Functional evaluation of a new bioartificial liver system in vivo and in vivo

AIM:To evaluate the functions of a new bioartificial liver(BAL)system in vitro and in vitro.MEHTODS:The BAL system was configurated byinoculating porcine hepatocyte spheroids into the cellcircuit of a hollow fiber bioreactor.In the experiments ofBAL in vitro,the levels of alanine aminotransferase(ALT),total bilirubin(TB),and albumin(ALB)in the circulatinghepatocyte suspension and RPMI-1640 medium weredetermined during 6 h of circulation in the BAL device.In the experiments of BAL in vitro,acute liver failure(ALF)model in canine was induced by an end-side portocavalshunt combined with common bile duct ligation andtransaction.Blood ALT,TB and ammonia levels ofALF in canines were determined before and after BALtreatment.RESULTS:During 6 h of circulation in vitro,therewas no significant change of ALT,whereas the TB andALB levels gradually increased with time both in thehepatocyte suspension and in RPMI-1640 medium.Inthe BAL treatment group,blood ALT,TB and ammonialevels of ALF in canines decreased significantly.CONCLUSION:The new BAL system has the ability toperform liver functions and can be used to treat ALF.

Detection of PERV by polymerase chain reaction and its safety in bioartificial liver support system

AIM： To establish a method detecting porcine endogenous retrovirus （PERV） in China experimental minipigs and to evaluate the safety of PERV in three individuals treated with bioartificial liver support systems based on porcine hepatocytes. METHODS： Porcine hepatocytes were isolated with two-stage perfusion method, then cultured in the bioreactor, which is separated by a semipermeable membrane （0.2μm） from the lumen through which the patients＇ blood plasma was circulated. After posthemoperfusion, patients＇ blood was obtained for screening. Additionally, samples of medium collected from both intraluminal and extraluminal compartments of the laboratory bioreactor and culture supernate in vitro was analyzed. The presence of viral sequences was estimated by polymerase chain reaction （PCR） and reverse transcriptase-polymerase chain reaction （RTPCR）. Finally, the infection of virus in the supernate of common culture was ascertained by exposure to the fetal liver cells. RESULTS： PERV-specific gag sequences were found in the porcine hepatocytes using RT-PCR. and were detected in all samples from the intraluminal, extraluminal samples and culture supernate. However, culture supernatant from primary porcine hepatocytes （cleared of cellular debris） failed to infect human fetal liver cells. Finally, RT-PCR detected no PERV infection was found in the blood samples obtained from three patients at various times post-hemoperfusion. CONCLUSION： The assays used are specific and sensitive, identified by second PCR. PERVs could be released from hepatocytes cultured in bioreactor without the stimulation of mitogen and could not be prevented by the hollow fiber semipermeable membrane, indicating the existence of PERV safety in extracorporeal bioartificial liver support system （EBLSS）.

Key challenges to the development of extracorporeal bioartificial liver support systems

BACKGROUND: For nearly three decades, extracorporeal bioartificial liver (BAL) support systems have been anticipated as promising tools for the treatment of liver failure. However, these systems are still far from clinical application. This review aimed to analyze the key challenges to the development of BALs. DATA SOURCE: We carried out a PubMed search of Englishlanguage articles relevant to extracorporeal BAL support systems and liver failure. RESULTS: Extracorporeal BALs face a series of challenges. First, an appropriate cell source for BAL is not readily available. Second, existing bioreactors do not provide in vivolike oxygenation and bile secretion. Third, emergency needs cannot be met by current BALs. Finally, the effectiveness of BALs, either in animals or in patients, has been difficult to document. CONCLUSIONS: Extracorporeal BAL support systems are mainly challenged by incompetent cell sources and flawed bioreactors. To advance this technology, future research is needed to provide more insights into interpreting the conditions for hepatocyte differentiation and liver microstructure formation.

A bioartificial transgenic porcine whole liver expressing human proteins alleviates acute liver failure in pigs

