Current Therapies for Immune Mediated Hemolytic Anemia in Dogs.

Autoimmune disease treatment in dogs currently relies on the use of broad-spectrum

immunosuppressive drugs, which are associated with unacceptable adverse effects in

some patients. Immune Mediated Hemolytic Anemia (IMHA) requires high doses and

long-term administration of these medications, often resulting in a terrible choice for the

pet owner between the disease, the medications or euthanasia (1) (2). Stem cell

therapy offers an alternative option for these pets.

Overview Immune Mediated Hemolytic Anemia (IMHA) is caused by antibody deployment against

red blood cells. The presence of these autoantibodies initiate destruction of the red

blood cells through one of two ways: complement activation results in hemolysis

(rupture) of the red blood cells within the blood stream, or macrophages in the spleen

and liver gradually destroy the red blood cells (3). Immunosuppressive drugs dampen

the immune response but do not address the underlying cause - the inciting antibody

production. Stem Cell Therapy addresses both the underlying cause of anti-red blood

cell antibody production and protects the antibody tagged red blood cells from

destruction. Stem Cells, unlike conventional therapeutics are alive and dynamically self-

modulate their response to immune disease thereby preventing over-reaction or

overdoses in therapy (4).

To best understand the mode of action of Allogenic Mesenchymal Stem Cell (AMSC)

therapy we need to review a detailed description of the pathogenesis of the immune

dysfunction underlying Immune Mediated Hemolytic Anemia (IMHA) in dogs.

Understanding IMHA Immune reactions against red blood cells happen when antibodies are made against the

proteins on the surface of these cells. These antibodies signal that these “tagged” cells

should be destroyed. This happens either in the bloodstream (intravascular hemolysis)

or within the liver and spleen (extravascular hemolysis). Once red blood cells are

destroyed, the clinical signs are mostly attributable to loss of oxygen to the tissues. The

by-products of red blood cell destruction affect the liver and kidney and the lungs

causing blood clots and multiple organ failure. Death occurs in approximately 25% of

extravascular cases and 75% of intravascular cases.

Steven D. Garner

Chief of Staff

Safari Veterinary Care Centers

2402 Marina Bay Drive

League City, TX 77573

281-332-5612

Why does the Body Produce Antibodies against its own Red Blood Cells?

Immune Mediated Hemolytic Anemia is caused by a malfunction of lymphocytes.

Lymphocytes are white blood cells produced in the bone marrow from the mother cell

called a lymphoblast. Figure 1. One group of cells called B- Lymphocytes (B-cells)

produce the abnormal antibodies that cause IMHA. Another group of cells called T-

Lymphocytes (T-cells) get specific orders from the Thymus gland and are supposed to

control how the B-cells produce antibodies. IMHA arises when the T-cells and B-cells

are out of balance.

Figure 1 shows the origin of all blood cells in the body. In general, the immune system has two divisions, the innate and the adaptive. The innate is non-specific and operates in conjunction with the adaptive immune system. Cells involved in the innate immune system are the eosinophil, neutrophil, basophil and macrophage. The adaptive includes the T lymphocytes and B lymphocytes because they are programed to be specific against specific enemies of the body.

What is the difference between Primary and Secondary IMHA?

Immune Mediated Hemolytic Anemia has primary and secondary types. Primary IMHA

occurs about 75% of the time where the antibodies are produced against “normal” red

blood cell proteins. Secondary IMHA occurs when there is another disease affecting the

red blood cells that initiates the IMHA. Factors such as infection, antibiotic

administration, exposure to toxins, tick borne diseases, lymphoma, hemangiosarcoma

or allergic reactions can cause the B-cells to attack the red blood cells with antibodies

resulting in secondary IMHA. Secondary IMHA is usually a temporary disease that

resolves when the offending substance or infection is removed, stopped, or treated.

Primary IMHA occurs when normal red blood cell proteins are attacked abnormally.

