws that were not injected (see Figure 1), confirming an anti-inflammatory effect. Similar findings have been confirmed repeatedly with many follow-up experiments and show that HIMF can be used to reduce inflammation in animals.33,34,35,36 The results also indicate that HIMF has anti-inflammatory effects when given orally.37

Figure 2. Effect of subcutaneous (SubQ), oral, intramuscular (IM), and intravenous (IV) hyperimmune milk factor (HIMF); control (no treatment); and aspirin on tissue swelling caused by carrageenan in the rat paw. Graph borrowed from Woods C, Gingerich D. Technical Brief: Pharmacology of MicroLactin™.

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The swelling in response to the carrageenan occurs in two distinct stages. The first phase of swelling occurs because of the actions of several inflammatory mediators (eg, bradykinin and histamine); the second phase occurs because of the actions of inflammatory cells, especially neutrophils. An additional experiment showed significant effects of HIMF during the second, but not the first, stage (see Figure 3). This indicates that HIMF appears to decrease the effect of inflammatory cells—neutrophils and macrophages—instead of inflammatory mediators.38

The Pleural Neutrophil Migration Inhibition Assay also utilized carrageenan to test the anti-inflammatory action of HIMF. In this test, scientists injected carrageenan into the pleural cavity (the space between the lungs and the chest wall of rats) causing neutrophils to rush to the area. Three milligrams of HIMF injected immediately into the rats’ abdomen (IP injection) inhibited neutrophil migration by more than 70% after four hours, whereas the control rats showed only 18% inhibition. This means that HIMF blocked 70% of the neutrophils that should have responded to the carrageenan. Therefore, HIMF appears to inhibit the number of neutrophils present at an inflammatory site. The ability of neutrophil to travel from the blood stream to inflamed tissues is called neutrophil migration.39

Figure 3. Volume (in uL) of tissue swelling in response to carrageenan injections during a rat paw edema test. The control group received saline, and the hyperimmune milk factor (HIMF) group received 40 mg of intravenous HIMF. The Wilcoxon sum of ranks test was used to determine a significant difference (* = p <0.01) between the 2 groups during the stage in which inflammatory cells act. Graph is adapted from Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:73. Figure1.

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Reverse Passive Arthus Reaction

In the reverse passive Arthus reaction (RPA), rats are injected with a protein found in egg whites (antigen) and rabbit antibodies to that protein. At the site of the injections, the antibodies bind to the antigens and create an intense inflammatory reaction. Because neutrophils are primarily responsible for the reaction, medications that inhibit inflammation in a RPA test are thought to affect the function of neutrophils.40

In an RPA experiment, rats treated with 20 mg of IV HIMF showed 81% fewer neutrophils, 44% less tissue swelling, and 69% less bleeding at the site of the injections than rats that had been injected with saline (see Figure 4).41 Because the neutrophil is responsible for the majority of the inflammation in an RPA test, the results of this study further support the idea that HIMF affects the function of neutrophils in inflammation.

Subcutaneous Sponge Implants

Scientists further tested the effects of HIMF on neutrophils by surgically implanting polyurethane sponges under the skin in rats. These subcutaneous (SQ) sponges act as foreign material—a trigger of inflammation. After the sponges were removed, the number of neutrophils and the amount of fluid (edema) were measured.42,43

In one study, researchers implanted bacteria-free sponges under the skin of rats that were simultaneously given 5, 10, 20, or 40 mg of IV HIMF. After their removal, the sponges from rats receiving the 20 and 40 mg doses contained dramatically fewer neutrophils and a mildly less fluid than sponges from the rats that did not receive medications (see Figure 5). The rats given 5 or 10 mg did not show a significant difference from the control group. The number of neutrophils was also significantly decreased when HIMF was given 30, 60, and 120 minutes after the implant.44,45 These studies indicate

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Figure 4. The effect of 20 mg of intravenous (IV) hyperimmune milk factor (HIMF) on the number of neutrophils, amount of tissue swelling, and bleeding seen at the injection sites of a reverse passive Arthus reaction in rats. A significant decrease in neutrophil number, tissue swelling, and bleeding were present (p < 0.01, n = 6 per group). Data collected from Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999.

