Winroof Software
Results:HCMV-positive cells were detected in islets and exocrine areas in the patient with fulminant T1DM. Greater numbers of macrophages and CD4 + and CD8 + T lymphocytes had infiltrated into HCMV-positive islets than into HCMV-negative islets, and 52.6% of HCMV-positive cells were also positive for IRF3. Α Cells expressed IRF3, ZBP1, or RIG-I.
No HCMV-positive cells were detected in the control subjects. HHV-6−positive, but not EBV-positive, cells were present in the patient and the control subjects. Fulminant type 1 diabetes mellitus (T1DM), characterized by the extremely rapid progression of hyperglycemia and ketosis/ketoacidosis caused by the destruction of almost all pancreatic β cells, has been established as a subtype of idiopathic T1DM (, ). The etiology is thought to be viral infection because preceding flulike symptoms are frequently observed. In addition, there have been several reports of elevated broadly reactive anti-enterovirus immunoglobulin A and anti-virus antibody to human herpesvirus 6 (HHV-6), mumps virus, Coxsackie virus B4, and Epstein-Barr virus (EBV) in fulminant T1DM (–). However, only enterovirus has been detected in the autopsy pancreas of a patient with fulminant T1DM (–).Drug-induced hypersensitivity syndrome (DIHS) is a severe adverse drug reaction caused by carbamazepine, allopurinol, or mexiletine; it is characterized by visceral organ involvement and the reactivation of various human herpesviruses, such as HHV-6, human cytomegalovirus (HCMV), and EBV.
Although the frequency of fulminant T1DM in DIHS is only 0.54%, this is much higher than the frequency in the general Japanese population (0.010%) , indicating that virus reactivation might be associated with the onset of fulminant T1DM.In this study, we performed immunohistochemical examination of an autopsy pancreas from a patient who developed fulminant T1DM after DIHS. Patient and Methods Case presentationA 69-year-old male was prescribed allopurinol for hyperuricemia.
After 2 weeks, he had exanthema, fever, and cervical lymphadenopathy, and elevated hepatic enzyme and C-reactive protein levels were observed in his blood test results. He was then admitted to a hospital. Although he was treated with prednisolone, the exanthematous eruption persisted. At 3 weeks after admission, his levels of plasma glucose, glycoalbumin, and serum C-peptide were 836 mg/dL, 15.7% (normal range, 12.4–% to 16.3%), and 0.15 ng/mL (normal range, 0.61 to 2.09 ng/mL), respectively. A diagnosis of diabetic ketoacidosis was made on the basis of an arterial blood pH of 6.998, bicarbonate level of 2.1 mmol/L, anion gap of 34.9 mEq/L, 3-hydroxybutyrate level of 8.1 mmol/L (normal range, 0 to 0.074 mmol/L), and lactic acid level of 5.5 mmol/L (normal range, 0.44 to 1.78 mmol/L).
After 1 week, his hemoglobin A1c level (NGSP) was 7.7%, and HCMV pp65 antigen was detected in his blood. He was diagnosed with fulminant T1DM after DIHS. After 3 weeks, he died of septicemia and heart failure. Control subjectsSeven patients with normal glucose tolerance who had undergone pancreatic resection between 2009 and 2013 in the Department of Gastroenterological Surgery, Osaka University Hospital, were evaluated as control subjects. The tissues examined in this study were identified as noncancerous lesions by hematoxylin and eosin staining. ImmunohistochemistryThe primary and secondary antibodies and chromogenic substrates used in this study are listed in Supplemental Tables 1–3. To measure relative β or α cell areas, pancreatic sections were stained for insulin or glucagon using an avidin-biotin complex (ABC) and 3,3-diaminobenzidine (DAB) tetrahydrochloride substrate.
Next, we used double immunofluorescence staining for HCMV and insulin, glucagon, or amylase.To evaluate a relationship between the infiltration of inflammatory cells into islets and HCMV infection, pancreatic sections were stained for glucagon and HCMV by immunofluorescence staining and for CD68 by the ABC-DAB method (triple staining). In addition, we performed double immunofluorescence staining for glucagon and HCMV or glucagon and CD4 or CD8 on serial sections. For control tissues, we performed double immunofluorescence staining for insulin and CD68 or CD3.To evaluate antivirus immune responses to the virus infection, we also determined the expression of interferon regulatory factor 3 (IRF3), a transcription factor essential for type I interferon (IFN) production, using ABC-DAB staining and double immunofluorescence staining with HCMV or glucagon.