Primary IMHA has two forms – Intravascular and Extravascular. Intravascular IMHA

has a much higher mortality rate (75%) and occurs when the antibodies activate

Complement. Extravascular IMHA occurs when nibbles are bitten off the surface of the

red blood cells by macrophages as they pass through the spleen and liver. This slower

process is still very serious but has only a 25% mortality rate.

The Importance of Complement

Complement is the name for a group of molecules present in the blood plasma that

enhances (complements) the immune system. These molecules when combined cause

intravascular hemolysis in IMHA. Complement activation is shown in Figure 2.

Figure 2. Complement activation starts when antibodies are attached to Red Blood Cells, ultimately creating the Membrane Attack Complex (knife) shown above. Once formed this knife cuts through the surface of the red blood cells see Figure 3.

Figure3. “Mr. Red Cell being attacked by activation of the complement cascade causing intravascular hemolysis in IMHA.

Figure 4. Depicts the stem cell as a superhero which produces Factor H that acts like Kevlar to protect the cell membrane from the Membrane Attack complex (knife).

NORMAL RBC➔ ANTIGEN COATED ➔ANTIBODY COATED ➔ COMPLEMENT ATTACK

Mr. Red Cell

STEM CELL ➔FACTOR H (KEVLAR) ➔PREVENTS COMPLEMENT ATTACK

Mr. Stem Cell

Factor H Protects Cells from Complement Attack

Factor H regulates complement. Factor H provides control by binding to the surfaces of

cells to protect cell surfaces from the cutting and puncturing of the knife-like

complement molecule. Like the Kevlar worn by law enforcement officials protects them

from knives and bullets, Factor H protects the cells from complement attack. Factor H is

produced by stem cells.

Stem Cells Produce Factor H

We have known for some time that Mesenchymal stem cells (MSCs) possess potent

immunosuppressive properties (5). But only recently did we understand that MSCs

produce Factor H (6). Mesenchymal stem cell therapy has been shown to stop the

damage from many auto-immune diseases and the mechanism for this effect has been

shown to be due to the production of Factor H by the mesenchymal stem cells and

subsequent protection against complement damage (7). MSCs production of Factor H

is constitutive (8). This means that Factor H is produced in relatively constant amounts

regardless of the environment or stimulation. This is an important justification for the use

of allogenic mesenchymal stem cells for the treatment of IMHA.

What is meant by Immune Tolerance? The purpose of the immune system is to remove foreign invaders. To do this it has to

recognize who is supposed to be in the body and who is alien and a potential threat to

the body. Red blood cells that are being attacked by antibodies in IMHA have been

mistakenly determined to be foreign. How did this happen and how can this problem be

fixed?

There are natural balances in the controls of the immune system that we are just

coming to understand. These balances involve the production of different types of T-

Lymphocytes (T-cells) depending on the need for immunity vs. the need to tolerate the

presence of a seemingly abnormal protein or substance. For example, our intestine

houses many species of bacteria that are arguably foreign to our bodies, yet our

immune system tolerates their presence. Mammals who maintain placental pregnancy

within their bodies must tolerate the foreign tissue of the developing fetus with different

DNA than the mother and not attack it as foreign. The surface of the skin and opening of

the mucous membranes harbor “normal” bacterial and fungal flora that must be

tolerated by the immune system. On the other hand, cells that are infected or damaged

or that have turned cancerous should be attacked and removed from the body by the

same immune system. It is this balance that is defective with IMHA.

Recent experiments involving the thymus gland have uncovered previously unknown

elements of the T-cell populations that help control these systems. The thymus is a

small gland in the chest between the left and right lungs at the level of the trachea that

has been found to be essential in the control of the immune system. T-cells are named

after the thymus because they are processed within this small organ. Mutant mice

which do not have a thymus gland spontaneously develop fatal, widespread, early-onset

autoimmunity (9). Removal of the thymus gland in three-day old mice led to the rapid

development of autoimmunity that was corrected by the infusion of T-cells from adult

mice. This research advanced our understanding of the creation and management of

the T-cell lineage and how it controls the immune system. Certain T-cell types regulate

or keep in check the aberrant or overactive activity of other T-cells. When the immune

system functions normally, the immune system produces a population of T-cells that are

regulatory called regulatory T cells or Tregs that are specialized for immune

suppression (10). In addition, it is becoming apparent that every immune reaction not

only creates specific B-Cells and T-Cells but also Tregs and the balance between the

populations of cells is critical for the proper control of the immune response. IMHA is an

example of an excessive attack on self-antigens as a result of improper immune

tolerance resulting from dysregulation or deficiency of the Tregs (1) (10).

