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Vol.IIssue: 1:

Proceedings of the Annual Symposium & Plenary Session on Regenerative Medicine (PASRM)

doi:10.46582/jsrm.0101007

(JSRM code: 001010700007)

Autologous Immune Enhancement Therapy (AIET) for a Case of Acute Myeloid Leukemia (AML) - Our Experience

Damodar S1, Terunuma H2, Sheriff AK3, Farzana L3, Manjunath S3, Senthilkumar R3, Shastikumar G3, Abraham S4

(PASRM** 2006-001)

1Narayana Hrudayalaya, Bangalore, India, 2 Biotherapy Institute, Tokyo, Japan, ,  3Nichi-In Centre for Regenerative Medicine, Chennai, India, 4Yamanashi University, Tamaho, Japan.

Background: Hematological malignancies such as AML conventionally require Bone Marrow transplantation, failing which Umbilical cord blood transplantation have been reported in a few cases. We have come across a patient in whom both the options were not feasible due non-availability of a matching donor or donor unit of umbilical cord blood in whom the AIET along with chemotherapy has been administered and we report herewith the early results.

 

Materials and methods: An 8-year-old girl was evaluated for fever of 2 weeks duration in November 2005. Blood investigations revealed anemia with leucocytosis with the presence of blasts with Auer rods. A bone marrow examination confirmed a diagnosis of acute myeloid leukemia–M1. She was started on induction chemotherapy with the pediatric BFM-AML 93 protocol. In theb post-induction chemotherapy period, the bone marrow was in remission. Since she did not have a sibling for a bone marrow transplant or fully or partially matching homologous umbilical cord blood unit, she was considered for AIET. In the first sitting cells were harvested from the bone marrow and cultured and re-infused to the patient after 3 weeks The first cell dose was 2.0 x 108 cells. During this period she received her consolidation chemotherapy. Subsequently cells were harvested from the peripheral blood prior to each cycle of chemotherapy and re-infused after 2-3 weeks. The subsequent cell doses were 7.5 x 107 cells, 2x 106 cells, and 6.3 x108 cells. The procedure followed was as per Terunuma et al., reported earlier and was aimed at expanding lymphocytes and activating them as well as expanding Natural Killer cells (NK) in vitro. Documentation of the cell count were done before and after expansion by Immuno-Pheno Typing and LAL endotoxin tests were done before every transfusion.

 

Results: IP typing showed a reasonably good expansion and activation of lymphocytes at all four expansions, but NK cell expansion was good only during the first and fourth transfusions. The quantity of MNCs harvested and their expansion in general was good after the first and fourth time compared to the 2nd and 3rd time. The patient had no adverse reaction during any of the AIET transfusions. Currently the patient is on maintenance chemotherapy with normal blood counts. The body weight before the starting of the first chemotherapy was 17.2 Kgs and now its 21.0 Kgs.

 

Conclusion: Autologous Immune Enhancement Therapy for the case mentioned above has been accomplished for the first time in India (as far our knowledge is concerned), and has had no side effects per se because of the transfusion. A longer follow-up of the same patient and administration of the AIET to several more patients of the same condition has to be done for further authentication.

 

Corresponding Author: Dr. Sharat Damodar, Consultant  Hematologist, Narayana Hrudayalaya, 258/A Bommasandra Industrial Estate, Anekal Taluk, Bangalore 560099, India. drsharat_damodar@yahoo.co.in; Tel: +91-80-27835000 to 018 , Fax: +91-80-27832648

 

(JSRM code: 001010700008)

Autologous Bone Marrow Stem Cell Therapy for an Ischemic Ulcer of the Lower Limb in a Diabetic Patient

Subrammaniyan SR1, Rajkumar M1, Amalorpavanathan J1, Shankar R1, Farzana L2, Manjunath S2, Senthil RS2, Sheriff AK2, Dominic J2*, Abraham S2 (PASRM*** 2006-002)

1Vijaya Hospital, Chennai, India, 2Nichi-In Centre for Regenerative Medicine, Chennai, India

*Formerly with 2Nichi-In Centre for Regenerative Medicine, Chennai, India

Background: Chronic limb ischemia is an outcome of peripheral arterial disease. If revascularization cannot be done, amputation is the only option left. Recent studies report that the injection of Bone marrow mononuclear cell concentrate with stem cells or Peripheral blood mononuclear cells rich in CD34+ cell content have resulted in angiogenesis, improved the functional activity of the ischemic limb and enhanced the healing of the ischemic ulcer. Here we report our experience with one such case. 

