In hepatocytes,

cell division is complex, because polyploidy and aneuploidy are extremely high in p53+/+ livers from mice3 and humans.4 Nonetheless, disruption of normal Aurka and Lats2—and to a lesser extent Foxm1 and Plk4—expression partially accounts for enrichment in mitotic segregation errors and enhanced polyploidy S1P Receptor inhibitor seen in p53-deficient liver. After PH, transcriptional activity of p53 and how it contributes to activation or repression of mitotic or cell cycle regulators is more difficult to interpret. There may be a partial compensation by TA-p73, which has been shown to play a role in liver tumor suppression in combination with p53.35 A fully delineated story of how hepatocytes survive, and even thrive, in spite of high levels of polyploidy and

aneuploidy is not yet clear. p53 and its downstream effectors contribute to polyploidization and mitotic fidelity, as shown here in vivo. Whether p53 regulation is connected to activation of the insulin receptor and AKT signaling, implicated in cytokinesis failure Selumetinib in vitro and formation of polyploid hepatic cells,36 is unknown. Further characterization of new hepato-specific cell cycle pathways and definition of regulatory mechanisms are critical to understanding development, homeostasis, regeneration, and pathology of the liver. Additional Supporting Information may be found in the online version of this article. “
“JNK plays Branched chain aminotransferase a key role in hepatotoxicity by binding and phos-phorylating Sab on the outer mitochondrial membrane (J Biol Chem 286, 35071-8, 2011, Cell Death Dis; 5:e989, Jan 9, 2014). The mechanism for how this event on the cytoplasmic face of the outer membrane leads to impaired mitochondrial electron transport, increased ROS, and APAP-induced necrosis is unknown. We focused our attention on dysregulation of tyro-sine kinases (Src) because mitochondrial Src activity is known to regulate multiple steps in electron transport in other contexts (Biochem J. 447, 281-9,2012). Methods: Isolated mouse liver mitochondria were exposed to pure activated JNK +/− ATP, with or without Src or protein tyrosine

phosphatase (Ptp) inhibitors. APAP (300mg/kg) or PBS was given by ip injection to C57BL/6N mice; mitochondria and cytoplasm were prepared at 1,2,4 hours and histology and serum ALT were assessed at 24 hours. Knockdown of target genes in liver was by adeno-shRNA. Results: Using resistance to proteinase K digestion, we identified intramitochondrial c-Src mainly in an activated form (P-419-c-Src). Upon exposure of isolated mitochondria to P-JNK plus ATP, P-c-Src levels markedly decreased while total c-Src was unchanged. The decrease of P-c-Src was accompanied by inhibition of oxygen consumption rate (OCR), which depended on Sab expression. Addition of Src inhibitors (PP2 or Src inhibitor 1) to normal mitochondria directly inhibited OCR.

The 2/3 PH in C57BL/6 mice caused a decrease in Axin1 expression that was detectable at 12 hours, lowest between 24 and 36 hours, and began to return at 48 hours after surgery (Fig. S8A,B). The expression changes in Axin1 suggest that Axin1 might be inhibited by lncRNA-LALR1 during liver regeneration. Taken together, these data showed that lncRNA-LALR1 activated the Wnt/β-catenin pathway in hepatocytes. LncRNA-LALR1 decreased the expression of Axin1, and the stability of the β-catenin destruction complex receded, which led to the decline in the levels of phosphorylated β-catenin (inactive); active β-catenin could no longer CX-4945 cell line stay bound and was released. This monomeric form

of β-catenin binds to proteins such as T-cell factor-4 (TCF-4) and lymphoid enhancement factor (LEF) and translocates to the nucleus to control the transcription of target genes, including c-myc and cyclin D1. Finally, lncRNA-LALR1 facilitated mouse cell cycle progression and hepatocyte proliferation (Fig. 7). We wondered whether the mechanism of lncRNA-LALR1 activates the Wnt/β-catenin pathway by suppressing Axin1. We performed a computational screen (http://jaspar.genereg.net; see more CTCFBSDB2.0[19]) and found a CTCF binding site within the AXIN1 promoter region (−1,892 bp upstream of the transcription start site of AXIN1). Recent studies have reported