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Figure 5. The effect of hyperimmune milk factor (HIMF) on neutrophil migration and fluid accumulation in subcutaneous sponges. * = p <0.01, n = 6 Graphs borrowed from Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:73. Figure5.

that HIMF decreases neutrophil migration and tissue swelling (edema) in the inflammatory response. HIMF can both prevent and treat inflammation.

Anti-inflammatory medications that suppress neutrophil migration are not always indicated in bacterial infections because neutrophils may be necessary to limit bacterial growth. The large bacterial load that can occur after the use of anti-inflammatory medications (such as cyclosporine and methylprednisolone) can cause a rebound effect when massive numbers of neutrophils then rush to the site. This exaggerated response after the use of anti-inflammatory drugs often creates excessive tissue damage and scarring. To see if HIMF is appropriate in patients with bacterial infections, scientists infiltrated SQ sponges with live Escherichia coli bacteria. The sponges of rats treated with 40 mg of IV HIMF showed significantly fewer neutrophils but more bacteria than the control group; the decreased number of neutrophils allowed the E. coli bacteria to grow unchecked. However, the rebound effect of tissue damage and scarring that can occur with anti-inflammatory drugs did not occur after HIMF testing. Therefore, HIMF is thought to have anti-inflammatory effects that work without causing rebound tissue damage.46,47,48

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Experimental Pyelonephritis

Kidney infections—also called pyelonephritis—cause significant inflammation, permanent tissue damage, and scarring. When tissue damage occurs within the kidney, it can easily be seen as indented scarring on the kidney’s surface. Researchers can therefore easily determine the amount of tissue damage that has occurred by visualizing these surface scars.

Scientists experimentally caused pyelonephritis in 26 rats by injecting E. coli into their kidneys. Half of the rats were given 40 mg of IV HIMF at the time of the kidney injections and then again 48 hours later. The other half served as the control group. The kidneys were then examined 4 or 21 days later to determine how much scarring, fluid accumulation, and bacteria were present.

While the kidneys of HIMF-treated rats contained higher bacterial counts, they also showed 22% less fluid accumulation and 24% less scarring than the control group. HIMF was able to not only inhibit inflammation (as evidenced by the reduction in tissue swelling) but was able to inhibit tissue damage (as evidenced by the reduction in scarring). By 21 days, however, the kidneys of both the HIMF-treated and control groups showed similar amounts of scarring, fluid accumulation, and bacteria. Similar to the SQ sponge studies, the rebound effect of increased tissue destruction after the use of anti-inflammatory drugs was not seen at this later date. Considering that only two doses of HIMF were given (at the induction of the infection and 48 hours later), it would have been interesting to know the extent of the tissue damage at 21 days if HIMF had been given daily throughout the course of the experiment.

Intravital Microscopy

After learning that HIMF decreases the number of neutrophils that accumulate at an inflamed site, scientists needed to understand how exactly this occurs. To understand this, scientists needed to watch neutrophils function in real-time. Intravital microscopy does this through the use of specialized microscopes that visualize the cells as they act in a living animal’s body.49 In the case of HIMF, intravital microscopy allows researchers to study neutrophil adhesion, a vital step in neutrophil migration. Neutrophil adhesion refers to a neutrophil’s ability to stick to the cells of the blood vessel wall. After adhesion, a neutrophil can then squeeze in between the vessel cells in order to pass from the blood into inflamed tissues.