The same method was used to examine the expression of Z-DNA binding protein 1 (ZBP1) and retinoic acid-inducible gene I (RIG-I), which are representative DNA and RNA sensors, respectively.To screen for multiple infections, we stained for HHV-6, EBV, and enterovirus. Morphometric analysisRelative β or α cell area, the area of tissue containing insulin- or glucagon-positive cells in the entire pancreatic section, was quantified digitally using the WinROOF software program (Mitani Corporation, Japan) as previously reported. We counted all HCMV-, insulin-, or glucagon-positive cells within one section for analysis of the double immunofluorescence staining.To evaluate the infiltration of inflammatory cells into islets, islets 150 μm in diameter were examined as analysis objects, and we regarded inflammatory cells found in the islet periphery and throughout the islet parenchyma as “infiltrating”. To evaluate the localization of HHV-6 and enterovirus, we examined islets consisting of four or more endocrine cells.
Statistical analysisData that were normally distributed are presented as the mean ± standard deviation unless otherwise noted. Data for continuous variables with a skewed distribution are presented as the median (minimum, maximum).
The number of inflammatory cells infiltrating islets with or without HCMV infection was analyzed by the Student t test if continuous and parametric and by the Mann-Whitney U test if continuous and nonparametric. Differences were considered significant for values of P.
HCMV infection and localization in the pancreas. (A) Representative image of HCMV infection in the pancreas of this case. Double immunofluorescence staining for (B) HCMV and insulin, (C) HCMV and glucagon, and (D) HCMV and amylase. (B) HCMV (green) was not detected in residual β cells (red); (C) however, it was detected in α cells (red). (D) High levels of HCMV (arrows) were detected in acinar cells (red).
Bars = (A, D) 200 μm, (B) 100 μm, and (C) 50 μm. Infiltration of inflammatory cellsWe separately evaluated the infiltration of inflammatory cells into HCMV-negative and HCMV-positive islets. The numbers of CD68 + cells infiltrating HCMV-negative and HCMV-positive islets with residual α cells were 4.2 ± 5.1 (n = 239) and 6.5 ± 6.5 (n = 11) ( P = 0.154), respectively (A–C).
The numbers of infiltrating CD4 + cells were 0 (0, 5) (n = 271) and 1 (0, 6) (n = 17) ( P = 0.0023), respectively (D–F), and the numbers of infiltrating CD8 + cells were 0 (0, 10) (n = 267) and 3 (0, 20) (n = 16) ( P = 0.0012), respectively (G–I). In the control subjects, the numbers of CD68 + or CD3 + cells in and around the islets (n = 16 ± 12) were 0 ± 0 and 0.07 ± 0.10, respectively.
Infiltration of CD68 +, CD4 +, and CD8 + cells. (A, B) Triple staining of the same section for HCMV (green, arrowhead), glucagon (red), and CD68 (DAB). Double immunofluorescence staining for (D, G) HCMV (green; arrowheads) and glucagon (red); (E) CD4 (green; arrows) and glucagon (red); and (H) CD8 (green; arrows) and glucagon (red) in serial sections (D, E and G, H). (C, F, I) Infiltration of macrophages and CD4 + and CD8 + T lymphocytes was increased in HCMV-positive islets (black circle and black bar) compared with HCMV-negative islets (white circle and white bar).
Bars = (A, B, G, H) 50 μm and (D, E) 100 μm. Error bars indicate standard deviation (C) and 95% confidence interval (F, I). N.s., not significant. Infiltration of CD68 +, CD4 +, and CD8 + cells. (A, B) Triple staining of the same section for HCMV (green, arrowhead), glucagon (red), and CD68 (DAB).
Double immunofluorescence staining for (D, G) HCMV (green; arrowheads) and glucagon (red); (E) CD4 (green; arrows) and glucagon (red); and (H) CD8 (green; arrows) and glucagon (red) in serial sections (D, E and G, H). (C, F, I) Infiltration of macrophages and CD4 + and CD8 + T lymphocytes was increased in HCMV-positive islets (black circle and black bar) compared with HCMV-negative islets (white circle and white bar). Bars = (A, B, G, H) 50 μm and (D, E) 100 μm. Error bars indicate standard deviation (C) and 95% confidence interval (F, I). N.s., not significant. IRF3, ZBP1, and RIG-I expressionIn this case, double staining for IRF3 and HCMV revealed that 52.6% of the HCMV-positive cells (n = 1559) were also positive for IRF3 (A–C).