Materials and Methods: A 68-year-old diabetic female patient with critical limb ischemia of the left lower limb with diffuse multiple critical stenosis of the only patent Tibial artery with a large ischemic ulcer with infection. The ulcer measured 30X12 cm at the posterior of the calf, exposing the Gastrocnemius and Achilles tendon and extending to the medial aspect of the foot measuring 14 X 10cm. A lateral extension of the wound was present as two places just above the ankle joint, each measuring 4.5 X 4.0 CM. The patient had been advised amputation of the lower limb elsewhere. She was administered Autologous Bone Marrow Stem Cell Therapy (ABMSCT) twice at an interval of one month. 110 ml of Bone marrow was aspirated each time under short general anesthesia, transported in Acid Dextrose Citrate and was processed for mononuclear cells (MNC) by ficoll density gradient centrifugation, as per cGMP Protocol. The MNC concentrate was injected at various sites in the Gastrocnemius muscle and the surrounding area after necessary wound debridement. First time the MNC concentration injected, contained 603 Million cells and the second time 786 Million cells. The patient was followed up regularly for all relevant parameters. Skin grafting was performed to the uncovered areas of the wound on the 57th day from the first stem cell injection. Angiography was done to adjudge the progress. 

Results: The healthy granulation gradually started appearing in the areas which were initially unhealthy and ischemic. Skin started growing from the edges of the wound and fully covered the lateral two wounds and 23% of the area of the wound on the medial side of the foot. Approximately 15% of the wound surface of the posterior of the calf was covered with the in growing skin at the time of skin grafting.

Conclusion: ABMSCT has been reported for treatment of CLI in many parts of the world, but not in such a large wound of the size that we have come across. As ABMSCT enhances the wound healing process in case of chronic ischemic wounds we recommend that it may be considered in cases similar to what we have experienced, before deciding on an amputation, to salvage the limb. 

over Image:

 

Pictures of one of the lateral extensions of the wound of the patient mentioned in the study.

 

 The picture on the left had been taken upon admission of the patient and the right one taken 57 days after administration of the Autologous Bone Marrow Stem Cell Therapy according to the author.

Corresponding Author: Dr. S.R. Subrammaniyan, Consultant  Vascular Surgeon, Vascular Surgery Department, Vijaya Hospitals, 180, NSK Salai, Vadapalani, Chennai-600026, India. vijayavascular@hotmail.com Tel: +91-44-23722122

(JSRM Code: 001010700009)

A Rabbit Model of Ex Vivo Cultivation and Transplantation of Autologous Limbal Epithelial Cells Grown in a Thermo-reversible Gelation Polymer (Mebiol Gel)

Sitalakshmi G 1 , Sudha B1, Vinay S1, Madhavan HN1, Krishnakumar S1, Mori Y2, Yoshioka H2 , Abraham S3 (PASRM** 2006-003)

1Vision Research Foundation, Sankara Nethralaya, Chennai, India, 2Waseda University, Tokyo, Japan, 3Yamanashi University- School of Medicine, Tamaho, Japan.

Purpose: To evaluate the efficacy of autologous expanded corneal epithelial cell transplants derived from harvested limbal epithelial stem cells cultured on a thermo-reversible polymer (Mebiol Gel) for the management of unilateral limbal stem cell disease (LSCD). Mebiol Gel is a copolymer composed of the thermo responsive polymer block [poly (N-isopropylacrylamide-co-n-butyl methacrylate) (poly NIPAAm-co-BMA)] and the hydrophilic polymer block [polyethylene glycol (PEG)].