Woodman et al. used intravital microscopy to study inflammation induced by platelet-activating factor (PAF), which causes a six-time increase in neutrophil adhesion. Rats that received 40 mg of IV HIMF showed 80-90% less neutrophil attachment in PAF-treated tissues than rats that did not receive HIMF (control group). HIMF also appeared to completely block neutrophils from passing through blood-vessel walls in response to PAF, whereas the rats untreated with HIMF showed a 12-time increase.50,51

More importantly, HIMF reversed neutrophil adhesion caused by PAF. This reversal is not a typical action of anti-inflammatory treatments.52 In their study, Woodman et al. administered PAF then waited 30 minutes before giving 40 mgs of HIMF; this delay allowed the region to become inflamed and neutrophils to attach. Ten minutes after the HIMF was given, there was a significant reduction in

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the number of neutrophils adhered to the blood vessel wall (see Figure 6). That is, the HIMF “peeled” the neutrophils off of the vessel walls!53,54

Woodman et al. used intravital microscopy to show that HIMF inhibits neutrophil attachment, prevents neutrophils from passing from the blood into inflamed tissues, and reverses neutrophil attachment. This keep neutrophils in the bloodstream where they do no harm, instead of allowing them to act at sites of inflammation. As the most common inflammatory cell in the body, neutrophils account for a significant amount of the inflammatory response.

Flow Cytometry

Flow cytometry detects the microscopic activity of cells. Among other things, it can help measure the activity of receptors on a cell’s surface. Woodman et al. used this technology to observe that neutrophils treated with HIMF exhibited fewer CD18 cell receptor proteins than untreated neutrophils. The CD18 protein is part of the integrin receptor found on neutrophils that firmly binds to a matching ICAM receptor on blood vessel cells. When neutrophils express fewer CD18 proteins, they cannot firmly adhere to blood vessel walls. These results indicate that the inhibition of the CD18 protein accounts for the significant decrease in neutrophil adhesion that that has been seen in previous studies.55,56

Transepithelial Electrical Resistance (TER)

Tight junctions prevent inflammatory cells, proteins, ions, and other molecules from passing in between cells. They are present between the cells in blood vessel walls and in other organs of the body. During the inflammatory process, inflammatory mediators cause an increase in vascular permeability, which causes blood-vessel tight junctions to open. The loose tight junctions then allow inflammatory cells (neutrophils, macrophages, and lymphocytes) to pass from the bloodstream into inflamed tissues. The increase in vascular permeability also allows plasma proteins to pass between blood vessel cells,

Figure 6. The effect of platelet-activating factor (PAF) and 40 mg of hyperimmune milk factor (HIMF) on the number of neutrophils attached to 100 μm vessel wall. The vessel was treated with PAF alone for 30 minutes. After 30 minutes, HIMF was added. Ten minutes after HIMF was added, there was a large reduction in the number of attached neutrophils. This is not a typical effect of anti-inflammatory treatments. Graph adapted from Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999, Figure 27A.

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drawing water with them. The increased amounts of proteins and water in the tissues account for the swelling (edema) seen with inflammation.

One way for scientists to assess the permeability of tight junctions is through the use of transepithelial electrical resistance (TER). Cells with intact tight junctions show a significant difference in electrical charge from one end of the cell to the other (polarization). The ability for tight junctions to block an electrical current from travelling from one side to the other (ie, the ability to maintain the cell’s polarization) is termed TER. When tight junctions fully open, the TER is lost.57

Stelwagen and Ormrod evaluated the effect of HIMF on tight junction permeability by measuring the TER of mammary and kidney cells. They treated these cells with a chemical (EGTA) that opens tight junctions. The cells treated with HIMF maintained their TER significantly more than those that were not treated (see Figure 7). Also, the cells that had been treated with HIMF recovered their TER faster than the control cells (see Figure 7). These results show that HIMF can protect tight junctions and therefore decrease vascular permeability and the movement of inflammatory cells out of the bloodstream.58

An additional experiment by Stelwagen and Ormrod showed that HIMF increased the TER value of mammary cells, which indicates that HIMF can actually stimulate the production and strength of tight junctions (see Figure 8). Because tight junctions form in mature cells that are not multiplying (growing), Stelwagen and Ormrod tested HIMF’s effect on cell growth. The researchers added HIMF to cultures of multiplying cells; the HIMF-treated cells multiplied less than untreated cells. Because HIMF inhibits cell multiplication, the effects on TER must be caused by “tighter” tight junctions and not because of an increased number of cells.59

Figure 7. The effect of hyperimmune milk factor (HIMF) on transepithelial electrical resistance (TER)—an indicator of intact tight junctions. Tight junctions open under the influence of EGTA. HIMF prevented the loss of TER significantly more than the untreated control group. Also, the HIMF-treated cells recovered their TER faster than the control group. ** = p<0.01 Graph borrowed from Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388.