What is the role of stem cells in the immune system

Mesenchymal stem cells exert powerful control over the creation and modulation of

Tregs through the release of powerful chemicals called cytokines. Mesenchymal stem

cells release the cytokine Transforming Growth Factor Beta (TGF-β) which induces the

development of Tregs (4) (9) (8) (11). This is an important justification for the use of

allogenic mesenchymal stem cells for the treatment of IMHA.

To understand the implications of treatment choices made for the benefit of pets with

IMHA it is best to understand how the immune system works normally when fighting

disease and how it is deranged in the case of IMHA.

How does the body differentiate between self and foreign material?

Lymphocytes are on constant surveillance – their job is to detect and remove invaders

and abnormal cells. All lymphocytes are formed in the bone marrow, but, where they

mature determines their function. B-cells mature in the bone marrow. T-cells mature in

the thymus. The thymus is like “Boot Camp” for T-cells. The T-cells have to memorize a

list of all the proteins in the body. This list is called the Major Histocompatibility Complex

I (MCH I). If the T-cell finds a cell with something not on the list, such as an infected cell

or a cancer cell, it knows to attack it. The thymus tests each T-cell to make sure they

have memorized the list and that they will not attack normal tissues. T-cells that pass

the MHC I test are labeled with a protein called CD8+. Some T-cells have a second list,

the Major Histocompatibility Complex II (MCH II). This list has additional information

about cells that process antigens. These T-cells are labeled CD4+ and they receive

their orders from Antigen Processing Cells (APCs) in the lymph nodes. Their job is to

tell the T-cells what to attack. Any T-cell that does not pass “Boot Camp” is killed. T-

cells that pass “Boot Camp” are termed naïve T-cells or TH0 cells. Only about 2%

survive “Boot Camp”.

What tells the T-cells to attack?

These CD4+ T-Cells cells (TH0 cells) that passed “Boot Camp” are ready for battle but

do not know who the enemy is or how to fight them. The process of activation of a TH0-

cell is a two-stage process. Stage one occurs when the TH0 cells are shown who their

enemy is by an Antigen Presenting Cell (APC). This is like being shown a photo of the

enemy. Once the enemy is known the TH0 cells are then fully activated in the second

stage of the activation process. This stage results in determining the type of “soldier”

that the Boot Camp graduate will become. This second stage activation and

programming determines the branch of the armed forces, the type of soldier and the

method for killing the enemy that will be employed once he is activated.

The military analogy is that the soldier receives an enemy photo from an APC then his

“orders” that directs his future in military service. “Orders” come in the form of cytokines.

These cytokines affect the cell’s DNA and make actual transformations in the cell type.

The cell will then take on a different role and will in turn secrete different cytokines that

will allow him to perform his mission against the enemy in the photo. This mission

usually involves recruiting and directing B-Cells to produce antibodies against the

enemy. The new T-Cell will also have the ability to direct other parts of the innate

immune system through the use of newly formed cytokines that have been unlocked in

his DNA.

What stops the T-cell attack once the job is done?