that the transcription factor CTCF can bind to the promoter region of target genes and inhibit their expression.[20] There was no significant difference in the CTCF mRNA and protein levels in lncRNA-LALR1-up-regulated CCL-9.1 cells compared to those in the control cells (data not shown). To determine whether lncRNA-LALR1 could change the binding of CTCF to the AXIN1 promoter region, D-malate dehydrogenase we performed ChIP analysis in lncRNA-LALR1-up-regulated CCL-9.1 cells and lncRNA-LALR1-down-regulated BNL CL.2 cells. We observed that overexpression of lncRNA-LALR1 increased the binding of CTCF at the AXIN1 promoter region in CCL-9.1 cells, and the binding declined in lncRNA-LALR1-down-regulated BNL CL.2 cells (Fig. 8A). These results confirmed that lncRNA-LALR1 could increase the binding of CTCF to the AXIN1 promoter region in hepatocytes. In addition, we

tested whether lncRNA-LALR1 could associate with CTCF. We performed RIP with an antibody against CTCF from extracts of BNL CL.2 cells and CCL-9.1 cells. We observed significant enrichment of lncRNA-LALR1 with the CTCF antibody compared with the nonspecific IgG control antibody (Fig. 8B). Next, we performed an in vitro RNA pulldown to validate the association between lncRNA-LALR1 and CTCF in BNL CL.2 cells and CCL-9.1 cells. This analysis confirmed that lncRNA-LALR1 physically associated with CTCF in vitro (Fig. 8C). Together, the RIP and RNA pulldown results demonstrate a specific association between CTCF and lncRNA-LALR1. The expression level of Axin1 was not statistically different in lncRNA-LALR1-down-regulated BNL CL.2 cells and lncRNA-LALR1-up-regulated CCL-9.

The method has therefore been made more stringent by the further requirement of selecting activation times, which warrant maximal rate of FX activation [20]. A relatively greater inter-laboratory variation is obtained on analyses of rFVIII preparations with both OS and CS methods as compared with plasma derived concentrates. Reason(s) Fluorouracil cell line for this

remain(s) to be identified. Direct proportionality between FVIII activity and FXa generation enables high resolution for the CS method at both high and low levels of FVIII activity. CS methods show strong correlation with OS methods for analysis of samples from haemophilia A patients both before and after FVIII concentrate infusion [13] and also in samples from VWD patients

[21]. Combined use of CS and OS methods is important in diagnosis of new haemophilia A patients, as discrepant results are obtained in certain subgroups. This has been shown in comparisons of OS and TS clotting methods and also for OS and CS methods [22]. TS and CS clotting methods showed lower FVIII activities and were in better agreement Cetuximab with clinical phenotype. Mutation analyses revealed point mutations on the A1, A2 and A3 domain interfaces causing the discrepancy. Interestingly, reversed findings have also been reported and mutations close to thrombin cleavage sites have been identified [23]. In the latter study thrombin was present in one CS reagent and it might be informative to perform analyses with the original CS method [13] which may be suitably modified to explore the initial FVIII activation phase. Altogether, the CS method has demonstrated wide applicability for determining FVIII activity in plasma samples and FVIII concentrates. Recognizing the diversity of FVIII both regarding its source and its formulation, a humble attitude is recommended on assay of FVIII activity, including careful optimization of preanalytical variables. Both classes of FVIII inhibitors, allo- and auto-antibodies, may present as

fully neutralizing inhibitors (type 1) or as inhibitors that only partially inhibit FVIII activity (type 2). The difference in kinetics between the two inhibitor types is probably related to their epitope specificity. Type 1 inhibitors, which occur predominantly Parvulin in haemophiliacs are directed against the FVIII A2 domain in >70% of patients, whereas type 2 inhibitors, which occur predominantly in acquired haemophilia, are directed against the VWF and phospholipid-binding FVIII C2 domain, making the epitope less accessible and resulting in incomplete FVIII inactivation. FVIII inhibitors manifest themselves by unexpected moderate-severe bleeding in individuals with previously normal haemostasis (autologous inhibitors) or by excessive bleeds, bleeding in unusual sites or low recovery and/or half-life of infused haemostatic products in haemophiliacs substituted with FVIII.