The rate of IRF3 positivity in α cells was 0.3% (n = 2872) ((D–F). In the control subjects, the number of IRF3-positive cells was 2.4 ± 2.8 (per section per subject) detected in the nonislet and nonexocrine regions. The rates of ZBP1 and RIG-I positivity in α cells were 0.4% (n = 2904) and 1.1% (n = 2987), respectively (G–L). In the control subjects, there was no expression of ZBP1 or RIG-I. Expression of IRF3, ZBP1, and RIG-I. Double immunofluorescence staining for (A) HCMV (red) and (B) IRF3 (green).
(C) HCMV-infected cells expressed IRF3 as shown in the merged image. Double immunofluorescence staining for (D) glucagon (red) and (E) IRF3 (green). (F) Some α cells expressed IRF3 as shown in the merged image. Double immunofluorescence for (G, J) glucagon (red) and (H) ZBP1 (green) or (K) RIG-I. (I, L) α cells expressing ZBP1 or RIG-I are shown in the merged images (arrows). Bars = (C, F, I) 10 μm and (L) 50 μm.
Expression of IRF3, ZBP1, and RIG-I. Double immunofluorescence staining for (A) HCMV (red) and (B) IRF3 (green). (C) HCMV-infected cells expressed IRF3 as shown in the merged image. Double immunofluorescence staining for (D) glucagon (red) and (E) IRF3 (green). (F) Some α cells expressed IRF3 as shown in the merged image. Double immunofluorescence for (G, J) glucagon (red) and (H) ZBP1 (green) or (K) RIG-I.
(I, L) α cells expressing ZBP1 or RIG-I are shown in the merged images (arrows). Bars = (C, F, I) 10 μm and (L) 50 μm. Presence of HHV-6, EBV, and enterovirus in the pancreasThe numbers of HHV-6−positive cells in one pancreatic section of this case and in the control subjects were 422 and 87 ± 87 , respectively. Double immunofluorescence staining for HHV-6 and glucagon or insulin revealed that all of the HHV-6 infected cells were present in nonislet areas (numbers of islets in the case and in the control subjects were 181 and 31 ± 14, respectively). No EBV-positive cells were detected in the case or in any control subjects. For enterovirus infection, only one VP1–positive cell was detected in one islet of two sections widely separated and , into which no CD8 + cells had infiltrated.
VP1-–positive cells were not detected in any control subjects. HHV-6 and enterovirus infection in the pancreas. HHV-6 infection was detected in the pancreas of (A) the case and (B) control subjects. (C) HHV-6 (green; arrow) was detected in nonislet lesions (insulin, red). Staining for (D) VP1 and (E) glucagon in serial sections of this case. (F) Enterovirus infection was detected in islets with no infiltration of CD8 + cells.
Bars = (A) 200 μm, (B, C) 100 μm, and (D–F) 50 μm. DiscussionIn the patient who developed fulminant T1DM after DIHS, many HCMV-positive cells were detected in islet cells and exocrine lesions of the autopsy pancreas. Although HCMV-positive β cells were not detected because of their almost complete destruction, there were significantly increased numbers of α cells expressing IRF3, ZBP1, or RIG-I; there were also increased numbers of infiltrating macrophages and CD4 + and CD8 + T lymphocytes in HCMV-positive islets compared with negative islets, indicating that β cells were injured by HCMV-specific immunoresponses.We detected HCMV infection in the islet cells of an autopsy pancreas with fulminant T1DM. To date, only enterovirus has been detected in the fulminant T1DM pancreas (–). In the current case, HCMV infection was detected in 0.2% of α cells, and the infiltration of inflammatory cells was increased in HCMV-positive islets compared with HCMV-negative islets, suggesting that the specific immunoresponse for HCMV was generated in HCMV-infected α cells and then spread to nearby β cells or possibly directly in HCMV-infected β cells.Because the relative β cell area was decreased by 97% compared with that of control subjects and few residual β cells remained, we could not detect HCMV-positive β cells in this case. It was previously reported that a human β cell could be infected with HCMV (, ).
HCMV-infected CM insulinoma cells showed enhanced expression of major histocompatibility complex class I and intercellular adhesion molecule-1, the production of inflammatory cytokines such as interleukin-6 and interleukin-8, and the activation of the peripheral blood mononuclear leukocytes. Although CM insulinoma cells are different than native β cells regarding the function of insulin secretion, they have a high homology with β cells concerning their basic cell structure, including surface markers that have a role in virus infection. Thus, it is possible that in this case β cells might have been infected with HCMV and injured by HCMV-specific immunoresponses, which is distinct from autoimmune T1DM characterized by β cell−specific immunoresponse. The other possible mechanism in fulminant T1DM in this case could be a bystander effect. Coxsackie B4 virus infection in nonobese diabetic mice, which carry the nonobese diabetic major histocompatibility complex allele to which presentation of the cross-reactive epitope is restricted, did not change the course of diabetes, but Coxsackie B4 infection of transgenic mice, which harbor a transgene encoding a diabetogenic T cell receptor specific to an islet antigen and not cross-reactive with Coxsackie B4, resulted in the rapid onset of diabetes.