Figure 8. The effect of hyperimmune milk factor (HIMF) on the formation of transepithelial electrical resistance (TER). Mammary epithelial cells were cultured with or without the presence of 2 mg/mL HIMF. * = p<0.05, ** = p <0.01 Graph borrowed from Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388.

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At the conclusion of their paper, Stelwagen and Ormrod speculate as to the evolutionary significance of an anti-inflammatory factor in milk. A major cause of sickness and death in newborns with digestive diseases is dehydration caused by the loss of fluids. An anti-inflammatory component of milk that prevents fluid loss through the tight junctions in the intestines would therefore protect newborn health.60

Mammary Gland Tests

Owens et al.61 tested the effects of HIMF on mammary gland infections (mastitis) in both mice and cows. In one study, they infected mice with large amounts of bacteria (Staphylococcus aureus Newbould 305) in order to determine the amount of bacteria that would kill half of the mice in the study (the bacteria’s lethal dose or LD50). The lethal dose of S. aureus was significantly higher in mice that had been treated with IV HIMF for seven days prior to becoming infected. That is, significantly more bacteria was needed to kill mice treated with HIMF (>2 X 1010 CFU) than mice that did not receive HIMF (5 X 1010 CFU). This study showed that HIMF can protect animals with bacterial infections. This protective effect differs from typical anti-inflammatory drugs in that most of these medications cause animals to be more susceptible to infections.

Other studies62 by Owens et al. also suggest a protective effect of HIMF. These researchers infused HIMF directly into the mammary glands of mice and then infected the glands with S. aureus. Glands treated with HIMF actually resisted the infection in three of the four mice (75%), while all of the glands of the control group became infected. The glands treated with HIMF also showed fewer inflammatory cells than the control group. Owens et al. also injected HIMF into the abdomens of mice (IP injections) for seven days before inducing mastitis with S. aureus. The untreated group showed 10 times more bacteria than the HIMF-treated mice. The HIMF mice also showed fewer inflammatory cells and larger percentage of mammary ducts than the control group (see Figure 9). These mice could therefore secrete larger amounts of milk than the control group. These studies show that HIMF has effects that extend beyond just anti-inflammatory relief. Contrary to other anti-inflammatory medications, HIMF appears to help protect animals from infections and may improve the tissue function of animals with infections.

Figure 9. The effect of hyperimmune milk factor (HIMF) on the microscopic composition of the mammary gland of mice. The mammary glands of HIMF-treated mice showed a larger percentage of milk ducts (blue portion), which would allow these mice to produce more milk than the control group. All values: p<0.05 Chart data obtained from Owens WE, Nickerson, SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology. 1992;30:233-246.

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To further understand the protection provided by HIMF in bacterial infections, Owens et al.63 tested the function of macrophages. Macrophages (which are inflammatory cells) kill bacteria and other foreign organisms by engulfing them—a process known as phagocytosis. In a laboratory (in vitro) test, opsonized S. aureus bacteria were added to cultures of cow mammary gland macrophages. After four hours, the number of bacteria eaten by macrophages were counted. Macrophages treated with HIMF phagocytized 10 times more bacteria (1430 CFU) than untreated macrophages (135 CFU) (see Figure 10). HIMF, therefore, appears to increase the ability of macrophages to destroy bacteria. This enhanced phagocytosis may explain the protective effects of HIMF on animals with bacterial infections.