Background:Preventing heterologous protein influx in patients is important when using xenogeneic bioartificial livers(BALs)to treat liver failure.The development of transgenic porcine livers synthesizing human proteins is a promising approach in this regard.Here,we evaluated the safety and efficacy of a transgenic porcine liver synthesizing human albumin(h ALB)and coagulation factor VII(h FVII)within a bioartificial system.Methods:Tibetan miniature pigs were randomly subjected to different interventions after surgeryinduced partially ischemic liver failure.Group A(n=4)was subjected to basic treatment;group B(n=4)was to standard medical treatment and wild-type porcine BAL perfusion,and group C(n=2)was to standard medical treatment and transgenic BAL perfusion.Biochemical parameters,coagulation status,survival time,and pathological changes were determined.Expressions of h ALB and h FVII were detected using immunohistochemistry and enzyme-linked immunosorbent assays.Results:The survival time in group A was 9.75±1.26 days;this was shorter than that in both perfused groups,in which all animals reached an endpoint of 12 days(P=0.006).Ammonia,bilirubin,and lactate levels were significantly decreased,whereas albumin and fibrinogen levels were increased after perfusion(all P<0.05).h ALB and h FVII were detected in transgenic BAL-perfused pig serum and ex vivo in the liver tissues.Conclusions:The humanized transgenic pig livers could synthesize and secrete h ALB and h FVII ex vivo in a whole organ-based bioartificial system,while maintaining their metabolism,detoxification,transformation,and excretion functions,which were comparable to those observed in wild-type porcine livers.Therefore,the use of transgenic bioartificial whole livers is expected to become a new approach in treating acute liver failure.

Isolation and short term cultivation of swine hepatocytes for bioartificial liver support system

BACKGROUND: The demand for the clinical use of hepa- tocytes is increasing. The aim of this study was to develop a method for procurement of high qualitative pig hepatocytes and to evaluate the state of freshly isolated and cultured hepatocytes. METHODS: The domestic extracorporeal circulating perfu- sion apparatus was used to isolate and harvest swine hepato- cytes by the two-step perfusion method with EDTA and collagenase. The viability, function and morphology of the freshly isolated and cultured cells were evaluated and ob- served by the trypan blue exclusion test, biochemical mea- surements, phase contrast microscopy and transmission electron micrography (TEM). RESULTS: The total yield of isolated hepatocytes reached to 1.5(±0.4)×l010 per liver with a viability of 92(±5)%, and the purity of hepatocytes reached to 98% Immediately after isolation, phase-contrast microscope and TEM showed that undamaged hepatocytes appeared bright, translucent and spherical in shape, with a characteristic well-contrasted border. After 24 hours, the concentrations of alanine aminotransferase (ALT), aspartate aminotrans- ferase ( AST ), lactate dehydrogenase ( LDH ), albumin (ALB), creatinine (Cr) and blood urea nitrogen (BUN) in the fluid of culture were declined significantly. CONCLUSION: This method of procuring swine hepato- cytes could get high quality cells with active metabolic function.

Artificial and bioartificial liver support:A review of perfusion treatment for hepatic failure patients

Liver transplantation and blood purification therapy,including plasmapheresis,hemodiafiltration,and bioartificial liver support,are the available treatments for patients with severe hepatic failure.Bioartificial liver support,in which living liver tissue is used to support hepatic function,has been anticipated as an effective treatment for hepatic failure.The two mainstream systems developed for bioartificial liver support are extracorporeal whole liver perfusion(ECLP)and bioreactor systems.Comparing various types of bioartificial liver in view of function,safety,and operability,we concluded that the best efficacy can be provided by the ECLP system.Moreover,in our subsequent experiments comparing ECLP and apheresis therapy,ECLP offers more ammonia metabolism than HD and HF.In addition,ECLP can compensate amino acid imbalance and can secret bile.A controversial point with ECLP is the procedure is labor intensive,resulting in high costs.However,ECLP has the potential to reduce elevated serum ammonia levels of hepatic coma patients in a short duration.When these problems are solved,bioartificial liver support,especially ECLP,can be adopted as an option in ordinary clinical therapy to treat patients with hepatic failure.

Hybrid bioartificial liver support in cynomolgus monkeys with D-galactosamine-induced acute liver failure

AIM: To evaluate a hybrid bioartificial liver support system (HBALSS) in cynomolgus monkeys with acute liver failure.

Evaluation of a novel choanoid fluidized bed bioreactor for future bioartificial livers

AIM: To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes.

Advances in cell sources of hepatocytes for bioartificial liver

BACKGROUND: Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells. DATA SOURCES: A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the Key words such as liver failure bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed. RESULTS: Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines the incorporation of BAL with GS-HepG2 cells or alginate encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved. CONCLUSIONS: Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functionalhepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

Comparison between bioartificial and artificial liver for the treatment of acute liver failure in pigs

