Join Us | Latest Articles | Contact

Journal Home


Editorial Board


Recent Articles


Submit to this journal


Special Issues


Current issue

International Journal of Surgery Research and Practice





DOI: 10.23937/2378-3397/1410043



Alternatives to Liver Transplantation in Pediatric Liver Diseases

Clara T Nicolas1,2* and Scott L Nyberg1,2


1William J Von Liebig Transplant Center, Mayo Clinic, Rochester MN, USA
2Department of Surgery, Mayo Clinic, Rochester MN, USA


*Corresponding author: Clara T Nicolas, MD, William J Von Liebig Transplant Center, Department of Surgery, Mayo Clinic, 200 1st Street SW, Rochester, MN, 55905, USA, Tel: 507-284-1606, E-mail: Nicolasmartinez.clara@mayo.edu
Int J Surg Res Pract, IJSRP-3-043, (Volume 3, Issue 2), Mini Review; ISSN: 2378-3397
Received: November 30, 2015 | Accepted: July 15, 2016 | Published: July 18, 2016
Citation: Nicolas CT, Nyberg SL (2016) Alternatives to Liver Transplantation in Pediatric Liver Diseases. Int J Surg Res Pract 3:043. 10.23937/2378-3397/1410043
Copyright: © 2016 Nicolas CT, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.



Abstract

Inherited metabolic disorders and acute liver failure (ALF) are often indications for liver transplantation in pediatric patients. Liver transplantation, however, is limited by the shortage of donor organs, as well as by the need for chronic immunosuppression. This review focuses on the latest advancements made in the field of liver regenerative medicine as possible future alternatives to pediatric liver transplantation or as a means of temporary liver function support. Cell transplantation offers great promise for the treatment and long-term correction of inherited metabolic disorders, especially when ex vivo gene therapy is combined with autologous hepatocyte or induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) transplant. Bioartificial liver (BAL) systems are currently being tested that may be able to bridge patients to either liver transplantation or endogenous liver regeneration, in the case of ALF. Still, further research is required before these forms of cell therapy are incorporated into clinical practice: the optimal cell type for both cell transplantation and BAL systems must be found, methods for the large-scale expansion of these cells must be created, and safety concerns pertaining to each cell type must be addressed.


Introduction

Liver transplantation is to date the only proven treatment for pediatric end-stage liver diseases, including biliary atresia and other cholestatic diseases, as well as acute liver failure (ALF) and a number of inherited metabolic disorders [1]. Although the success of this operation has improved significantly in the past few decades, and the scarcity of organs has been to some extent circumvented by the utilization of split-liver grafts and living-related donors [2], it requires life-long immunosuppression, with the medical complications and growth restrictions that this entails [3]. Alternatives to liver transplantation are actively being sought after, and cell therapy has shown promise for the treatment of both inherited metabolic disorders and acute liver failure.


Inherited metabolic disorders

Metabolic disorders are the second most common indication for pediatric liver transplantation after biliary atresia [4]. They can be divided into 1) diseases that result in structural liver damage with liver failure or cirrhosis, such as α1-antitrypsin deficiency, and 2) diseases that are due to an enzymatic defect expressed solely or predominantly in the liver, but that result in injury of other organ systems, such as Crigler-Najjar type 1 syndrome [5]. In these diseases cell transplantation offers the potential for long-term correction of the metabolic deficiency [6].

Furthermore, primary hepatocyte or stem cell-derived hepatocyte-like cell (HLC) transplantation, delivered into the liver via the portal vein, is less invasive than orthotopic liver transplantation, and as the native liver is not removed the transplanted cells need not replace all hepatic functions, but only improve the single enzyme deficiency [7]. For the treatment of metabolic disorders, cell transplantation aims at the addition of cells rather than at the replacement of diseased cells.