In another experiment, Owens et al.64 tested the effects of HIMF on mastitis in cows by introducing S. aureus into their udders. Udders treated with 5 mg of HIMF 24 hours before the introduction showed fewer infections (5 out of 10) than udders treated with saline (7 out of 10). Milk from HIMF-treated udders also showed smaller bacterial counts than the milk from saline-treated udders during the first three days following the infections; however, the HIMF could not cure the infection. This study further supports the idea that HIMF can increase an animal’s ability to defend itself against bacterial infections.

Host-versus-Graft and Graft-versus-Host Assays

Most people are aware that organ and bone marrow transplants (grafts) can be rejected by the recipient (host). This is because the immune system (lymphocytes in particular) of the recipient attacks the transplant because it sees the transplant as a foreign organism (non-self). Scientists duplicate this process in the host-versus-graft assay in order to test the function of lymphocytes (a type of white blood cell). In this assay, researchers inject a rat with lymphocytes from its F1 hybrid offspring. Because these lymphocytes appear foreign to the parent, the parent’s T-lymphocytes attack it causing the mouse’s lymph nodes to swell. The lymph nodes are weighed to determine the magnitude of the immune response.

Less known than organ transplant rejections is that bone marrow and blood cell transplants (grafts) can actually attack the recipient (host). These immune cells see their new body as non-self and

Figure 10. The effect of 2000 μg/mL hyperimmune milk factor (HIMF) on the ability for macrophages to engulf (phagocytize) bacteria. The results indicate that HIMF increases the ability of macrophages to destroy bacteria. This may account for HIMF’s protective effects for animals with bacterial infections. Graph data obtained from Owens WE, Nickerson, SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology. 1992;30:233-246.

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attack the host. Researchers replicate this process in the graft-versus-host assay by injecting the parent’s lymphocytes into its F1 hybrid offspring.

In a host-versus-graft assay, Ormrod and Miller65 gave 20 mg of IV HIMF to host rats 48, 24, and 3 hours prior to injecting lymphocytes from their untreated F1 offspring. HIMF suppressed the host-versus-graft reaction by 30%. In a graft-versus-host assay, Ormrod and Miller66 injected HIMF-treated rat host lymphocytes into their F1 hybrid offspring. No change in the graft-versus-host reaction was seen. These studies indicate that HIMF affects the function of lymphocytes within the body. These studies also showed that the spleen’s weight and cell count increased after HIMF use, suggesting that HIMF causes lymphocytes to accumulate in the spleen.

A substance called concanavalin A causes lymphocytes to multiply in number. During these experiments, Ormrod and Miller67 applied it to the lymphocytes that they removed from the rats’ bodies in order to increase the number of lymphocytes that could be used in the experiment. When the lymphocytes were untreated with HIMF, they multiplied as expected. When the concanavalin A was applied to lymphocytes from HIMF-treated rats, however, the lymphocytes did not multiply. This finding further supports the idea that HIMF interferes with the ability for lymphocytes to function normally.

Collagen-Induced Arthritis Model in Mice

The most common way to test treatments for rheumatoid arthritis is through the use of the collagen-induced mouse model. In this test, scientists inject type II collagen into the skin of mice. The collagen triggers rheumatoid arthritis, symptoms of which typically appear in 21-28 days. Trained technicians assess the degree of arthritis by scoring the amount of redness, tissue swelling, and joint stiffness exhibited by the mice. The study is blinded so that the technicians do not know which mice receive the medications and which mice are in the control group.68

Gingerich et al.69 mixed HIMF, Stolle’s Immune Milk, a whey protein isolate (WPI Plus), or a salt solution (control) into the daily food of 40 mice. Stolle’s Immune Milk, HIMF, and WPI Plus—all of which are made from the milk of hyperimmunized cows—significantly decreased the occurrence and the severity of the arthritis seen. This study indicates that HIMF has similar anti-inflammatory effects as Stolle’s Immune Milk. Other studies of Stolle’s Immune Milk have showed that it relieves the symptoms of rheumatoid arthritis;70 prevents the age-related decline of the immune system;71 prevents certain infections in immunocompromised patients;72 protects lung airways and tissue from toxic chemicals (like cigarette smoke);73 and reduces atherosclerosis, blood pressure, and cholesterol levels.74