Materials and Methods: In 12 rabbits, a limbal biopsy was taken and thereafter ocular surface damage was created on the same eye by removal of corneal and limbal epithelium. The limbal biopsies were cultured on the Mebiol Gel as explant cultures for 3 weeks to produce confluent epithelial cells, harvested non-enzymatically by reducing the temperature to 40C. Three weeks later, vascularised pannus was removed and expanded autologous limbal epithelial cells were placed in a drop fashion to cover the entire damaged corneal surface. The rabbits were sacrificed at the end of the study and the damaged corneas were harvested for histological and RT-PCR study for the limbal phenotype (p63 & ABCG2), transient amplifying cell markers (Integrin β 1 & Connexin 43) and corneal phenotype (K3 & K12) markers. Outcome measures such as corneal vascularization (V), corneal haze (H), fluorescein staining (F) and histopathology (P) were evaluated between 6-8 months. Each outcome was scored based on the severity of each variable as judged on photography and histopathology. Two rabbits (controls) underwent transplantation with Mebiol gel alone without cultured cells.

Results: Reparative surgery was a total success (score, 8-10) in 7 (58.3%), partial success (score, 6-7) in 2 (16.7%) and failure (score, <5) in 3 (25%). Histological and RT PCR study documented successful growth of corneal epithelium onto the recipient surface. RT PCR studies showed the limbal phenotype in the limbus and cornea phenotype in the central portion. Transient amplifying cell marker is present in both limbus and cornea. Reparative surgery was a failure in controls.

Conclusion: The results of the rabbit model suggest that autologous limbal epithelial cells grown in thermo-reversible gel polymer may restore a nearly normal ocular epithelial surface in eyes with unilateral LSCD.

Corresponding Author: Dr.G.Sitalakshmi, Dept of corneal services, Vision Research Foundation, Sankara Nethralaya, 18, College Road, Chennai - 600006, India. Tel: +91-44 28271616; sitalakshmi.sharma@gmail.com

 

(JSRM Code: 001010700010)

Towards Cell Therapeutics for Bullous Keratopathy Using Corneal Endothelial Precursor Cells; Successful In Vitro Expansion After Transportation in Indian Conditions and Characterization

Nelson J1, John S1, Philip T1, Kaliyamurthy J1, Farzana L2, Manjunath S2 Sheriff AK2, Abraham S2, 3, Yoshioka H 4, Mori Y 4, Amano S5 (PASRM** 2006-004)

1Joseph Eye Hospital, Trichy, India, 2Nichi-In Centre for Regenerative Medicine, Chennai, India, 3Yamanashi University, Tamaho, Japan, 4 Waseda University, Tokyo, Japan, 5Tokyo University-School of Medicine, Tokyo Japan

Background: Bullous Keratopathy affects thousands of people in India every year, for which many experimental studies have been reported on cell therapy using corneal endothelial precursors. Successful culture of the endothelial cells and precursors taken from cadaver eyes, processed immediately after harvesting it from corneal buttons have been reported from developed nations under stringent processing conditions. But corneal endothelium removed from the button, transported at varying temperature conditions for long and then successfully cultured have not yet been reported anywhere. In Indian conditions we have done this work keeping in mind, a feasible model of a centrally equipped laboratory which can serve remote places within its reach making such cell therapeutics using in vitro cultured endothelial precursors available in distant places also.

 

Materials & Methods: Endothelial layer from the corneal button were removed by an experienced ophthalmologist in Trichy and was transported to the NCRM laboratory, Chennai. The transportation was done at varying temperature conditions ranging from 22 Deg C (Train coach) to 30 Deg C (Outside temperature) taking an average of 10 hrs till processing, using two methods of preservation. GroupI; Specimens were suspended in DMEM + BFGF+EGF + B27+ Collagenase type1 (Basal culture medium). Group II: Specimens embedded in TGP above which the same basal culture medium was added. Upon arrival in the laboratory, specimens were trypsinized and grown as a sphere suspension culture for 2 wks. Gr. I in the basal culture medium and Gr.II in TGP topped with basal culture medium. Viable cells in both groups counted immediately upon processing and thereafter at different intervals during the process of culture expansion and subjected to characterization by RT-PCR.

 

Results: Corneal endothelial precursors cells could be grown successfully as sphere forming assay. Immediately after processing following the transportation, viability of cells in Gr.II were significantly higher. The no. of single cells forming spheres was more in Gr. II, whereas in Gr. I aggregation of developing spheres were common. Spheres from both the groups proven positive for neuronal marker B-3Tubulin and negative for Cytokeratins K3 and K12, thereby proving that they are nothing but corneal endothelial precursor cells.

