Three cases of Immune Mediated Hemolytic Anemia (IMHA) are described that have been treated with allogenic mesenchymal stem cell therapy. Each case had received traditional immunosuppressive medications and two cases had received multiple transfusions. Clinical improvement was noted immediately after stem cell administration as documented by rising packed cell volumes and improved clinical signs. There were no adverse reactions to the stem cell therapy.
Three cases of Immune Mediated Hemolytic Anemia are described that have been treated with allogenic mesenchymal stem cell therapy. Each case had received raditional immunosuppressive medications and two cases had received multiple transfusions. Clinical improvement was noted immediately after stem cell administration as documented by rising packed cell volumes and improved clinical signs. There were no adverse reactions to the stem cell therapy.
Primary Immune Mediated Hemolytic Anemia develops in two forms, intravascular and extravascular (1) .The intravascular form is precipitated by the formation of anti-erythrocyte IgM antibodies that activate complement which results in intravascular hemolysis. Extravascular red cell destruction occurs in the spleen and liver as a result of macrophage mediated removal of IgG tagged whole red blood cells (1) . Ultimately IMHA is thought to be a disease of lack of immune tolerance to red blood cell surface antigens. Immune tolerance is mediated through a subset of T-lymphocytes with immune regulatory cytokine secretion (Treg cells) (2) (3) (4) . Traditional therapy for IMHA involves blood transfusions as needed to maintain homeostasis while immunosuppressive medications are used to halt the anti-red blood cell antibody production and suppress the reticuloendothelial system (5) (6) . This therapy is deemed effective as a cure in about 1/3 of the cases, while another 1/3 of the cases become symptom free if immunosuppressive medications are given and the final 1/3 of the cases die from the effects of rapid uncontrollable hemolysis. This last group of pets usually succumbs within the first two weeks of the disease and are most likely associated with the intravascular form of the disease (7) (8) . Allogenic mesenchymal stem cell therapy has been documented to have anti-inflammatory properties (9) (10) (11) , mediate autoimmune disease, and stimulate the production of Treg cells in the presence of inflammation (12) (13) . Stem cells also produce Factor H which is the primary regulator of complement (14) (15) (16) .Stem cell therapy seems to be an effective tool in stopping the immune destruction in IMHA. Allogenic mesenchymal stem cells are non-immunogenic, easily accessible, can be expanded to clinical scales in a short period of time, and can be biopreserved and shipped for point-of care delivery to the pet with minimal loss of potency enabling these donor cells to be used in clinical cases that are too sick or too time sensitive for autologous fat processing.
The dog has no previous history of disease and was completely normal 72 hours prior to initial presentation. Two nights prior he vomited and was given carprofen, propectalin and canned pumpkin by the owner. Presented to the primary care veterinarian the next day for lethargy. Primary care veterinarian diagnosed IMHA, started prednisone, azathioprine, clopidogrel, cerenia and sucralfate and referred to the referral hospitals. There, he was diagnosed with nonregenerative anemia and laboratory confirmation of PCV of 13%, and elevated total bilirubin. An immediate transfusion of pRBC 125ml was given then repeated 6 hours later. Additional diagnostic tests did not identify secondary causes of anemia, therefore, primary extravascular immune-mediated hemolytic was diagnosed. However, his total bilirubin was 11 suggesting intravascular hemolysis. He was on the following immunosuppressive medications: Azathioprine (12.5mg PO q24h), Cyclosporine (25mg PO q12h), Prednisone (5mg PO q12h) and intestinal protectants and appetite stimulants. Over the next 10 days the pet received multiple transfusions: His PCV varied wildly day to day from 12 to 25 necessitating six successive transfusions. The clinicians attempted therapeutic plasmapheresis with partial completion. The pet developed Transfusion Related Acute Lung Disease (TRALI), was subsequently place into an oxygen cage. Allogenic mesenchymal stem cells were administered by IV infusion (6 million cells in 0.9% NaCl) on day 7, day 10 and day 14 post presentation. Following stem cell therapy, no additional transfusions were given. The pet was discharged on the same immunosuppressive medications. Laboratory confirmation of regenerative anemia. Three months later, three weeks after azathioprine was discontinued, the pet’s PCV dropped to 17%. Another transfusion and two more doses of stem cells resolved the relapse.