05). It is known, because of the high prevalence of HBV infection in the Chinese population, the vast majority of HCC occurs in patients with HBV and liver cirrhosis, and HBV-related HCC has become one of the main disease burdens in

China. To date, early diagnosis of HCC, especially in liver cirrhosis based on the emergence of small nodular lesions, is always a difficult problem for clinicians. Small HCC, known as a tumor of 5 cm or less in diameter in patients with single HCC, often have no obvious clinical symptoms and signs. It is believed that patients with liver cirrhosis are an ideal target population for HCC surveillance.33 Liver transplantation is an effective treatment for small, unresectable HCCs in patients with cirrhosis.34 Therefore, if we could differentiate the small buy Y-27632 HCC and liver cirrhosis, the survival rate of HCC patients would be greatly improved. In this study, the cohorts of liver cirrhosis and HCC patients selected were all infected with HBV, and all HCC cases were based on the liver cirrhosis, thus representing a realistic clinical scenario in which a diagnostic test for HCC needs

to be applied. Clusterin, first discovered as serum apolipoprotein J with chaperoning properties for protein stabilization, was virtually expressed in all tissues, and found in all human fluids.11,35 It is involved in numerous physiological processes that are important for carcinogenesis and/or tumor growth, including apoptotic cell death, cell cycle regulation, Ruxolitinib mw DNA repair, cell adhesion, tissue remodeling, lipid transportation, membrane recycling and immune system regulation.12 It was reported that

clusterin have altered expression in different cancer tissues, and it is apparent that this protein plays a significant role in the tumorigenesis of several types of human cancer, including HCC.30,31 In patients serum of lung cancer,36 colorectal carcinoma,37 bladder cancer38 and endometrial adenocarcinoma,39 upregulated serum clusterin was examined as compared with that in control healthy subjects. Depsipeptide In other types of human cancer, such as breast cancer40 and esophageal squamous cell carcinoma,17 however, downregulated serum clusterin was frequently observed. These results suggest a possible diagnostic role of this marker in different human cancers. To date, however, the expression levels of serum clusterin in HCC and its potential diagnostic significance is still not clear. In the present study, we used a method of sandwich ELISA to examine the serum clusterin concentrations in a cohort of HCC patients and control subjects (i.e., healthy subjects, HBV carriers, chronic hepatitis B and liver cirrhosis patients). The results demonstrated that there were no significant differences of serum clusterin levels between healthy subjects and HBV carriers.

05). It is known, because of the high prevalence of HBV infection in the Chinese population, the vast majority of HCC occurs in patients with HBV and liver cirrhosis, and HBV-related HCC has become one of the main disease burdens in

China. To date, early diagnosis of HCC, especially in liver cirrhosis based on the emergence of small nodular lesions, is always a difficult problem for clinicians. Small HCC, known as a tumor of 5 cm or less in diameter in patients with single HCC, often have no obvious clinical symptoms and signs. It is believed that patients with liver cirrhosis are an ideal target population for HCC surveillance.33 Liver transplantation is an effective treatment for small, unresectable HCCs in patients with cirrhosis.34 Therefore, if we could differentiate the small Selleckchem Venetoclax HCC and liver cirrhosis, the survival rate of HCC patients would be greatly improved. In this study, the cohorts of liver cirrhosis and HCC patients selected were all infected with HBV, and all HCC cases were based on the liver cirrhosis, thus representing a realistic clinical scenario in which a diagnostic test for HCC needs

to be applied. Clusterin, first discovered as serum apolipoprotein J with chaperoning properties for protein stabilization, was virtually expressed in all tissues, and found in all human fluids.11,35 It is involved in numerous physiological processes that are important for carcinogenesis and/or tumor growth, including apoptotic cell death, cell cycle regulation, selleck kinase inhibitor DNA repair, cell adhesion, tissue remodeling, lipid transportation, membrane recycling and immune system regulation.12 It was reported that

clusterin have altered expression in different cancer tissues, and it is apparent that this protein plays a significant role in the tumorigenesis of several types of human cancer, including HCC.30,31 In patients serum of lung cancer,36 colorectal carcinoma,37 bladder cancer38 and endometrial adenocarcinoma,39 upregulated serum clusterin was examined as compared with that in control healthy subjects. Buspirone HCl In other types of human cancer, such as breast cancer40 and esophageal squamous cell carcinoma,17 however, downregulated serum clusterin was frequently observed. These results suggest a possible diagnostic role of this marker in different human cancers. To date, however, the expression levels of serum clusterin in HCC and its potential diagnostic significance is still not clear. In the present study, we used a method of sandwich ELISA to examine the serum clusterin concentrations in a cohort of HCC patients and control subjects (i.e., healthy subjects, HBV carriers, chronic hepatitis B and liver cirrhosis patients). The results demonstrated that there were no significant differences of serum clusterin levels between healthy subjects and HBV carriers.