 

Conclusion: We could evolve a method by which endothelial cells removed as a layer from corneal button could be transported in Indian conditions in a safe and efficient method yielding viable cells and further successful in vitro expansion of corneal endothelial precursor cells. After further confirmatory steps, if the cells could be used for cell therapeutics in patients suffering from Bullous Keratopathy, this would be a major breakthrough and help thousands of patients recover their vision in the country.

 

Corresponding Author: Dr. C.A. Nelson Jesudason, Joseph Eye Hospital - Institute of Ophthalmology, Tiruchrappalli

 

(JSRM Code: 001010700011)

Potential of Regenerative Medicine in Articular Cartilage Injury

Arumugam S1

1Sri Ramachandra Medical College & Research Institute (Deemed University), Chennai, India

(PASRM** 2006-005)

Introduction: Successful regeneration of functional and durable articular cartilage is a public health priority. Internationally, knee osteoarthritis alone is expected to be the fourth leading cause of disability in women and the eighth leading cause in men.  For those who live long enough, pain and dysfunction resulting from some degree of cartilage injury and degeneration seem a virtual certainty.

 

Joint cartilage shows a very limited capacity for self-repair. Currently following operative treatments are used;

1) Micro fracture , where an awl is use to penetrate the subchondral bone plate in a systematical manner arthroscopically. Though some hyaline like cartilage are formed the major part of repair tissue consists of fibrous cartilage.

2) Mosaicplasty ,where cylindrical cartilage grafts from non-affected parts of the joint are obtained and moved into the defect. The procedure requires open surgery. Mosaicplasty raises some concerns First, to what extent do harvesting of cartilage plugs damage the knee? Second, the plugs cannot be made to fit exactly in to the defect so some regeneration of tissue in between plugs has to take place. The tissue generated is likely to be of a fibrous cartilage type.

Role of tissue engineering: The pivotal work of Brittberg el al (1994) from Sweden opened new avenues in the field of  management of articular cartilage injury. They took cartilage from the edge of a knee joint, grew it in culture and returned it to the defect by using a periosteal flap to create watertight chamber into which the chondrocytes were injected. This technique is called autologous chondrocyte implantation (ACI). 

A newer development in chondrocyte tissue engineering includes the MACI (matrix induced autologous chondrocyte cell implantation) technique. This method overcomes certain problems encountered in ACI for example harvesting of the periosteum and injection of cells, which may cause sub optimal results. Matricel membranes have been developed which are porcine in origin and have been found to be biodegradable. These are Type III collagen and I and do not produce any host immune response. Preclinical studies in rabbits have shown that MACI restores hyaline cartilage within 6 months of implantation.

 

The chondrocyte cell culture and tissue engineering at the international level has progressed beyond the research laboratories and are on the threshold of clinical trials. If a successful procedure for regeneration of cartilage can be developed in India it would have a major impact in helping a millions of patients in our country.

 

Corresponding Author: Dr. S. Arumugam, Associate Professor & Head, Dept. of Arthroscopy and Sports Medicine, Sri Ramachandra Medical College & Research Institute (Deemed University), Chennai-600116, India. arumugam@srmc.edu Tel: +91-44-24765557

 

(JSRM Code: 001010700012)

Treatments of Liver Failure in Japan

Fujii H1, Matsuda M1

 

1 Yamanashi University-School of Medicine, Tamaho, Japan

(PASRM** 2006-006)

Liver failure is a fatal disease. Common causative disease of liver failure in Japan are end-stage liver disease due to cirrhosis and /or hepatocellular carcinoma (HCC), fulminant hepatitis and postoperative liver failure.

 As for postoperative liver failure, operative mortality after hepatic resection for HCC decreased from 2.3% in 1990-1991 to 0.6% in 1998-1999 in Japan. According to a nationwide survey of patients with potentially fatal fulminate hepatitis in 1998, the etiology was hepatitis A viral (HAV) infection in 4%, hepatitis B viral (HBV) infection in 44%, and nonA-nonB in 41 in Japan.

Most of the Japanese doctors believe that plasma exchange (PE) and continuous hemodiafiltration (CHDF) are the most effective therapies for the treatment of acute liver failure, but randomized controlled studies are needed to determine their effects.

 Liver transplantation is the only established treatment for liver failure, however, donor shortage remain problematic. Furthermore, cadaveric liver tranplantations are unsuited to the social mores of Japanese culture. In 1988 living donor liver transplantation was performed for the first time in Japan. Many Japanese patients with fulminant hepatic failure or end-stage cirrhosis were treated by living donor liver transplantation nowadays.