Past two days prior to presentation owners relayed that she was not her normal self and lethargic. Pet presented to referral veterinary medical service for history of anemia and thrombocytopenia. Hct 24.9% Platelet count 59,000. Abdominal ultrasound performed revealed hepatosplenomegaly otherwise unremarkable. Saline autoagglutination was positive. Started on immunosuppressive drugs: Prednisolone (10mg PO q12h), Mycophenolate (125mg PO q12h), and Intestinal protectants. The PCV dropped to 14% over the next 24 hours and pRBC were given. The thrombocytopenia resolved but over the next 7 days five transfusions were given almost every day as the PCV varied from 14 to 27.
Cyclosporine (50mg PO Q12h) was added 7 days into therapy. Allogenic mesenchymal stem cells were administered IV at the dose of 1 million cells per kg in 0.9% NaCl on day 9 and day 13 post presentation. The pet did not receive another transfusion after the stem cell therapy and has rapidly improved in clinical presentation as well as PCV. She was discharged on the same immunosuppressive drugs and has gradually been weaning them. Weekly evaluations show spherocytosis is still present indicating immune hyperactivity still present.
Pet presented to the primary veterinarian one day after the pet stopped eating. PCV 21% with 15 to 20 spherocytes per oil immersion field typical for IMHA. This pet had previously had IMHA and was no longer on medications (taken off azathioprine 1 month prior to this event). Over the next 10 days the pet was treated with dexamethasone, cyclosporine (50mg PO q12h) and intestinal protectants. No transfusions were given. 9 days post presentation the pet was given 10 million allogenic mesenchymal stem cells. The PCV went up 3 points within 24 hours. The next stem cell injection was given three days later and the PCV went from 17% to 25% in 24 hours. He was discharged on the same medications the next day.
The therapeutic strategy for managing severe cases of Immune Mediated Hemolytic Anemia(IMHA) as demonstrated in Case #1 and Case #2 is using blood transfusions to maintain homeostasis while allowing enough time for an immunosuppressive protocol to take effect. Less severe cases (Case #3) the strategy is to manage the IMHA with immunosuppressive drugs alone. In all three cases, clinical improvement as evidenced by increased PCV values was achieved shortly after the administration of allogenic mesenchymal stem cells. No further transfusions were required and the pet’s clinical signs rapidly improved. The addition of allogenic mesenchymal stem cell therapy to the therapy regime apparently accelerated the return to normalcy. On the other hand, in all three cases, the timing of the administration of the stem cells also coincided with the expected time of efficacy of the administration of the immunosuppressive drugs. The clinical impression, however, was that the stem cells were in fact the determining factor in the success of these cases. It should be noted that Case #1 relapsed three weeks after the discontinuation of azathioprine and recovered rapidly upon administration of blood, stem cells and azathioprine without the typical lag time for azathioprine to take effect, suggesting the effects were due to the stem cells. The rationale for using stem cell therapy was questioned by many of the referral veterinarians and several refused to administer the cells on these grounds. Stem cell therapy in the treatment of IMHA offers therapeutic advantages focused at the root of the IMHA disease.
Research has shown that stem cells possess anti-inflammatory and immunomodulatory properties, primarily by modulating the type and function of T-Lymphocytes (17) (18) (19) (20) (21) (22) (23) (24) (12) . In addition complement activation and subsequent hemolysis is a major factor in IMHA mortality (6) (2) (1) (8) and was the mechanism for the rapid destruction of the transfused cells in Cases #1 and #2. Mesenchymal stem cells constitutively produce Factor H which is the primary regulator of complement activation (16) . Factor H production from intravenous stem cell infusions effectively shut down intravascular hemolysis secondary to complement activation thereby potenitially saving the lives of the pets affected by this form of IMHA. Stopping production of the billions of anti-red blood cell antibodies per second is strategic for winning the battle in IMHA. Immunosuppressive drugs are the traditional linchpin functioning as antimetabolites against antibody producing lymphocytes. Stem cells augment this strategy by producing Treg cells that produce a variety of cytokines that have the ability to kill these cells, effectively stopping the anti-red blood cell IgG antibody production responsible for the extravascular hemolysis (spherocytes) noted in Case #3 of this report (2) . The processing of red cell surface proteins as “not-self” by macrophages and dendritic cells in the reticuloendothelial system is the underlying origin for anti-red blood cell antibody production in IMHA. Stem cells make Treg cells by producing the soluble protein Transforming Growth Factor Beta (TGF- β ) (11) (13) . TFG- β converts T-lymphocytes to Treg lymphocytes (25) and Treg cells have the ability to direct the macrophages and dendritic cells to recognize the red blood cell antigens again as “self” (2) . In fact these Treg cells also produce memory Treg cells that remember that the red cell antigens are “self” in the future, effectively having the potential to cure IMHA. The therapeutic effect of stem cells on IMHA needs further evaluation for dose of cells, timing of administration and when to discontinue therapy.