AIM： To characterize and evaluate the therapeutic efficacy of bioartificial liver （BAL） as compared to that of continuous hemodiafiltration （CHDF） with plasma exchange （PE）, which is the current standard therapy for fulminant hepatic failure （FHF） in Japan. METHODS： Pigs with hepatic devascularization were divided into three groups： （1） a non-treatment group （NT; n = 4）; （2） a BAL treatment group （BAL; n = 4）, （3） a PE ＋ CHDF treatment group using 1.5 L of normal porcine plasma with CHDF （PE ＋ CHDF, n -- 4）. Our BAL system consisted of a hollow fiber module with 0.2 i^m pores and 1 × 10^10 of microcarrier-attached hepatocytes inoculated into the extra-fiber space. Each treatment was initiated 4 h after hepatic devascularization. RESULTS： The pigs in the BAL and the PE ＋ CHDF groups survived longer than those in the NT group. The elimination capacity of blood ammonia by both BAL and PE ＋ CHDF was significantly higher than that in NT. Aromatic amino acids （AAA） were selectively eliminated by BAL, whereas both AAA and branched chain amino acids, which are beneficial for life, were eliminated by PE ＋ CHDF. Electrolytes maintenance and acid-base balance were better in the CPE ＋ CHDF group than that in the BAL group. CONCLUSION： Our results suggest that PE ＋ CHDF eliminate all factors regardless of benefits, whereas BAL selectively metabolizes toxic factors such as AAA. However since PE ＋ CHDF maintain electrolytes and acid-base balance, a combination therapy of BAL plus CPE ＋ CHDF might be more effective for FHF.

Progress in bioreactors of bioartificial livers

BACKGROUND: Bioartificial liver support systems are becoming an effective therapy for hepatic failure. Bioreactors, as key devices in these systems, can provide a favorable growth and metabolic environment, mass exchange, and immunological isolation as a platform. Currently, stagnancy in bioreactor research is the main factor restricting the development of bioartificial liver support systems. DATA SOURCES: A PubMed database search of English-language literature was performed to identify relevant articles using the keywords 'bioreactor', 'bioartificial liver', 'hepatocyte', and 'liver failure'. More than 40 articles related to the bioreactors of bioartificial livers were reviewed. RESULTS: Some progress has been made in the improvement of structures, functions, and modified macromolecular materials related to bioreactors in recent years. The current data on the improvement of bioreactor configurations for bioartificial livers or on the potential of the use of certain scaffold materials in bioreactors, combined with the clinical efficacy and safety evaluation of cultured hepatocytes in vitro, indicate that the AMC (Academic Medical Center) BAL bioreactor and MELS (modular extracorporeal liver support) BAL bioreactor system can partly replace the synthetic and metabolic functions of the liver in phase I clinical studies. In addition, it has been indicated that the microfluidic PDMS (polydimethylsiloxane) bioreactor, or SlideBioreactor, and the microfabricated grooved bioreactor are appropriate for hepatocyte culture, which is also promising for bioartificial livers. Similarly, modified scaffolds can promote the adhesion, growth, and function of hepatocytes, and provide reliable materials for bioreactors. CONCLUSIONS: Bioreactors, as key devices in bioartificial livers, play an important role in the therapy for liver failure both now and in the future. Bioreactor configurations are indispensable for the development of bioartificial livers used for liver failure, just as the modified scaffold materials available for bioreactors are favorable to the construction of effective bioartificial livers.

Bioartificial liver assist devices in support of patients with liver failure

Bioartificial liver assist devices (BALs) offer anopportunity for critical care physicians and transplantsurgeons to stabilize patients prior to orthotopic livertransplantation. Such devices may also act as a bridgeto transplant, providing liver support to patientsawaiting transplant, or as support for patients post liv-ing-related donor transplant. Four BAL devices thatrely on hepatocytes cultured in hollow fiber membranecartridges (Circe Biomedical HepatAssist (r), VitagenELADTM, Gerlach BELS, and Excorp Medical BLSS)are currently in various stages of clinical evalua-tion. Comparison of the four devices shows that severalunique approaches based upon the same overall systemarchitecture are possible. Preliminary results of theExcorp Medical BLSS Phase I safety evaluation at theUniversity of Pittsburgh, after treating four patients(F, 41, acetominophen-induced, two support periods;M, 50, Wilson's disease, one support period; F, 53, a-cute alcoholic hepatitis, two support periods; F, 24,chemotherapy-induced, one support period) are pre-sented. All patients presented with hypoglycemia andtransient hypotension at the start of extracorporealperfusion. Hypoglycemia was treated by IV dextroseand the transient hypotension responded positively toIV fluid bolus. Heparin anticoagulation was used onlyin the second patient. No serious or adverse eventswere noted in the four patients. Moderate biochemicalresponse to support was noted in all patients. Morecomplete characterization of the safety of the BLSSrequires completion of the Phase I safety evaluation.