 Bioartificial liver supports are being considered as the second line of treatment for liver failure.

 

Corresponding Author: Dr. Hideki Fujii, Professor & Head, Ist Dept. of Surgery, Yamanashi University-School of Medicine, 1110, Shimokatoh, Tamaho 409-3898, Japan. hfujii@yamanashi.ac.jp Tel: +81-55-2731111

 

(JSRM Code: 001010700013)

Burden of Acute Liver Failure in India and Treatment Options

Jayanthi V1

 

1Stanley Medical College, Chennai, India

(PASRM** 2006-007)

Acute hepatic failure (AHF) in India almost always presents with encephalopathy within 4 weeks of the onset of the first symptom of hepatic injury. It is commonly due to viral hepatitis or drug use. Infectious diseases such as sepsis, severe malarial fever, or typhoid fever may present rarely with AHF. Unlike the West, viral hepatitis is the cause in approximately 95-100% of patients in India and therefore this constitutes a more homogeneous population than AHF in the West.

In northern India, hepatitis E (HEV) and hepatitis B (HBV) viruses are the important viral causes of AHF; approximately 60% of cases are caused by these two viruses. At a tertiary referral centre from the northern part of India, Acharya et al reported Hepatitis B virus core mutants as important agents in hepatitis B related AHF. Approximately, 50% of the patients were women and one-quarter of them were pregnant, in their series. Thus it is apparent, that pregnant women who contract viral hepatitis constitute a high-risk group for the development of AHF. However, the outcome of AHF in this group is similar to that in non-pregnant women and men. No association with any particular virus has been identified among sporadic cases of AHF. Approximately one-third of AHF patients survive with aggressive conservative therapy, while death occurred within 72 h of hospitalization in the remaining two thirds. Cerebral oedema and sepsis (Both fungal and gram-negative bacteria) were the major fatal complications.

Another publication by Dhiman et al, highlighted the prognostic factors of AHF under the 3 categories of hyperacute, acute and subacute hepatic failure. They studied early indicators of prognosis by multivariate analysis in 204 consecutive patients with AHF admitted with hepatic encephalopathy over five years on classifiying them into hyperacute, acute and subacute hepatic failure. The etiology was virus related in 186 (91.1%), drug induced in 15 (7.4%), Wilson's disease in one (0.5%), acute Budd-Chiari syndrome in one (0.5%), and malignant infiltration in one (0.5%).

Sixty (32.3%) patients with viral hepatitis survived. Univariate analysis showed that the interval between onset of encephalopathy and onset of jaundice, grade of encephalopathy, raised intracranial pressure, prothrombin time, and serum bilirubin levels on admission correlated with outcome in these patients. Multivariate logistic regression analysis showed that the presence of raised intracranial pressure at the time of admission, prothrombin time >100 sec on admission, age (>50 yr), and onset of encephalopathy seven days after onset of jaundice were associated with poor prognosis. Forty seven (37.0%) of 129 patients with hyperacute survived compared with 9 (22.5%) of 40 with acute and 4 (21.1%) of 19 with subacute liver failure (P = NS). Raised intracranial pressure was more frequent in patients with hyperacute (48.8%) than in patients with acute (32.5%) and subacute liver failure (15.8%; P = 0.01), while clinically detectable ascites was more frequent in patients with subacute (78.9%) compared with hyperacute (19.7%) and acute liver failure (37.5%; P< 0.0001).

Khuroo from Kashmir, studied the early prognostic indicators for acute liver failure in endemic zones for hepatitis E virus. Of the total of 180 [69 males and 111 females: age (mean +/- SD) 31.1 +/- 14.7 years] with acute liver failure, 131 (72.8%) patients died. Hepatitis E virus was the aetiological cause in 79 (43.9%) patients, in one-third the cause remained unknown. Of 83 women in childbearing age, 49 (59.0%) were pregnant, 33 (67.3%) of them were in the third trimester. Forty-seven (95.8%) pregnant women had HEV infection. The four variables which predicted the adverse outcome on multivariate analysis were non-hepatitis-E aetiology, prothrombin time >30 s, grade of coma >2 and age >40 years in that order of significance. Pregnancy per se or duration of gestation did not adversely affect the prognosis.