This indicates that Coxsackie B4 virus might induce tissue damage through bystander T cell activation. Similarly, HCMV infection in islet cells might induce the activation of bystander lymphocytes, including autoreactive T cells, which might contribute to β cell death in this case.It was suggested that antivirus immunoresponses mediated by IRF3 expression had a function in the HCMV infection of pancreatic islets. Type I IFN, a representative factor of the antivirus immunoresponse, is important for the infiltration and activation of macrophages and CD4 +/CD8 + T cells. In this case, the expression of IRF3 was detected in 52.6% of HCMV-infected cells and some α cells, suggesting that type I IFN anti-virus immunoresponses occurred in HCMV-infected islet cells. Generally, following the infection of a DNA virus such as HCMV, the expression of IRF3 occurs via ZBP1, which is a DNA sensor ; in this case, however, the expression of RIG-I, an RNA sensor, was detected in α cells in addition to ZBP1. Similar to a study that reported that HCMV-infected fibroblasts expressed RIG-I , double immunofluorescence staining for HCMV and RIG-I in the adrenal gland of this case revealed that 13.7% of HCMV-positive cells expressed RIG-I (data not shown).
In a previous study of a fulminant T1DM patient, RIG-I was expressed in the islet cells of the enterovirus-infected pancreas; however, activation of the DNA sensor pathway was not observed. By contrast, because both ZBP1 and RIG-I were detected in the islet cells in this case, HCMV infection might be involved in IRF3 expression via both DNA and RNA sensors, partly sharing a common pathway with the enterovirus-infected subject who developed fulminant T1DM.We evaluated infiltrating CD68 +, CD4 +, and CD8 + cells as markers for macrophages, helper T cells, and cytotoxic T cells, respectively. Insulitis is occasionally diagnosed with infiltrating CD45 + cells as a marker for immune cells. CD45 is a leukocyte common antigen, and inflammation mediated by CD45 + cells, mainly CD68 + macrophages, was found in type 2 diabetic pancreases.
Thus, this definition might not distinguish pancreases retrieved from individuals with T1DM from those with type 2 diabetes.Although HHV-6 and EBV are reactivated in sequence in DIHS and enterovirus may be related to the onset of fulminant T1DM, none of these viruses is likely to be related to the onset of fulminant T1DM, at least in this case. IFN and checkpoint inhibitors have been reported to be related to the development of T1DM, but these drugs were not used in this patient.In conclusion, more HCMV-positive cells were detected in the autopsy pancreas of a patient who developed fulminant T1DM after DIHS. Furthermore, α cells expressing IRF3, ZBP1, or RIG-I and significantly increased numbers of inflammatory cells were observed in HCMV-positive islets compared with HCMV-negative islets. These findings suggest that the immunoresponse caused by HCMV infection was associated with β cell injury. Z-DNA binding protein 1.AcknowledgmentsA.I. Is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
The author thanks Dr. Hidetoshi Eguchi, Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, for the resected pancreatic tissues of the control subjects; Dr. Yasuko Mori, Division of Clinical Virology, Graduate School of Medicine, Kobe University, for the HHV-6B antigen; and Misako Kobayashi, Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, for her excellent technical assistance.This work was supported by KAKENHI Grant Number JP15K09429.Author Contributions: S.Y. Researched data and wrote the manuscript.
Contributed to the discussion and reviewed/edited the manuscript. S.U., J.K., and H.I. Contributed to the discussion. Contributed to the discussion and reviewed the manuscript. Were engaged in the treatment of this case.Disclosure Summary: The authors have nothing to disclose.
Author contributions: Hirabaru M and Eguchi S contributed equally to this work; Hirabaru M, Mochizuki K and Eguchi S designed the study; Hirabaru M, Eguchi S, Mochizuki K, Takatsuki M, Soyama A, Kosaka T, Kuroki T, Shimokawa I and Eguchi S performed the study and analyzed the data; and Hirabaru M and Eguchi S wrote the manuscript.Correspondence to: Susumu Eguchi, MD, PhD, Department of Surgery, Nagasaki University Graduate School of Biomedical sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.Telephone: +316 Fax: +319. Recently, there have been reports from liver biopsies that showed the progression of liver fibrosis in liver transplant patients after the cessation of immunosuppression.