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Rat Lungs Exposed to Cigarette Smoke

One such experiment of Stolle’s Immune Milk studied the effect of daily cigarette smoke on the lungs of rats. Wilborn et al.75 gave rats either water, normal milk, or Stolle Immune Milk for the duration of the experiment, including the two weeks prior to smoke exposure. For two weeks, rats were placed daily inside a cigarette smoke machine, in which they breathed the smoke of 2 cigarettes (20 minutes per day). The exposure to cigarette smoke caused the rats who drank water or normal milk to develop inflammation around their airways, lung damage, and debris within the lungs. The rats that drank Stolle Immune Milk showed markedly less inflammation and essentially no smoke debris. Compared to the control rats, the macrophages in the rats who drank Stolle Immune Milk showed evidence of greater than 50% more phagocytosis, more internal (lysosomal) activity, and greater regeneration. Stolle Immune Milk activated macrophages to work more effectively to eliminate harmful smoke debris, allowing the rats to have clearer airways with easier respiratory function. Together, the findings indicate that the Stolle Immune Milk can protect lung tissues from toxic substances.76

Abbreviations:

CFU = colony forming units (a method of counting bacterial numbers) EGTA = ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N;-tetraacetic acid HIMF = hyperimmune milk factor ICAM = intercellular adhesion molecules IM = intramuscular (in to the muscle) IP = intraperitoneal (into the abdomen) IV = intravenous (into the vein) PAF = platelet-activating factor RPA = reverse passive Arthus reaction

Figure 11. The arthritis score of mice treated with oral salt solution (control), Stolle (SMBI) Immune Milk, protein-free fraction (HIMF), or whey fraction (WPI Plus). The occurrence and severity of arthritis symptoms (redness, tissue swelling, and joint stiffness) were significantly decreased with Stolle milk (P <0.05), HIMF (P <0.05), and WPI Plus (P = 0.017). Graph borrowed from Woods C, Gingerich D. VPL Technical Brief: Pharmacology of MicroLactin®.

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SQ = subcutaneous (under the skin) TER = transepithelial electrical resistance

1 Allen J, Hector D. Benefits of breastfeeding. New South Wales Public Health Bulletin. 2005;16:42-46. 2 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 3 Ormrod DJ, Miller TE. The anti-inflammatory activity of a low molecular weight component derived from the milk of hyperimmunized cows. Agents and Actions. 1991;32(3/4):160-166. 4 Beck LR. Method of treating inflammation using bovine milk. US Patent #4284623. Aug1981. 5 Ormrod DJ, Miller TE. The anti-inflammatory activity of a low molecular weight component derived from the milk of hyperimmunized cows. Agents and Actions. 1991;32(3/4):160-166. 6 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 7 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 8 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. 9 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 10 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. 11 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 12Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 13 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 14 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 15 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 16 Woodman R, Fuhrer P, Beck L, Kubes P. The effects of hyperimmunized milk factor (HIMF) on neutrophil adhesion in vivo [Abstract]. Society for Leukocyte Biology, 29th National Meeting, Charleston South Carolina. 1992. 17 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 18 Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388. 19 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 20 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 21 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 22 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 23 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 24 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 25 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 26 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72.