Allogenic mesenchymal stem cells are sourced from canine donors’ adipose tissues. The cells are processed, cultured and expanded following protocols described elsewhere. The cells are from a single donor and are “fresh” cells in that they come from actively growing cultures of less than 5 generations or passages. These cells are not recently reconstituted cryopreserved cells. Heparin is added to theculture medium (4U/ml) 12 hours prior to harvest to enhance therapeutic effectiveness and reduce the first pass effect (26) (27) . The cells are then washed with PBS twice to remove residual heparin. Alternatively, the pet may be pretreated with heparin (200U/kg). The cells are then harvested from their cultures, counted with an automated cell counter, tested for viability and placed in a single use vial for the specific pet. The cells are dosed at 1 million cells per kg. Overnight shipping to the point-of-care facility results in minimal loss of potency, enabling these donor cells to be used in clinical cases that are too sick or too time sensitive for autologous fat processing. They are then diluted and administered intravenously over a 30-minute time period.
The author, Dr. Garner owns and operates the stem cell laboratory at Safari Veterinary Care Centers in League City, Texas. As such he acknowledges a financial interest in stem cells provided for use in the cases in this report.
1. Canine Immune-Mediated Hemolytic Anemia: Pathophysiology, Clinical Signs, and Diagnosis. Andrea Balch, Andrew Mackin. 2007, compendium.
2. Novel immunotherapies for immune-mediated haemolytic anaemia in dogs and people. James W Swann, Oliver A Garden. London : Elsevier, October 15, 2016, The Veterinary Journal, Vol. 207, pp. 13-19.
3. New Insights in the Pathogenesis of Autoimmune Hemolytic Anemia. Barcellini, Wilma. Milan, Italy : s.n., September 7, 2015, Transfusion Medicine and Hemotherapy, Vol. 42, pp. 287-293.
4. Immunomodulatory drugs and their application to the management of canine immune-mediated disease. N. T. Whitley, M.J. Day. s.l. : British Small Animal Veterinary Association, 2011, Journal of Small Animal Practice, Vol. 52, pp. 70-85.
5. Canine Immune-Mediated Hemolytic Anemia: Treatment and Prognosis. Andrea Balch, DVM, MS, DACVIM,Andrew Mackin, BVMS, DACVIM. s.l. : Compendium, April 2007, Compendium.
6. DAY, M. J. & MACKIN, A. J. (2008). Immune-mediated haematological disease. In Clinical Immunology of the Dog and Cat. 2nd. Edn. London, UK : Manson Publishing, 2008. pp. 94-121.
7. Immune-mediated haemolytic anaemia – a retrospective study – focus on treatment and mortality. ALLYN, M. E. & TROY, G. C. 2007, Journal of Veterinary Internal Medicine, Vol. 11, p. 131.
8. Idiopathic Immune-Mediated Hemolytic Anemia: Treatment Outcome and Prognostic Factors in 149 Dogs. C.J. Piek, G. Junius, A. Dekker, E. Schrauwen, R.J. Slappendel, and E. Teske. Utrecht, The Netherlands : s.n., 2008, J Vet Intern Med, Vol. 22, pp. 366-373.
9. Immunobiology of mesenchymal stem cells. S Ma, N Xie, W Li, B Yuan, Y Shi*, and Y Wang. s.l. : Macmillan Publishers Limited, 2014, Cell Death and Differentiation, Vol. 21, pp. 216-225.