Kar et al, from Delhi, studied the HGV infection in acute viral hepatitis, fulminant hepatic failure (FHF) and in normal healthy blood donors. HGV-RNA was detected in 6 (37.5%) of 16 patients with fulminant hepatic failure, in 7 (19.4%) of 36 acute viral hepatitis, and two (4%) in 50 control blood donors. In both AVH and FHF, HGV was more frequently detected in (8/13; 61.5%) patients co-infected with other hepatotropic viruses and the most common co-infections were found to be HEV (6/8; 75%) and HBV (5/8; 62.5%). The authors concluded that frequency of hepatitis G virus is found to be certainly higher (37.5%) in fulminant hepatic failure than that in any other type of viral hepatitis in India. But since the virus is often detected in co-infection with either hepatitis B or E virus, which are known potential hepatitis agents, the role of HGV as an independent hepatitis agent is uncertain.

Pregnancy and AHF: Studies from India, Iran, Africa and Middle East have found the incidence of fulminant hepatitis to be higher in pregnancy. Malnutrition superimposed on the normal demands of pregnancy and inversion of T and B lymphocytes in early pregnancy have been postulated to be the contributing factors. Beniwal et al from North India studied 97 consecutive pregnant patients in third trimester with acute viral hepatitis (AVH) or fulminant hepatic failure (FHF). Hepatitis E virus (HEV) was the causative agent in 47.4% of the cases of viral hepatitis and 52.6% were caused by non-E viruses (HAV-5.2%,HBV-7.2%,HCV-0%,non A-E 47.4%). HEV was responsible for 36.2% of the cases of AVH and 75% of the cases of FHF. The mortality was 24.7% (24/97). All of them had FHF. Eighteen of 24 cases (75%) who expired were HEV positive. The mortality rate was 39.1% in HEV group and 11.7% in non HEV group. Majority of patients (87.5%) who expired had died undelivered. Hepatitis E was the commonest etiological agent in those who had fulminant disease during pregnancy and was associated with high mortality rate.

In a study of pregnant women with liver disease from Chennai, in 2004, none of the patients developed AHF. Viral hepatitis was the most common cause for jaundice. In a series of 290 cases of jaundice complicating pregnancy, Lahiri from Kolkata reported that 90% were due to viral hepatitis. Bhosale et al reported the incidence of viral hepatitis to be 0.28%. Viral marker study revealed that 45% women were affected with HEV, 21.4% with HBV, 19 % with HAV. Unlike the Chennai study, Bhosale reported that 57% women were referred in a state of encephalopathy with coma leading to high mortality. HEV alone was responsible for 52% of mortality. The over all maternal mortality was 59.2% and perinatal mortality of 51.5%.

In summary, HEV infection alone is responsible for 47.4% of the cases of viral hepatitis in pregnant females in the third trimester. This is corroborative with the fact that HEV infection accounts for 50-70% of all patients with sporadic viral hepatitis in India. In pregnant females in third trimester with viral hepatitis, the prevalence of HEV infection is reportedly between 40-57%. HAV infection was less common (0% vs 5.2%) and HBV infection more common (34.6% vs 7.2%) in central India. HCV infection was not seen in any case as was also observed by other groups.

Among the HEV positive pregnant females, the mortality rate was 39.1%. The mortality rate is in the range of 30-45% and may be as high as 70%. Majority of the cases die undelivered.

Thus uniformly, viral hepatitis is the most common cause of jaundice in pregnancy, fulminant hepatic failure, atleast from southern and eastern parts of India are rare. 

AHF in children: In a study of 36 children admiited as AHF from Mumbai, a viral aetiology could be established in 22 children (61.1%). Hepatitis A (n=12), Hepatitis B (n=3), Hepatitis A and B (n=2), and Hepatitis A and E (n=4). Two children had enteric fever (1 with associated HEV), 2 had Wilson's disease, 1 had Indian Childhood Cirrhosis (ICC) and 2 had drug induced hepatitis. Etiological diagnosis was not possible in 8 children (22%). Fourteen children (39%) died. Poor outcome was associated with spontaneous bleeding, raised prothrombin time, lower transaminases and higher bilirubin on admission.(Bendre et al)