Primary hepatocyte transplantation has been used to treat a number of metabolic disorders in both adults and children, including familial hypercholesterolemia, Crigler-Najjar syndrome type 1 (CNS1), urea cycle defects (UCD), infantile Refsum disease, glycogen storage disease type Ia, and progressive familial intrahepatic cholestasis, with clinical improvement and partial correction of the metabolic abnormality in most cases [8].In children, most experience in liver cell transplantation has been acquired in the treatment of CNS 1 and UCD [9]. The success of hepatocyte transplantation in CNS 1 is easily monitored through reduction of plasma bilirubin [10], and its beneficial effects have been reported to last up to 11 months [11]. Management of hyperbilirubinemia in a CNS 1 infant patient was also achieved through transplantation of hepatic progenitor cells [12]. In UCD, periods of hyperammonemia and clinically relevant crises were shown to be reduced during an observation period of up to 13 months [13]. Individual results are encouraging, but controlled clinical trials are necessary to evaluate the overall significance of hepatocyte transplantation for the treatment of metabolic diseases [14]. Furthermore, with allogeneic hepatocyte transplantation the issues inherent to rejection and immunosuppression remain [15], and its use is limited by the available supply of liver tissue [16]. Autologous hepatocytes can also be used, but this involves performing a liver resection.

An alternative is the production of autologous stem cell-derived HLCs. With the development of stem cell technology, and especially human induced pluripotent stem cells (hiPSCs), the treatment of hereditary liver disease can be taken a step further: patient-specific therapies can be created by combining genetic correction with autologous cell transplantation [17,18]. This allows for the bypassing of the two main issues inherent to treatment with embryonic stem cells (ESCs): ethical concerns raised by the destruction of embryos, and the possibility of immune incompatibility. Disease-free autologous hiPSCs are generated through ex vivo gene therapy [19], and the genetically-corrected hiPSCs may then be differentiated and used for transplantation. A patient-specific, disease-free line of hiPSCs can be obtained in 4-5 months [20]. To date, α1-antitrypsin deficiency and familial hypercholesterolemia have both been genetically corrected in hiPSCs [21,22]. This can also be done using autologous hepatocytes, but as discussed earlier these must be obtained through liver resection. Clinically, successful ex vivo gene therapy and autologous hepatocyte transplantation has been performed only once, for familial hypercholesterolemia [23]. More recently, a combination of ex vivo gene therapy with a lentiviral vector encoding FAH and autologous hepatocyte transplantation was used to correct hereditary tyrosinemia type 1 in an FAH-deficient pig model [24]. In contrast to hepatocyte transplantation [25], there are no established large animal models of human metabolic disease treated successfully with stem cell-derived HLCs [26].

Genetically-corrected autologous hepatocyte or stem cell-derived HLC transplantation may be the logical next step in the treatment of inborn errors of metabolism. However, several important limitations to widespread clinical use of cell transplantation for correction of metabolic deficiencies still exist. Results, although promising, are still modest, and evidence of long-term efficacy is lacking. This may in part be due to the low levels of engraftment seen in cell transplantation. Some diseases, such as hereditary tyrosinemia type I and α1-antitrypsin deficiency, inherently provide a natural selective advantage for the transplanted cells [27,28], but in other cases injury to the recipient liver or other methods to increase engraftment may be necessary [29,30]. Furthermore, and although cell transplantation is far less invasive than orthotopic liver transplantation, it has on rare occasion been associated with complications including portal vein thrombosis [31,32]. Finally, there are other limitations to cell therapy that are specific to the use of stem cells particularly: stem cell-derived HLCs have not yet reached a full degree of functional maturity, and the issue of their potential for tumorigenicity must be addressed [33].

Cell transplantation may in the future take the place of liver transplantation for the treatment of inherited metabolic disorders in children. Before this happens, however, the ideal cell type for this therapy must be identified, and a method for efficient, large-scale production of cells, as well as for their successful engraftment after transplantation, must be developed. More information is necessary on the dosage of cells required in children, taking into account that restoration of around 10% of original enzyme activity is usually sufficient to achieve metabolic control [34], and on the optimal method of delivery [35]. Further research on the use and behavior of stem cell-derived HLCs is also necessary in order for them to be safely incorporated into clinical practice. These advancements may beused not only for inborn errors of metabolism, but also for the treatment of hepatocellular carcinoma and chronic liver disease in adults [36]. In the case of inherited metabolic disorders specifically, ex vivo gene therapy followed by autologous cell transplantation holds great promise for the treatment of single-gene abnormalities.