10. Mesenchymal stromal cells and the innate immune response. Katarina Le Blanc, Lindsay C. Davies. s.l. : Elsevier, 2015, Immunology Letters, pp. 140-146.
11. Secretion of immunoregulatory cytokines by mesenchymal stem cells. Dobroslav Kyurkchiev, Ivan Bochev, Ekaterina Ivanova-Todorova, Milena Mourdjeva, Tsvetelina Oreshkova, Kalina Belemezova, Stanimir Kyurkchiev. 5, s.l. : Baishideng Publishing Group Inc., World J Stem Cells, Vol. 6, pp. 552-570. 1948-0210.
12. Mesenchymal stem cells as therapeutics. Parekkadan B, Milwid JM. 2010, Annual Review Biomed Eng, Vol. 70, pp. 325–330.
13. Activation, homing, and role of the mesenchymal stem cells in the inflammatory environment. Lukáš Zachar, Darina Bačenková,Ján Rosocha. [ed.] University Hospital of L. Pasteur. Košice, Slovak Republic : Dove Press , 12 15, 2016, Journal of Inflammation Research, Vol. 9, pp. 231-240.
14. Complement factor H and the hemolytic uremic syndrome. John P. Atkinson, Timothy H.J. Goodship. s.l. : The Rockefeller University Press, June 11, 2007, Journal of Experimental Medicine, pp. 1245-1248.
15. Mesenchymal Stem Cells Inhibit Complement Activation by secreting Factor H. Zhidan Tu, Z, Qing Li ,Feng Lin. [ed.] Inc. © Mary Ann Liebert. 2010, Stem Cells and Development, Vol. 19 Number 11.
16. Mesenchymal Stem Cells Control Complement C5 Activation by Factor H In Lupus Nephritis. Haijun Ma, Chang Liu, Bingyu Shi, Zhuoya Zhang, Ruihai Feng, Minghao Guo, Liwei Lu, Songtao Shi, Xang Gao, Wanjun Chen, Lingyun Sun. s.l. : Elsiever, 2018, EBioMedicine, Vol. 32, pp. 21-30.
17. Human mesenchymal stem cells modulate allogeneic immune cell responses. Aggarwal S, Pittenger MF. 2005, Transplantation, Vol. 105, pp. 1815–1822.
18. Anti-inflammatory effects of mesenchymal stem cells: novel concept for future therapies. Iyer S, Rojas M. 2008, Expert Opin Biol Ther, Vol. 8, pp. 569-582.
19. Uccelli A, Moretta L, Pistoia V. Uccelli A, Moretta L, Pistoia V. 2008, Nat Rev Immunol, Vol. 8, pp. 726– 736.
20. Stem cells and cell therapies in lung biology and lung diseases. Weiss DJ, Bertoncello I, Borok Z, Kim C, Panoskaltsis-Mortari A,Reynolds S et al. 2011, Proc Am Thorac Soc, Vol. 8, pp. 223–272.
21. MSCs inhibit monocyte-derived DC maturation and function by selectively interfering with the generation of immature DCs: central role of MSC-derived prostaglandin E2. Spaggiari GM, Abdelrazik H, Becchetti F, Moretta L. 2009, Blood, Vol. 113, pp. 6576–6583.
22. Human leukocyte antigen-G5 secretion by human mesenchymal stem cells is required to suppress T lymphocyte and natural killer function and to induce CD4þCD25highFOXP3þ regulatory T cells. Selmani Z, Naji A, Zidi I, Favier B, Gaiffe E, Obert L et al. 2008, Stem Cells, Vol. 26, pp. 212–222.
23. Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications. Yi T, Song SU. 2012, Arch Pharm Res, Vol. 35, pp. 213–221.
24. The chemokine system in diverse forms of macrophage activation and polarization. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. 2004, Trends Immunol, Vol.
25, pp. 677–686. 25. Conversion of Peripheral CD4+CD25- Naive T Cells to CD4+CD25+ Regulatory T cells by TGF-B Induction of Transcription Factor Foxp3. WanJun Chen, Wenwen Jin, Neil Hardegen, Ke-jian Lei, Li Li,Nancy Marinos, George McGrady, and Sharon M. Wahl. 2003, The Journal of Experimental Medicine, Vol. 198 Number 12, pp. 1875–1886.