In conclusion, viral hepatitis is the most common cause of AHF, atleast in northern parts of India. Similar reports are not available for the southern states. Acute on chronic liver disease is a more common clinical presentation in south India. There is a need to develop simple, clinical prognostic markers for AHF in the Indian subcontinent, in order to identify patients suitable for liver transplantation, since patients with AHF, despite the presence of sepsis, overt clinical features (i.e. fever, leucocytosis) may be absent and objective documentation of the presence of sepsis can be achieved by repeated culture of various body fluids. Also, the uniform classification of hyperacute, acute and subacute recommended may not be applicable for the Indian subcontinent (Acharya et al), where the rapidity of onset of encephalopathy does not seem to influence survival.

The ultimate management of patients with AHF would be liver transplantation, when conservative supportive resuscitative measures fails. Today, there are several centres in India, like the Apollo Hospitals, New Delhi, Global Hospital, Hyderabad and Christian Medical College Vellore which are performing liver transplants. Transplants are being done at the rate of roughly two to three every year when atleast 60,000 persons in the country need a liver transplant. The approximate cost for liver transplant varies from Rs 10 to Rs 45 lakhs followed by post transplant expenditure

Paucity of donors is one of the major handicaps hampering the growth and success of liver transplantation in the country. Even in the UK and US, there are as many as 4,000 recipients waiting for a liver transplant. There are several problems in obtaining a donor liver. Brain-dead patients almost always land up in neurological ICUs, since the majority are victims of road accidents or internal haemorrhage. In effect, the process of certifying the patient brain-dead has to begin with the neurologist. But in the end, this initiative gets lost and the real glory goes only to the transplant surgeon. 

India's cadaver transplant programme is suffering from a chronic dearth of body parts that has ensured it never really takes off. After years of spirited campaigning, the Transplantation of Human Organs Act, passed in 1994, while banning trade in organs allowed doctors to certify patients brain-dead. It was a landmark. With better surgical techniques and a greater understanding of the body's immune system, it aroused in most doctors a sense of actually being able to help patients with failed organs instead of just looking on helplessly. 

MARS has also served as a bridge to liver TX. There are very few centres in India wherein MARS is available and is often shared between centres performing liver transplant. It is an expensive modality of management. 

Another major breakthrough has been in identifying the role of a tiny population of stem cells, called as "true" stem cells in management of these patients, in the UK. One-thousandth of the cells circulating in the blood are CD34 cells. Of these, 10 per cent are a sub-population of these CD34 cells, which are true stem cells, can differentiate into any specialised cells. While CD34 cells have certain limitations as stem cells, the sub-population can differentiate into all kinds of specialised cells. These when injected into the affected area can take over the function which a diseased cell can no longer perform. What makes the discovery significant is that the world over the number of donors is much less than those requiring a transplant. In India, the problem is even more pronounced. Habibullah et al, from India has done pioneering work on stem cell transplant and today liver cell therapy (both extra and intrahepatic source of stem cells) has been projected as a bridge and an alternative to ortho transplant. The author has reported success in isolated cases and both preclinical and clinical applications are in progress. If successful, the technique would solve the problem of finding donors, lower the cost and will not require a high level technology for treatment.

References:

1. Acharya SK, Panda SK, Saxena A, Gupta SD.Acute Hepatic Failure in India: A Perspective From the East. J Gastroenterol Hepatol. 2000;15:473-479

2. Khuroo MS. Aetiology and prognostic factors in acute liver failure in India. J Viral Hepat 2003; 10: 224-31

3. Das, Kunal, Kar, P. and Das, B.C. Prevalence of new DNA transfusion transmitted virus (TTV) in acute virus hepatitis and fulminant hepatic failure: A study from North India. J Gastroenterol Hepatol. 2004; 19: 406.

4. Beniwal M, Kumar A, Kar P et al. Prevalence and severity of acute viral hepatitis and fulminant hepatitis during pregnancy: a prospective study from north Inida. Indian Journal of Medical Microbiology. 2003; 21:184-185

5. Kapoor S, Gupta RK, Das BC, Kar P. Clinical implications of hepatitis G virus (HGV) infection in patients of acute viral hepatitis & fulminant hepatic failure. Indian J Med Res. 2000 Oct;112:121-7.