Acute liver failure

ALF is an emergent situation with high mortality rates and a very limited time frame to locate and prepare a donor liver suitable for transplantation [37]. In this context, cell therapy may serve as a bridge to liver transplantation by supporting hepatic function while waiting for a donor organ [38]. This may be achieved through two methods: liver cell transplantation or bioartificial liver (BAL) support systems. Liver cell transplantation has been reported in at least ten pediatric patients, with hyperammonemia reduction, coagulation improvement, and hepatic encephalopathy regression seen in the majority of patients [9].

BAL systems remove toxic substances from the blood through albumin dialysis and at the same time perform synthetic liver functions through the incorporation of live, functioning hepatocytes into the device [39]. To date, none of the tested BAL devices have demonstrated survival benefit in a randomized controlled trial despite improvement in clinical and biochemical parameters [40,41], but research in this field is still very active, with a spheroid reservoir BAL recently being shown to improve survival in a porcine model of drug-overdose ALF [42]. Porcine hepatocyte spheroids have also been used in a BAL built for pediatric use and have displayed successful ammonia detoxification [43], but this device has not been clinically tested. Further investigation of BAL systems in the clinical and pediatric settings is warranted.

Several different cell types have been tested in BAL devices; to date, none has demonstrated clear superiority over the others. Primary hepatocytes show a tendency to lose function and apoptose in vitro, which may be partially overcome by culture in a spheroid configuration [44], but human hepatocytes are not easily accessible and porcine hepatocytes used in the HepatAssist device are associated with concerns of xenozoonosis. HepG2/C3A immortalized hepatoblastoma-derived human cells have also been used in the ELAD device, but the issue of their possible tumorigenesis has not yet been resolved. A future solution to this problem may be the expansion of hepatocytes in large-scale animal bioreactors: animal models of tyrosinemia type 1 have been created that could allow for liver repopulation with human hepatocytes due to the graft’s selective advantage over the native fumarylacetoacetate (FAH)-deficient cells [45,46]. Finally, hepatocytic induction of fibroblasts into hiHeps has recently yielded promising results in a BAL device demonstrating improved survival in a porcine ALF model [47].

Although liver transplantation is the only proven therapy for patients unlikely to recover from ALF, a large retrospective United Network for Organ Sharing (UNOS) data analysis showed that 5-year patient and graft survivals in children with ALF were significantly lower than in children transplanted for biliary atresia [48]. Furthermore, recovery without transplantation occurs in 15%-20% of patients with severe hepatic encephalopathy [1]. This means that endogenous regeneration takes place in the liver that in some cases is capable of restoring hepatic function, so that cell therapy may be able to eliminate the need for liver transplantation in selected patients [49]. When transplantation is necessary, bioengineered liver grafts may in the future allow us to bypass the shortage of donor organs while eliminating the need for chronic immunosuppression [50,51].


Conclusions

Cell therapy has shown promise as an alternative to orthotopic liver transplantation for the treatment of inherited metabolic disorders and ALF in pediatric patients. Primary hepatocyte transplantation has been used in children with CNS 1 and UCD with encouraging results, and in the future genetically corrected autologous stem cell-derived HLC transplantation may offer a long-term solution to single-gene metabolic disorders. In both metabolic disorders and ALF, cell therapy may serve as a bridge to liver transplantation by supporting normal liver function while a suitable donor organ is found, and in ALF cell-based therapeutics may in some cases also serve as a bridge to spontaneous endogenous regeneration, bypassing the need for liver transplantation altogether. Cell therapy for ALF includes cell transplantation as well as BAL systems. However, several important challenges must be overcome before these practices are incorporated into the clinical setting. Namely, the optimal cell type for each modality of cell therapy must be determined, and mechanisms set in place for the obtainment of cell quantities sufficient for large-scale clinical application, with efficient in vitro culture and in cell transplantation successful in vivo engraftment. Furthermore, these treatments must demonstrate safety in humans and within the framework of pediatrics.