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27 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 28 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 29 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 30 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 31 Morris CJ. Carrageenan-induced paw edema in the rat and mouse. In: Winyard PG, Willoughby DA, eds. Inflammation Protocols. Vol 225. Totowa, NJ: Humana Press Inc; 2003:115-121. 32 Beck LR. Method of treating inflammation using bovine milk. US Patent #4284623. Aug1981. 33 Beck LR. Method of treating inflammation using bovine milk. US Patent #4284623. Aug1981. 34 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 35 Ormrod DJ, Miller TE. The anti-inflammatory activity of a low molecular weight component derived from the milk of hyperimmunized cows. Agents and Actions. 1991;32(3/4):160-166. 36 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 37 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 38 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 39 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 40 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 41 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 42 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 43 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 44 Ormrod DJ, Miller TE. A low molecular weight component derived from the milk of hyperimmunized cows suppresses inflammation by inhibiting neutrophil emigration. Agents and Actions. 1992;37:70-79. 45 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 46 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 47 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 48 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 49 Kim P, Yun SH. Intravital microscopy analysis. In: Bhushan B, ed. Encyclopedia of Nanotechnology. Springer Netherlands; 2012:1162-1172. 50 Woodman R, Fuhrer P, Beck L, Kubes P. The effects of hyperimmunized milk factor (HIMF) on neutrophil adhesion in vivo [Abstract]. Society for Leukocyte Biology, 29th National Meeting, Charleston South Carolina. 1992. 51 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 52 Beck LR, Fuhrer JP. Milk lymphocyte anti-adhesion factor, and its role as an anti-microbial. International Dairy Federation. 1993;62-72. 53 Woodman R, Fuhrer P, Beck L, Kubes P. The effects of hyperimmunized milk factor (HIMF) on neutrophil adhesion in vivo [Abstract]. Society for Leukocyte Biology, 29th National Meeting; Charleston, South Carolina. 1992 54 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999. 55 Woodman R, Fuhrer P, Beck L, Kubes P. The effects of hyperimmunized milk factor (HIMF) on neutrophil adhesion in vivo [Abstract]. Society for Leukocyte Biology, 29th National Meeting, Charleston South Carolina. 1992 56 Beck LR, Fuhrer JP. Anti-inflammatory factor, method of isolation, and use. US Patent #5980953. Nov1999.

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57 Sonoda S, Spee C, Barron E, Ryan SJ, Kannan R, Hinton DR. A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells. Nature protocols. 2009;4(5):662-673. 58 Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388. 59 Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388. 60 Stelwagen K, Ormrod DJ. An anti-inflammatory component derived from milk of hyperimmunized cows reduces tight junction permeability in vitro. Inflammation Research. 1998;47:384-388. 61 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 62 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 63 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 64 Owens WE, Nickerson SC, Washburn PJ. Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection. Veterinary Immunology and Immunopathology. 1992;30:233-246. 65 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 66 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 67 Ormrod DJ, Miller TE. Milk from hyperimmunized dairy cows as a source of a novel biological response modifier. Agents and Actions. 1993;38(Special Conference Issue):C146-C149. 68 Rosloniec EF, Cremer M, Kang AH, Myers LK, Brand DD. Collagen-induced arthritis. Curr Protoc Immunol. Apr2010; Chapter 15:Unit 15.5.1-25. 69 Gingerich DA, Strobel JD, Brown A, Fuhrer JP. Efficacy of SMBI milk bioactive factors in a collagen-induced arthritis model in mice. Summary of presentation at Japan Rheumatism Association; Tokyo, Japan. 1997. 70 Stolle RJ, Beck LR. Prevention and treatment of rheumatoid arthritis. US Patent #4732757A. 1991. 71 Beck LR, Ishida A, Yoshikai Y, Murosaki S, Kubo C, Hidaka Y, Nomoto K. Use of hyperimmune milk to prevent suppression of T-lymphocyte production. US Patent #6056978. May 2000. 72 Beck LR, Ishida A, Yoshikai Y, Murosaki S, Kubo C, Hidaka Y, Nomoto K. Use of hyperimmune milk to prevent suppression of T-lymphocyte production. US Patent #6056978. May 2000. 73 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 74 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 75 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999. 76 Wilborn WH, Hyde BM, Beck LR, Fuhrer JP. Milk from hyperimmunized cows stimulates lysosomal activity in rat lung macrophages. Summary of presentation at The Lovelace Respiratory Research Institute Symposium on Respiratory Immunology; Santa Fe, NM. 1999.