Evaluation of a novel hybrid bioartificial liver based on a multi-layer flat-plate bioreactor

AIM: To evaluate the efficacy and safety of a hybrid bioartificial liver (HBAL) system in the treatment of acute liver failure. METHODS: Canine models with acute liver failure were introduced with intravenous administration of D-galactosamine. The animals were divided into: the HBAL treatment group (n = 8), in which the canines received a 3-h treatment of HBAL; the bioartificial liver (BAL) treatment group (n = 8), in which the canines received a 3-h treatment of BAL; the non-bioartificial liver (NBAL) treatment group (n = 8), in which the canines received a 3-h treatment of NBAL; the control group (n = 8), in which the canines received no additional treatment. Biochemical parameters and survival time were determined. Levels of xenoantibodies, RNA of porcine endogenous retrovirus (PERV) and reverse transcriptase (RT) activity in the plasma were detected. RESULTS: Biochemical parameters were significantly decreased in all treatment groups. The TBIL level in the HBAL group was lower than that in other groups (2.19 ± 0.55 mmol/L vs 24.2 ± 6.45 mmol/L, 12.47 ± 3.62 mmol/L, 3.77 ± 1.83 mmol/L, P < 0.05). The prothrombin time (PT) in the BAL and HBAL groups was significantly shorter than the NBAL and control groups (18.47 ± 4.41 s, 15.5 ± 1.56 s vs 28.67 ± 5.71 s, 21.71 ± 3.4 s, P < 0.05), and the PT in the HBAL group was shortest of all the groups. The albumin in the BAL and HBAL groups significantly increased and a significantly higher level was observed in the HBAL group compared with the BAL group (27.7 ± 1.7 g/L vs 25.24 ± 1.93 g/L). In the HBAL group, the ammonia levels significantly decreased from 54.37 ± 6.86 to 37.75 ± 6.09 after treatment (P < 0.05); there were significant difference in ammonia levels between other the groups (P < 0.05). The levels of antibodies were similar before and after treatment. The PERV RNA and the RT activity in the canine plasma were all negative. CONCLUSION: The HBAL showed great efficiency and safety in the treatment of acute liver failure.

No transmission of porcine endogenous retrovirus in an acute liver failure model treated by a novel hybrid bioartificial liver containing porcine hepatocytes

BACKGROUND： A novel hybrid bioartificial liver（HBAL） was constructed using an anionic resin adsorption column and a multi-layer flat-plate bioreactor containing porcine hepatocytes co-cultured with bone marrow mesenchymal stem cells（MSCs）. This study aimed to evaluate the microbiological safety of the HBAL by detecting the transmission of porcine endogenous retroviruses（PERVs） into canines with acute liver failure（ALF） undergoing HBAL.METHODS： Eight dogs with ALF received a 6-hour HBAL treatment on the first day after the modeling by D-galactosamine administration. The plasma in the HBAL and the whole blood in the dogs were collected for PERV detection at regular intervals until one year later when the dogs were sacrificed to retrieve the tissues of several organs for immunohistochemistry and Western blotting for the investigation of PERV capsid protein gag p30 in the tissue. Furthermore, HEK293 cells were incubated to determine the in vitro infectivity.RESULTS： PERV RNA and reverse transcriptase activity were observed in the plasma of circuit 3, suggesting that PERV particles released in circuit 3. No positive PERV RNA and reverse transcriptase activity were detected in other plasma. No HEK293 cells were infected by the plasma in vitro. In addition, all PERV-related analyses in peripheral blood mononuclear cells and tissues were negative.CONCLUSION： No transmission of PERVs into ALF canines suggested a reliable microbiological safety of HBAL based on porcine hepatocytes.

Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver

AIMTo develop a simplified bioartificial liver (BAL) device prototype, suitable to use freshly and preserved liver Microorgans (LMOs) as biological component. METHODSThe system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hematocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and ViaSpan® solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase (LDH) release and ammonia detoxification capacity. RESULTSThe concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and ViaSpan® were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls (49.3% ± 8.8%, P ® cold preserved LMOs, respectively (n = 6, P CONCLUSIONThis prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.

Recent advances in the application of cultured hepatocytes into bioartificial liver

INTRODUCTIONOver the past 3 decades,various experimentalliver support systems have been studied.Early artifi-cial liver support systems included hemodialysis,ex-tracorporeal liver perfusion,human cross-circulation,charcoal hemoperfusion,hepatodialysis,fresh blood,plasma exchange transfusion etc.Thesesystems were developed to remove toxic substancesfrom the blood.Clinical trials showed that thesedetoxification systems could promote the recovery

A reliable graded acute liver failure model in rats: treatment with internal bioartificial liver