References
  1. Spada M, Riva S, Maggiore G, Cintorino D, Gridelli B (2009) Pediatric liver transplantation. World J Gastroenterol 15: 648-674.

  2. Hackl C, Schlitt HJ, Melter M, Knoppke B, Loss M (2015) Current developments in pediatric liver transplantation. World J Hepatol 7: 1509-1520.

  3. Ng VL, Fecteau A, Shepherd R, Magee J, Bucuvalas J, et al. (2008) Outcomes of 5-year survivors of pediatric liver transplantation: report on 461 children from a north american multicenter registry. Pediatrics 122: e1128-1135.

  4. Kayler LK, Rasmussen CS, Dykstra DM, Punch JD, Rudich SM, et al. (2003) Liver transplantation in children with metabolic disorders in the United States. Am J Transplant 3: 334-339.

  5. Hansen K, Horslen S (2008) Metabolic liver disease in children. Liver Transpl 14: 713-733.

  6. Yu Y, Fisher JE, Lillegard JB, Rodysill B, Amiot B, et al. (2012) Cell therapies for liver diseases. Liver Transpl 18: 9-21.

  7. Mazariegos G, Shneider B, Burton B, Fox IJ, Hadzic N, et al. (2014) Liver transplantation for pediatric metabolic disease. Mol Genet Metab 111: 418-427.

  8. Dhawan A, Mitry RR, Hughes RD (2006) Hepatocyte transplantation for liver-based metabolic disorders. J Inherit Metab Dis 29: 431-435.

  9. Meyburg J, Schmidt J, Hoffmann GF (2009) Liver cell transplantation in children. Clin Transplant 23: 75-82.

  10. Ambrosino G, Varotto S, Strom SC, Guariso G, Franchin E, et al. (2005) Isolated hepatocyte transplantation for Crigler-Najjar syndrome type 1. Cell Transplant 14: 151-157.

  11. Fox IJ, Chowdhury JR, Kaufman SS, Goertzen TC, Chowdhury NR, et al. (1998) Treatment of the Crigler-Najjar syndrome type I with hepatocyte transplantation. N Engl J Med 338: 1422-1426.

  12. Khan AA, Parveen N, Mahaboob VS, Rajendraprasad A, Ravindraprakash HR, et al. (2008) Treatment of Crigler-Najjar Syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia. Transplant Proc 40: 1148-1150.

  13. Meyburg J, Das AM, Hoerster F, Lindner M, Kriegbaum H, et al. (2009) One liver for four children: first clinical series of liver cell transplantation for severe neonatal urea cycle defects. Transplantation 87: 636-641.

  14. Meyburg J, Hoffmann GF (2008) Liver cell transplantation for the treatment of inborn errors of metabolism. J Inherit Metab Dis 31: 164-172.

  15. Oldhafer F, Bock M, Falk CS, Vondran FW (2016) Immunological aspects of liver cell transplantation. World J Transplant 6: 42-53.

  16. Dhawan A, Puppi J, Hughes R, Mitry R (2010) Human hepatocyte transplantation: current experience and future challenges. Nat Rev Gastroenterol Hepato 7: 288-298.

  17. Garate Z, Davis BR, Quintana-Bustamante O, Segovia JC (2013) New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells. Hum Gene Ther 24: 571-583.

  18. Choi SM, Kim Y, Shim JS, Park JT, Wang RH, et al. (2013) Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells. Hepatology 57: 2458-2468.

  19. Eggenschwiler R, Loya K, Wu G, Sharma AD, Sgodda M, et al. (2013) Sustained knockdown of a disease-causing gene in patient-specific induced pluripotent stem cells using lentiviral vector-based gene therapy. Stem Cells Transl Med 2: 641-654.

  20. Raya A, Rodriguez-Piza I, Navarro S, Richard-Patin Y, Guenechea G, et al. (2010) A protocol describing the genetic correction of somatic human cells and subsequent generation of iPS cells. Nat protoc 5: 647-660.

  21. Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, et al. (2011) Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. Nature 478: 391-394.

  22. Fattahi F, Asgari S, Pournasr B, Seifinejad A, Totonchi M, et al. (2013) Disease-corrected hepatocyte-like cells from familial hypercholesterolemia-induced pluripotent stem cells. Mol Biotechnol 54: 863-873.