Different soldier types are required because the body must defend different fronts (the

gut, the skin, mucous membranes, respiratory tract etc.) from different invaders: viruses,

fungi, worms, bacteria and cancer. In addition, once the war is over, there must be

peacekeepers (Tregs) who stop the battle and prepare the body for repair and

regeneration. For example, TH1 lymphocytes respond to interleukin 12. In response to

these chemical orders they secrete interferon and interleukin 2 which allows them to

control the less trained cells of the immune system. TH1 cells act on macrophages,

cytotoxic T cells and IgG antibody producing cells. When Tregs malfunction, we get

autoimmune disease. TH1 malfunction is the cause of Type 4 hypersensitivity, flea

allergy in dogs and Type I diabetes. Appendix 1 shows the names of many different cell

types or soldiers, their chemical signals and the diseases that result when they

malfunction.

How do Treg Lymphocytes Control Immune Reactions?

Our newest understanding is that upon the issuance of “orders” to attack a certain

enemy, countervailing orders are also issued simultaneously to create Treg cells

directed to protect the same enemy. In addition, these Treg cells have certain

advantages over the regular army version of the T-cells. Treg cells could be considered

Seal Team 6 type cells as they are faster, multiply quicker, have a higher affinity for the

target and can outmaneuver the typical T-cell (10). They can even kill the T-cells or B-

cells if necessary. They are there to regulate the war to prevent the soldiers from going

too far in their mission. They are an essential component of the balance of the immune

system and when the immune system goes awry, they bring it back into order.

Tregs function to suppress the immune responses to beneficial microbes in the gut and

act to protect the developing fetus from harm. Tregs also have been found to protect

cancer cells from being destroyed and excessive Treg activity is being actively

researched as a method for cancer therapy.

The Role of Drug Therapy in IMHA Most of the medications used against IMHA are considered anti-metabolites. That is,

they inhibit metabolism as their mode of action. They turn off DNA synthesis, or

synthesis of one of the essential elements of DNA function or inhibit the communication

between the outer parts of the cell and the DNA. These medications do this to all cells

of the body equally without regard for the knock-on effects to other tissues they might

cause (liver damage, blood clot formation loss of immune function etc.).

Lets consider the common therapies for IMHA and IMTP, their mode of action, side

effects and the results of their use.

1. Glucocorticoids Glucocorticoids (prednisolone, prednisone, methylprednisolone and dexamethasone) bind receptors in

all cells of the body that turn off parts of the DNA (12). This results in reduced release of the chemicals

that cause cell death, reduced release of inflammatory chemicals, reduction in the the number of

receptors for antibodies which blocks cell destruction (13). The part of the DNA that processes foreign

antigens is blocked resulting in reduced immune function (14).

The side effects of glucocorticoids are well documented; steroid resistance occurs in up to 30% of

patients (15). We also see thrombosis (blood clots) (16), Gastrointestinal ulceration, liver disease, and

reduced resistance to disease. Signs of Cushing’s disease are seen with prolonged use of glucocorticoids.

The efficacy of glucocorticoids is not well documented. “To the best of our knowledge, glucocorticoids

have never been subjected to rigorous evaluation by randomized double-blinded, placebo-controlled

trials for treating canine immune-mediated disease (2)”. Glucocorticoid therapy itself carries

considerable morbidity, and treatment failure (2). Nevertheless, withholding glucocorticoids to compare

the effect of another immunosuppressive drug against placebo is cited as unethical (17). 6-month

survival from IMHA is reported to be 72% when used with azathioprine (18) (3).

2.Azathioprine Azathioprine is a T-cell inhibitor. It has an 11-day lead time which may be too long for severe or acute

cases. The side effects include bone marrow suppression (19), acute pancreatitis, hepatopathy and

gastrointestinal distress (20). There are no prospective controlled studies. A few large retrospective

studies lend limited support to its use (21) (2).

3. Cyclophosphamide (Cytoxan) Cyclophosphamide kills T-lymphocytes and regulatory T-cells. It is commonly used for cancer

chemotherapy. Side-effects include hemorrhagic cystitis and bone marrow suppression (2). Efficacy

studies show increased morbidity (makes conditions worse) Does not justify its use as an

immunosuppressive agent (2).