6. Sachin V. Bendre, Ashish R. Bavdekar, Sheila A. Bhave, Anand N. Pandit, S.D. Chitambar and V.A. Arankalle. Fulminat hepatic failure: etiology, viral markers and outcome. Indian pediatrics 1999;36:1107-12.

Corresponding Author: Dr. V. Jayanthi, Consultant Hepatologist, Stanley Medical College, Chennai-600001, India. drjayanthi35@yahoo.co.in

(JSRM Code: 001010700014)

Cell Therapeutics for Acute Liver Failure Using Foetal Hepatic Progenitors; In Vitro Expansion and In Vivo Implantation in Animal Models

Dedeepiya V1, Baskar S1, Parveen N2, Khan AA2, Habibullah CM2, Yoshioka H3, Mori Y3, Abraham S4

 

1Nichi-In Centre for Regenerative Medicine, Chennai, India, 2Centre for Liver Research & Diagnostics, Owaisi Hospital, Hyderabad, India, 3Advanced research center for science and engineering, Waseda University, Japan, 4Yamanashi University, Faculty of Medicine, Tamaho, Japan.

(PASRM** 2006-008)

Background: Acute liver failure which affects close to 1,20,000 patients in India every year still lacks a definitive treatment. Though foetal hepatocyte transplantation reported earlier have been successful it is marred with two major obstacles: (i) Lack of timely and adequate quantity of availability of foetal hepatocytes and (ii) short life span of the implanted cells in situ. These two problems have been approached by us using a Thermogelation polymer based foetal cell culture technique. Earlier studies have revealed that similar technology and processing method (i) allows in situ regeneration of the resected liver portion (Kubota et al) and (ii) allows the proliferation of the hepatic progenitors and stem cells into the resected area of the liver in situ which form a three dimensional organized structure of the entire damaged portion resulting in normal liver region as before the resection; these two in animal studies(Nagaya et al).

 

Materials & Methods: I. In vitro study: Hepatocytes were harvested from aborted fetal liver by two-step collagenase digestion method. The cells (5x106) were cultured in Mebiol Gel, a thermoreversible gelation polymer gel (TGP) and  Matrigel in the same ratio . Once the gel gets solidified 1 ml of DMEM was added and the incubated at 37ºC in 5% CO2 incubator. The cells were observed every day for 7 days. The growth parameters in terms of proliferation, functional capability of the cultured cells viz., ureagenesis, albumin secretion and formazan formation were analysed. II. Invivo animal study: Hepatocytes harvested by two step collagenase digestion method of rat liver were embedded in TGP and transplanted intra peritoneally into acute liver failure rat models induced by D-Galactosamine. The efficacy of the cells embedded in Mebiol Gel was studied by assessing blood parameters and histopathological findings at different time points.

Results:  In vitro study revealed that the decrease in AFP and increase in albumin in the cultured cells and the cells cultured in TGP are functionally more active than cells cultured in Matrigel and TGP based culture yielded more number of mitochondria per unit area, proven by the amount of formazan formed.  The ureagenesis study demonstrated that the cells are able to detoxify well in cells cultured in Mebiol. In the animal study, the acute liver failure condition reverted back within 3 days of transplantation. All the blood parameters returned back to normal. The survival rate >70% as compared to the positive control where in only D-Gal was given. The histopathological findings revealed that the hepatocytes survived well even after 30 days of transplantation and there was no infiltration of lymphocytes in and around the recovered TGP embedded cells.

 Conclusion: (i) The foetal hepatocytes undergo proliferation and maturation in TGP much better in both quantity and quality when compared to Matrigel and therefore this method can be used for foetal hepatocyte progenitor expansion, which when done in a large scale a possibility of foetal hepatiocyte stem cell bank would be feasible. (ii) The intraperitoneal transplantation of hepatocytes embedded in Mebiol Gel resulted in prolonged survival and function of the cells and was able to support acute liver failure in animal models thus giving a hope that when applied in humans, it could successfully provide liver support in severe acute liver failure when transplanted intraperitoneally.

 

Corresponding Author: Dr. V. Dedeepiya Devaprasad, Nichi-In Centre for Regenerative Medicine, Vijaya Health Centre Premises, 175, NSK Salai, Vadapalani, Chennai-600026, India. drddp@ncrm.org; Tel: +91-44-42321322 


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