Children first treated with FVIII at age <11 months demonstrated a trend towards an increased risk of inhibitor formation, although it should be noted that this was not confirmed after adjusting for genetic factors [28]. More recently, contrasting to these first studies, it has been demonstrated that the age at first exposure did not influence

directly the risk of inhibitor development. First the CANAL study (Concerted Action on Neutralizing Antibodies in severe haemophiLia A), a multicentre retrospective cohort study, investigated the relationship between inhibitor development and treatment characteristics in previously untreated patients (n = 366) with severe haemophilia A (residual FVIII activity of <0.02 IU mL−1) [23]. Data LY294002 chemical structure from this study initially showed that an early age at the date of first exposure to FVIII was associated with an increased

risk of inhibitor development (see Table 2). However, further analysis demonstrated that this association largely disappeared after adjustment for intensity of treatment, using early surgical procedures, early major peak treatment moments and high dosing of FVIII related to a higher risk of inhibitor development [23]. The effects of FVIII exposure at an early age were also investigated in a study by Chalmers et al. [25]. The study results reflected the findings from CANAL and showed that exposure to FVIII during the neonatal period was not directly associated with a higher incidence of inhibitors than in those treated later during the first year of life [25]. check details As outlined by data from the CANAL study, intensity of treatment has been implicated as a factor responsible for increasing the risk for inhibitor development [23]. In another multicentre cohort study carried out by Gouw et al., intensive treatment periods (peak treatment moments and surgical Fossariinae procedures) were shown to increase the risk of inhibitor formation [29]. Furthermore, reduced duration between exposure days (EDs) was significantly associated with increased risk of

inhibitor development (>100 days between EDs adjusted relative risk 1.0 vs. 2.5 [1.3–4.9] and 2.7 [1.1–6.9] for 10–100 days and <10 days respectively P for trend = 0.01) [29]. Whereas the problem of inhibitor has been demonstrated as particularly relevant in severe haemophilia, intensity of treatment has also been shown to increase the risk of inhibitor development in patients with mild haemophilia. Sharathkumar et al. found that the incidence of inhibitor development was more than four times higher in patients administered with full intense FVIII therapy (administered via continuous infusion) compared with patients receiving any exposure to FVIII (57% vs. 14% respectively) [30].

[13-15] In co-culturing system, neutrophil-derived reactive oxygen species stimulates collagen synthesis in human HSCs, whereas treatment with various antioxidants attenuates it.[13] In addition, activating rat HSCs can recruit neutrophils by producing neutrophil-attracting chemokines such as MIP-2 and cytokine-induced neutrophil Vemurafenib datasheet chemoattractant.[14, 15] Furthermore, recent studies suggest that interleukin-17 (IL-17) produced by several type cells including neutrophils has potent roles to recruit and activate neutrophils, which is closely related with liver fibrosis of both human and mice.[16-18] Activation of HSCs

and liver fibrosis are negatively or positively regulated by lymphocyte population

or its inflammatory cytokines such as IL-10 and IL-17, respectively. For example, increased numbers of CD8+ T cells and decreased CD4+/CD8+ ratio are associated with induction of liver fibrosis in mice and human.[19, 20] Adoptive transfer of CD8+ T cells to SCID mice showed more liver injury and fibrosis induced by CCl4 than those of mice transferred with CD3+ or CD4+ T lymphocytes, whereas CD8+ T cell-mediated liver fibrosis was attenuated by IL-10.[19] In terms of the effects of CD4+ T cells on liver fibrosis, the role of IL-17-producing CD4+ T cells (Th17 cells) has been extensively investigated http://www.selleckchem.com/products/Maraviroc.html in the pathogenesis MycoClean Mycoplasma Removal Kit of liver fibrosis. IL-17 cytokines including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F are central players not only