4. Cyclosporin (Atopica) Cyclosporin blocks T cell DNA transcription. It also blocks Interleukin 2 formation. The side-effects of

cyclosporine are gastrointestinal upsets and anorexia. Cyclosporin has good studies showing that it is

effective for atopy (22) and anal furunculosis (23). Prospective controlled studies for other conditions

are lacking (2).

5. Human Intravenous immunoglobulin Human Intravenous immunoglobulin competitively inhibits the binding of canine immunoglobulin G to

macrophages (24). It works from day 1 but only lasts 3 weeks (25).The side effects include increased

blood clotting and it increases inflammation in healthy dogs (26). Although IVIG has been used for

various conditions in dogs, efficacy data are limited to case reports and two small controlled studies.

6. Vincristine Vincristine increases platelet numbers. The main side-effect is myelosuppression in higher doses, and it

has been shown to cause abnormal platelet function in vitro (27).

7. Leflunomide Leflunomide is only available in some countries. It inhibits T and B cell proliferation. It has profound anti-

inflammatory effects. It may induce regulatory T cells (2). Leflunomide is used when pets are refractory

to glucocorticoids or the side-effects are intolerable (28). Controlled studies in the dog are confided to

renal transplantation.

8. Mycophenolate mofetil Mycophenolate mofetil blocks purine biosynthesis which blocks T cell and B cell proliferation. It may kill

activated T- cells (29). It has low toxicity and side-effects are limited to gastrointestinal effects. Is being

used for refractory myasthenia gravis, IMHA and pemphigus vulgaris (2).

9. Clodronate (LC) Clodronate is used for management of hypercalcemia from vitamin D intoxication in dogs. It kills

macrophages and dendritic cells that eat antibody tagged red blood cells. This drug is well-tolerated and

has few side effects. Controlled trials of LC are in progress at Colorado State University (CSU) for

management of IMHA in dogs (30).

10. Plasmapheresis Plasmapheresis is the partial removal of autoantibodies, immune complexes and complement

components from the blood. It can increase the chances of blood clots in the lungs. Most commonly

used for IMHA as a rescue therapy. Has some utility in this regard in dogs. Plasmapheresis has been

studied in Immune Mediated Thrombocytopenia Patients and no patients showed a response (31). As

such it is no longer recommended in humans for chronic IMHA or IMTP.

11. Splenectomy Splenectomy is the removal of the spleen as the organ most responsible for the destruction of antibody

coated red blood cells. Surgery is usually done when medical therapy has failed. Complications include

bleeding, infection, thrombosis, prolonged hospitalization, readmission to the hospital and therapeutic

failure. Results are extremely variable (32). No prospective studies available.

12. Allogenic Mesenchymal Stem Cell Therapy Allogenic mesenchymal stem cell therapy is the intravenous use of adipose derived stem cells from the

same species. In intravascular IMHA where red blood cells are destroyed by complement activation (3)

Stem cells block Complement attacks by secreting Factor H (27)(28)(29) (30). Regulatory T-cells increase

red cell tolerance (2). Lack of red cell tolerance causes IMHA. Stem Cells create Regulatory T-cells by

production of TGF-β (31). Stem Cells down regulate the production of antibodies by B-cells and down

regulate macrophage consumption of red blood cells. The stem cells’ response is modulated by the

relative need and will not affect the rest of the immune system’s ability to fight disease. Allogenic Stem

Cells are not allergenic or recognized by the immune system. They have been shown safe when given

intravenously in numerous human Phase I and Phase II Clinical Trials (33), (34), (4).

Randomized clinical trials for IMHA therapy in dogs with Stem Cells are lacking. It should be noted that

neither are there randomized controlled trials for the use of Parachutes to prevent death from

gravitational challenges. Gordon C S Smith’s article explores the need for common sense and

observation as an alternative to this method of determination of effectiveness of a therapeutic protocol.

Such is the case with stem cell therapy (35). More general research has shown that stem cells possess

anti-inflammatory and immunomodulatory properties; primarily by modulating the type and function of

T-Lymphocytes (36), (37), (38), (39), (40), (41), (42), (43), (44) (45). Unlike pharmaceutical treatments

that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and

signals at variable concentrations in response to local microenvironmental cues (4).