  23. Grossman M, Rader DJ, Muller DW, Kolansky DM, Kozarsky K, et al. (1995) A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia. Nat Med 1: 1148-1154.

  24. Hickey RD, Elgilani F, Mao SA, Glorioso JM, Amiot B, et al. (2015) Autologous hepatocyte transplantation after ex vivo gene therapy in a large animal model of metabolic liver disease. Hepatology 62: Abstract 2.

  25. Weber A, Groyer-Picard MT, Franco D, Dagher I (2009) Hepatocyte transplantation in animal models. Liver Transpl 15: 7-14.

  26. Meyburg J, Hoffmann GF (2010) Liver, liver cell and stem cell transplantation for the treatment of urea cycle defects. Mol Genet Metab 100 Suppl 1: S77-S83.

  27. Overturf K, Al-Dhalimy M, Tanguay R, Brantly M, Ou CN, et al. (1996) Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I. Nat Genet 12: 266-273.

  28. Ding J, Yannam GR, Roy-Chowdhury N, Hidvegi T, Basma H, et al. (2011) Spontaneous hepatic repopulation in transgenic mice expressing mutant human alpha1-antitrypsin by wild-type donor hepatocytes. J Clin Invest 121: 1930-1934.

  29. Jirtle RL, Michalopoulos G (1982) Effects of partial hepatectomy on transplanted hepatocytes. Cancer Res 42: 3000-3004.

  30. Yamanouchi K, Zhou H, Roy-Chowdhury N, Macaluso F, Liu L, et al. (2009) Hepatic irradiation augments engrfatment of donor cells following hepatocyte transplantation. Hepatology 49: 258-267.

  31. Baccarani U, Adani GL, Sanna A, Avellini C, Sainz-Barriga M, et al. (2005) Portal vein thrombosis after intraportal hepatocytes transplantation in a liver transplant recipient. Transpl Int 18: 750-754.

  32. Meyburg J, Hoerster F, Schmidt J, Poeschl J, Hoffmann GF, et al. (2010) Monitoring of intraportal liver cell application in children. Cell Transplant 19: 629-638.

  33. Nicolas C, Wang Y, Luebke-Wheeler J, Nyberg SL (2016) Stem Cell Therapies for Treatment of Liver Disease. Biomedicines 4.

  34. Meyburg J, Hoffmann GF (2005) Liver transplantation for inborn errors of metabolism. Transplantation 80: S135-S137.

  35. Meyburg J, Alexandrova K, Barthold M, Kafert-Kasting S, Schneider AS, et al. (2009) Liver cell transplantation: basic investigations for safe application in infants and small children. Cell Transplant 18: 777-786.

  36. Nicolas CT, Wang Y, Nyberg SL (2016) Cell therapy in chronic liver disease. Curr Opin Gastroenterol 32: 189-194.

  37. Karakayali H, Ekici Y, Ozcay F, Bilezikci B, Arslan G, et al. (2007) Pediatric liver transplantation for acute liver failure. Transplant Proc 39: 1157-1160.

  38. Nyberg SL (2012) Bridging the gap: advances in artificial liver support. Liver Transpl 18: S10-14.

  39. Bañares R, Catalina MV, Vaquero J (2014) Molecular adsorbent recirculating system and bioartificial devices for liver failure. Clin Liver Dis 18: 945-956.

  40. Demetriou AA, Brown RS Jr, Busuttil RW, Fair J, McGuire BM, et al. (2004) Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure. Ann Surg 239: 660-667.

  41. Reich DJ (2015) The Effect of Extracorporeal C3A Cellular Therapy in Severe Alcoholic Hepatitis - The VTI-208 ELAD Trial. AASLD San Francisco, CA.

  42. Glorioso JM, Mao SA, Rodysill B, Mounajjed T, Kremers WK, et al. (2015) Pivotal preclinical trial of the spheroid reservoir bioartificial liver. J Hepatol 63: 388-398

  43. Lorenti A, Barbich M, de Santibanes M, Ielpi M, Vazquez JC, et al. (2003) Ammonium detoxification performed by porcine hepatocyte spheroids in a bioartificial liver for pediatric use: preliminary report. Artif Organs 27: 665-670.