in various adaptive immune responses to certain pathogens but also in autoimmune diseases.[21] Except IL-17E, IL-17 family cytokines can be produced by Th17 cells (dominant cell type), CD8+ T cells, γδ T cells, NK cells, and neutrophils.[16] Interestingly, recent several studies have suggested that IL-17 plays important roles in exacerbating liver fibrosis in both human and mice.[17, 18] These studies demonstrated that IL-17-stimulated human HSCs recruited neutrophils via chemokine production such as IL-8 and GROα,[17] and it directly stimulated collagen production in primary murine HSCs and human HSC cell line LX-2 via STAT3 activation,[18] leading to accelerated liver fibrosis. In summary, IL-17 and its producing CD4+ T cells are involved in promoting the liver fibrogenesis via several mechanisms. NKT cells are a subtype of lymphocytes that shares cell surface receptors of both NK and T cells.[22] Mouse liver lymphocytes contain approximately 30% NKT cells, while human liver lymphocytes contain up to 10%.[7, 22] Recent studies demonstrate that NKT cells promote liver fibrosis by producing inflammatory cytokines such as IL-4 and IL-13, leading to activation of HSCs in several murine models including HBV transgenic mice and xenobiotics-induced liver injury.

“
“Sequences of molecular events that initiate and advance the progression of human colorectal cancer (CRC) are becoming clearer. Accepting that these events, once they are in place, accumulate over time, rapid disease progression might be expected. Yet CRC usually develops slowly over decades. Emerging insights suggest that the tumor cell microenvironment encompassing fibroblasts and endothelial and immune this website cells dictate when, whether, and how malignancies

progress. Signaling pathways that affect the microenvironment and the inflammatory response seem to play a central role in CRC. Indeed, some of these pathways directly regulate the stem/progenitor cell niche at the base of the crypt; it now appears that the survival and growth of neoplastic cells often relies upon their subverted engagement of these pathways. Spurned on by the use of gene manipulation technologies in the mouse, dissecting and recapitulating these complex molecular interactions between the Everolimus supplier tumor

and its microenvironment in the gastrointestinal (GI) tract is a reality. In parallel, our ability to isolate and grow GI stem cells in vitro enables us, for the first time, to complement reductionist in vitro findings with complex in vivo observations. Surprisingly, data suggest that the large number of signaling pathways underpinning the reciprocal interaction between the neoplastic epithelium and its microenvironment converge on a small number of common transcription factors. Here, we review the separate and interactive roles of NFκB, Stat3, and Myb, transcription factors

commonly overexpressed or excessively activated in CRC. They confer molecular links between inflammation, stroma, the stem cell niche, and neoplastic cell growth. A functional link between inflammation, the tumor microenvironment, and cancer progression is now accepted. Historically, gastrointestinal (GI) cancers were among the first, where compelling associations between chronic inflammation, the tumor microenvironment, and progression had been noted. Such associations were based on the elevated risk for development of CRC associated with long-term inflammatory bowel disease (IBD),1 and more generally with autoimmune disorders affecting Oxaprozin the GI tract.2 Evidence indicates that the persistent cycles of tissue damage and repair lead to molecular events that drive precursors lesions to cancer.3 However, pharmaceutical intervention with non-steroidal anti-inflammatory drugs in patients with chronic inflammation reduced colorectal cancer (CRC) risk not only in the general population,4,5 but also in those individuals with genetic predispositions for this malignancy.6 Here, prostaglandins provide a molecular rationale and therapeutic target assumed to be cyclooxygenase (Cox)-2, the rate-limiting enzyme for prostaglandin synthesis, although constitutively-active Cox-1 might also play a role.

“
“Sequences of molecular events that initiate and advance the progression of human colorectal cancer (CRC) are becoming clearer. Accepting that these events, once they are in place, accumulate over time, rapid disease progression might be expected. Yet CRC usually develops slowly over decades. Emerging insights suggest that the tumor cell microenvironment encompassing fibroblasts and endothelial and immune selleckchem cells dictate when, whether, and how malignancies

progress. Signaling pathways that affect the microenvironment and the inflammatory response seem to play a central role in CRC. Indeed, some of these pathways directly regulate the stem/progenitor cell niche at the base of the crypt; it now appears that the survival and growth of neoplastic cells often relies upon their subverted engagement of these pathways. Spurned on by the use of gene manipulation technologies in the mouse, dissecting and recapitulating these complex molecular interactions between the MI-503 research buy tumor