How do Drugs act on the Immune System to treat IMHA? The drugs listed above all interfere with DNA in some way. The reason these

medications work for IMHA is that IMHA is caused by lymphocytes and lymphocytes are

among the most rapidly dividing cells of the body. Lymphocytes use their DNA more

and are therefore, more affected by substances that damage DNA than other cells.

Tregs divide faster, have a higher metabolic rate and are more affected than other

lymphocytes by these medications (10). These medications do nothing to cure the

disease, or to stop the creation of the offending antibodies. The medications only act to

stop the manufacture of the antibodies after they have been created. In some cases,

the side-effects of the drugs are more damaging than the disease.

The Case for Stem Cells in the Treatment of IMHA Unlike these anti-metabolites, Mesenchymal Stem Cells are site regulated. Stem cells

secrete bioactive factors and signals at variable concentrations in response to local

micro-environmental cues and actually offer a cure for IMHA (4). This is another

important justification for the use of allogenic stem cells for the management of IMHA.

The importance of Regulatory T Cells in Preventing Disease

There is a large body of research demonstrating the importance of TH1 and TH2 subsets

of T-cells in preventing disease. TH1 cells produce Interferon (IFN)-γ which stimulates

cell mediated immunity against tumor cells or intracellular bacterial or viral infected

cells. In contrast TH2 cells produce many cytokines including interleukin (IL)-4, IL-5, IL-9

and IL-13 which activate B-cells allowing them to transform into the plasma cell clones

that produce antibodies. More recently, other subsets of cells such as the TH17 cells

that play a role in the battle against infectious agents at mucosal barriers. And TH9 cells

that are responsible for the response against helminthes (parasitic worms). The killer T-

cells all have their list of good versus bad cells, but they do not act until the receive their

orders in the form of chemical messages (cytokines) from the Antigen Processing Cells.

The cytokines tell the cells who to attack and with what weapons. Stem cells produce

these cytokines that determine the type of T-cell and the “orders” for the action of these

T-cells based on the needs of the animal. Stem Cell Therapy is the delivery of stem

cells in the appropriate numbers and into the appropriate location to effect change to the

benefit of the patient.

Stem Cell Function in IMHA

We know stem cells have multiple actions. Stem cells can home in on areas of

inflammation and get to where the disease is. Stem cells are bioactive, their action is

dynamic in response to the disease. Stem cells are potent anti-inflammatory agents.

Stem cells can turn off the inappropriate immune reaction without leaving the patient

susceptible to other infection. Stem cells correct the underlying problem not just treat

the symptoms. Stem cells act immediately; there is no lead-in time. Stem cells are safe.

Appendix 1

Naïve T- Cell

T-Cell Subset “Soldiers”

Cytokine “Orders

Received”

Cytokine Produced

“Orders Given”

Cellular Command

Aberrant Overreaction

TH1 IL-12 IFN-γ, IL-2 Macrophages Cytotoxic T cells IgG B Cells

CD4 T Cells

Type 4 hypersensitivity, Type I diabetes Tuberculin reaction

TH2 IL-4, IL-2 IL-4, IL-5 IL-9, IL-10 Humoral Immunity

development

Eosinophils Basophils Mast Cells IgE B Cells

Allergic rhinitis, atopy, asthma Type 2 and Type 3 hypersensitivity, Immune Mediated Hemolytic Anemia

TFH IL-21, IL-4 Antibody

production

TH9 IL-9 Allergy

TH17 IL-17 Inflammation Extracellular

pathogens and fungi. Maintain

mucosal barriers

Neutrophils Rheumatoid arthritis

THαβ IFN-αβ IL-10 Virus NK cells CD8 Killer T cells

Myasthenia gravis, Grave’s disease

Treg17 TGF-β plus IL-6 IL-17 and IL-10 Suppression of Autoimmune

TH3 TGF-β Tolerance

Treg IL-10 Suppression

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