  44. Brophy CM, Luebke-Wheeler JL, Amiot BP, Khan H, Remmel RP, et al. (2009) Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology 49: 578-586.

  45. Azuma H, Paulk N, Ranade A, Dorrell C, Al-Dhalimy M, et al. (2007) Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nature biotechnology 25: 903-910.

  46. Hickey RD, Mao SA, Glorioso J, Lillegard JB, Fisher JE, et al. (2014) Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease. Stem Cell Res 13: 144-153.

  47. Shi XL, Gao Y, Yan Y, Ma H, Sun L, et al. (2016) Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Res 26: 206-216.

  48. Futagawa Y, Terasaki PI (2004) An analysis of the OPTN/UNOS Liver Transplant Registry. Clin Transpl 315-329.

  49. van de Kerkhove MP, Hoekstra R, Chamuleau RA, van Gulik TM (2004) Clinical application of bioartificial liver support systems. Ann Surg 240: 216-230.

  50. Uygun BE, Soto-Gutierrez A, Yagi H, Izamis ML, Guzzardi MA, et al. (2010) Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med 16: 814-820.

  51. Uygun BE, Yarmush ML, Uygun K (2012) Application of whole-organ tissue engineering in hepatology. Nat Rev Gastroenterol Hepatol 9: 738-744.

International Journal of Anesthetics and Anesthesiology (ISSN: 2377-4630)
International Journal of Blood Research and Disorders   (ISSN: 2469-5696)
International Journal of Brain Disorders and Treatment (ISSN: 2469-5866)
International Journal of Cancer and Clinical Research (ISSN: 2378-3419)
International Journal of Clinical Cardiology (ISSN: 2469-5696)
Journal of Clinical Gastroenterology and Treatment (ISSN: 2469-584X)
Clinical Medical Reviews and Case Reports (ISSN: 2378-3656)
Journal of Dermatology Research and Therapy (ISSN: 2469-5750)
International Journal of Diabetes and Clinical Research (ISSN: 2377-3634)
Journal of Family Medicine and Disease Prevention (ISSN: 2469-5793)
Journal of Genetics and Genome Research (ISSN: 2378-3648)
Journal of Geriatric Medicine and Gerontology (ISSN: 2469-5858)
International Journal of Immunology and Immunotherapy (ISSN: 2378-3672)
International Journal of Medical Nano Research (ISSN: 2378-3664)
International Journal of Neurology and Neurotherapy (ISSN: 2378-3001)
International Archives of Nursing and Health Care (ISSN: 2469-5823)
International Journal of Ophthalmology and Clinical Research (ISSN: 2378-346X)
International Journal of Oral and Dental Health (ISSN: 2469-5734)
International Journal of Pathology and Clinical Research (ISSN: 2469-5807)
International Journal of Pediatric Research (ISSN: 2469-5769)
International Journal of Respiratory and Pulmonary Medicine (ISSN: 2378-3516)
Journal of Rheumatic Diseases and Treatment (ISSN: 2469-5726)
International Journal of Sports and Exercise Medicine (ISSN: 2469-5718)
International Journal of Stem Cell Research & Therapy (ISSN: 2469-570X)
International Journal of Surgery Research and Practice (ISSN: 2378-3397)
Trauma Cases and Reviews (ISSN: 2469-5777)
International Archives of Urology and Complications (ISSN: 2469-5742)
International Journal of Virology and AIDS (ISSN: 2469-567X)
More Journals

Contact Us

ClinMed International Library | Science Resource Online LLC
113 Barksdale Professional Center, Newark, DE 19711, USA
Email: contact@clinmedlib.org
Tel: +1-302-294-0935  

Feedback

Get Email alerts
 
Creative Commons License
Open Access
by ClinMed International Library is licensed under a Creative Commons Attribution 4.0 International License based on a work at https://clinmedjournals.org/.
Copyright © 2017 ClinMed International Library. All Rights Reserved.