and its microenvironment in the gastrointestinal (GI) tract is a reality. In parallel, our ability to isolate and grow GI stem cells in vitro enables us, for the first time, to complement reductionist in vitro findings with complex in vivo observations. Surprisingly, data suggest that the large number of signaling pathways underpinning the reciprocal interaction between the neoplastic epithelium and its microenvironment converge on a small number of common transcription factors. Here, we review the separate and interactive roles of NFκB, Stat3, and Myb, transcription factors

commonly overexpressed or excessively activated in CRC. They confer molecular links between inflammation, stroma, the stem cell niche, and neoplastic cell growth. A functional link between inflammation, the tumor microenvironment, and cancer progression is now accepted. Historically, gastrointestinal (GI) cancers were among the first, where compelling associations between chronic inflammation, the tumor microenvironment, and progression had been noted. Such associations were based on the elevated risk for development of CRC associated with long-term inflammatory bowel disease (IBD),1 and more generally with autoimmune disorders affecting buy Nutlin-3 the GI tract.2 Evidence indicates that the persistent cycles of tissue damage and repair lead to molecular events that drive precursors lesions to cancer.3 However, pharmaceutical intervention with non-steroidal anti-inflammatory drugs in patients with chronic inflammation reduced colorectal cancer (CRC) risk not only in the general population,4,5 but also in those individuals with genetic predispositions for this malignancy.6 Here, prostaglandins provide a molecular rationale and therapeutic target assumed to be cyclooxygenase (Cox)-2, the rate-limiting enzyme for prostaglandin synthesis, although constitutively-active Cox-1 might also play a role.

The family of serotonin receptors is subdivided into seven subgroups. These receptors

have been grouped according to their genetic and structural similarities and also according to the intracellular signaling pathways associated with each receptor. Serotonin regulates hepatic function and response to injury, blood flow, and proliferation of hepatocyte.14 In further studies, we could not detect any negative impact of serotonin in a model of ischemia/reperfusion injury. In contrast, we identified a new role for serotonin in tissue repair following ischemic injury.15 We therefore hypothesize that serotonin rescues liver regeneration after implantation of a small graft without enhancing the inherent ischemic damage, and thereby prevents SFS syndrome. 5-HT, 5-hydroxytryptamine; Dabrafenib supplier 5-HT2B, serotonin receptor-2B; AST, aspartate aminotransferase; DOI, α-methyl-5-HT; IL-6, interleukin-6; OLT, orthotopic liver transplantation; PCNA, proliferating cell nuclear antigen; PTX, pentoxifylline; SEC, sinusoidal endothelial cell; SFS, small-for-size; TNF-α, tumor necrosis factor α. Male inbred C57BL/6 mice were purchased from Harlan, Netherlands, IL-6−/−

mice with C57BL/6 background were obtained from http://www.selleckchem.com/products/VX-770.html Jackson Laboratory and used as syngeneic transplant donors and recipients. Animals were kept in accordance with the guidelines of the University of Zurich Animal Care Committee. The protocol of the study was approved by the Cantonal Veterinary office of Zurich. All mice were kept in a temperature-controlled environment

with a 12-hour light/dark cycle and with free access to food and tap water. We performed 30% partial OLTs in mice using techniques described previously.10 Some mice received a 25% OLT graft consisting of the right liver lobe. The recipient mice were divided into two groups: (1) α-methyl-5-HT (DOI, an agonist of the serotonin receptor and (2) a control group. DOI (1 mg/kg dissolved in saline) was given intravenously immediately following reperfusion of the partial Nintedanib (BIBF 1120) liver graft. Subsequently, recipients were injected subcutaneously twice a day for 2 days postoperatively. In control recipients, the same amount of vehicle solution was administered. Recipients were sacrificed at 1 hour, 3 hours, 2 days, or 7 days postoperatively. Hepatic regeneration, aspartate aminotransferase (AST) levels in serum, transcript levels of 5-HT2 receptors, IL-6, TNF-α in liver grafts, histology, scanning electron microscopy, and intravital microscopy were evaluated. In separate series of experiments, the recipient survival rates of 7 days after transplantation were tested. Some animals were treated with an antagonist of the 5-HT2B receptor: SB206553 (3 mg/kg) was injected subcutaneously into the donor and recipient before surgery and twice a day for 2 days after transplantation. Tissues were immersion-fixed in 4% buffered formalin and embedded in paraffin wax, then sectioned, and stained with hematoxylin-eosin.