BACKGROUND: Appropriate animal models are impor- tant for studying acute liver failure. This study was to assess a new suitable rat model for acute liver failure. METHODS: After the right influent hepatic vessels were clamped for a period of time (45, 60 or 90 minutes respec- tively), the animal model was established by removal of the clamp for restoring blood flow of the right lobes while im- mediately removal of the median, left lateral and caudate lobes. Animal survival rate was observed in the following 14 days in each group. To study the pathophysiological chan- ges of the model, some biochemical parameters in 5 con- secutive days were evaluated in the 60-minute group. Inter- nal bioartificial liver was transplanted in the peritoneal cavi- ty to test the reversibility of the model. RESULTS: The survival rate of the models decreased, as the ischemia time of the right lobes prolonged to zero in the 90-minute group, to 50% in the 60-minute group and to 100% in the 45-minute group on the fifth day after opera- tion. The levels of ammonia, alanine aminotransferase, al- kaline phosphatase, total bilirubin and prothrombin were elevated dramatically 12 to 24 hours after operation in the 60-minute group. When internal bioartificial liver was transplanted, the survival rate increased significantly in ad- dition to the levels of ammonia and total bilirubin. CONCLUSION: A period time of ischemic injury in the right lobe followed by 70% liver resection can produce a graded acute hepatic failure model in rats.

Prevalence of porcine endogenous retrovirus in Chinese pig breeds and in patients treated with a porcine liver cell-based bioreactor

AIM： To determine the prevalence of porcine endogenous retrovirus （PERV） in various pig breeds raised in China including Chinese experimental mini-pigs by PERV-reverse transcriptase （PERV-RT enzyme）. Moreover, the potential for infection of PERV was investigated in patients treated with a bioreactor based on porcine liver cells （n = 3）. METHODS： Pig serum, liver and muscle cell-free supernatants were collected from various Chinese pig breeds. Porcine hepatocytes were isolated with a two-step perfusion method. Three patients with acute or chronic liver failure were treated with a bioartificial liver support system （BALSS） for 8-12 h and serum samples were collected from the patients before, immediately after and 30 d after treatment. RESULTS： The activities of PERV-RT enzyme in pig liver and muscle cell-free supernatants were higher than in normal human controls. PERV-TR enzyme activity did not increase in patients before and after 1 mo of treatment. PERV-RT activities were not significantly different when compared with pre-treatment group （1.544±0.155576）, the post-treatment groups （1.501±0.053507, 1.461±0.033808 and 1.6006667±0.01963 for 0, 14 and 30 d post-treatment, respectively, P〉0.05）, and normal control group （1.440±1.0641, P〉0.05）. RT enzyme activity in Chinese experimental mini-pigs was higher than in normal human control group （1.440±1.0641 U/mL, P〈0.05）, and not significantly different （P〉0.05） when compared with the pig breeds except in the muscle supernatants. All the samples including muscle and liver cell supernatants from the Chinese mini-experimental pigs and the four domestic Chinese pig breeds contained PERVs. CONCLUSION： These results suggest that the risk of PERV infection through BALSS containing porcine liver cells without immunosuppressants may be quite low. Although there were PERVs in Chinese experimental mini-pigs and porcine liver cell culture suspensions, we did not find any evidence of persistent PERV infection in patients treated with this porcine hepatocyte-based bioartificial liver.

Hepatic reconstruction from fetal porcine liver cells using a radial flow bioreactor

AIM： To examine the efficacy of the radial flow bioreactor （RFB） as an extracorporeal bioartificial liver （BAL） and the reconstruction of liver organoids using embryonic pig liver cells. METHODS： We reconstructed the liver organoids using embryonic porcine liver cells in the RFB. We also determined the gestational time window for the optimum growth of embryonic porcine liver cells. Five weeks of gestation was designated as embryonic day （E） 35 and 8 wk of gestation was designated as E56. These cells were cultured for one week before morphological and functional examinations. Moreover, the efficacy of pulsed adminisbation of a high concentration hepatocyte growth factor （HGF） was examined. RESULTS： Both cell growth and function were excellent after harvesting on E35. The pulsed administration of a high concentration of HGF promoted the differentiation and maturation of these fetal hepatic cells. Microscopic examination of organoids in the RFB revealed palisading and showed that bile duct-like structures were well developed, indicating that the organoids were mini livers. Transmission electron microscopy revealed microvilli on the luminal surfaces of bile duct-like structures and junctional complexes, which form the basis of the cytoskeleton of epithelial tissues. Furthermore, strong expression of connexin （Cx） 32, which is the main protein of hepatocyte gap junctions, was observed. With respect to liver function, ammonia detoxification and urea synthesis were shown to be performed effectively. CONCLUSION： Our system can potentially be applied in the fields of BAL and transplantation medicine.