Herein, we focused on activated hepatic stellate cells expressing alpha smooth muscle actin (α-SMA) to understand the correlation between immunosuppressant medication and liver fibrosis. The study enrolled two pediatric patients who underwent living donor liver transplantation and ceased immunosuppressant therapy. The number of α-SMA-positive cells in the specimens obtained by liver biopsy from these two patients showed a three-fold increase compared with the number from four transplanted pediatric patients who were continuing immunosuppressant therapy. In addition, the α-SMA-positive area evaluated using the WinRooF image processing software program continued to increase over time in three adult transplanted patients with liver fibrosis, and the α-SMA-positive area was increasing even during the pre-fibrotic stage in these adult cases, according to a retrospective review. Therefore, α-SMA could be a useful marker for the detection of early stage fibrosis. INTRODUCTIONLiver transplantation is an established treatment for hepatic failure.
Recent developments in surgical techniques, anesthesia and perioperative management have contributed to a decrease in early mortality after liver transplantation. However, the mortality in patients with chronic hepatic failure has remained unchanged. Some of the causes of the poor prognosis for these patients include renal disorders, vascular disorders, malignant tumors, and the use of immunosuppressant medication-.
Therefore, a reduction in such medication may reduce the mortality rate.Despite many reports describing patients who have acquired immune tolerance, the characteristics of patients with immune tolerance are still unknown. Clinical immune tolerance refers to the state of maintaining normal organ graft function even after the cessation of immunosuppressant medication,. In practice, the cessation of immunosuppressant medication varies depending on each patient’s condition and must be individualized; although some patients have a favorable postoperative course and can successfully achieve a reduction of immunosuppressant medication, other patients have no choice but to stop the treatment, such as in the case of infection with the Epstein-Barr virus (EBV).
Tony Jaa in The Protector (2005) Tony Jaa and Lateef Crowder at an event for The. Ong-Bak: The Thai Warrior. This is first Thai film to have done so. 
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The probability of adult patients acquiring immune tolerance has been reported to be 8%-33%-, and this rate has been suggested to be much higher in pediatric patients,.However, liver transplant recipients with no abnormalities in hepatic function after the cessation of immunosuppressant medication have recently been reported to developed hepatic fibrosis, with the hepatic fibrosis improving after resumption of the medication. Therefore, there is a need to understand the mechanism(s) of hepatic fibrosis induced by withdrawal of immunosuppression. We have herein focused on hepatic stellate cells (HSCs), which may be involved in hepatic fibrosis. HSCs constitute a large portion of the hepatic interstitium, representing 5%-8% of the total number of liver cells. In the healthy liver, HSCs are quiescent, but can be activated by factors, including TGFβ1 and IFNγ, that are released by Kupffer cells (KC) and T cells after injury or stimulation,.
The appearance of alpha smooth muscle actin (α-SMA) in the activated HSCs can be detected using α-SMA immunostaining. Activated HSCs undergo apoptosis at sites of acute inflammation but induce sinusoidal sclerosis, leading to the development of sinusoidal portal hypertension at sites of chronic inflammation. The activated HSCs have also been suggested to be responsible for the expression of type I collagen and the progression of fibrosis.
We therefore predict that an immune response may cause fibrosis in patients who have discontinued immunosuppressant medication, however the mechanism underlying this response remains to be determined.We performed immunohistological analysis to determine the mechanism underlying the fibrosis associated with immunosuppressant medication in two pediatric patients who were doing well with good graft function without immunosuppression for several years after receiving living donor liver transplantation (LDLT). PatientsA total of 163 patients underwent LDLT in our department from August 1997 to May 2012. Among them, 12 were pediatric patients who were less than 18 years of age, and 2 of these pediatric patients had ceased immunosuppressant medication for a long period. One patient was an 18-year-old male who underwent LDLT for biliary atresia (BA) at 5-years of age. In this case, immunosuppression (IS) was stopped according to the weaning protocol because of his good condition 68 mo after the LDLT. Another patient was an 11-year-old female who underwent LDLT for BA at 11-mo of age. Her IS was stopped non-electively because of EBV infection 3 mo after the LDLT.
A total of eight liver biopsies were performed in these two patients. As a control, this study also included four pediatric patients who did not have hepatic function abnormalities or fibrosis and continued their immunosuppressant medication (no-tolerance cases).
To examine whether the findings in these pediatric cases were also relevant to adult patients with fibrosis, three randomly selected patients with liver fibrosis not due to hepatitis C were evaluated.Specimens were collected by ultrasound-guided core needle biopsy. Each specimen was stained with hematoxylin eosin, and the severity of fibrosis was determined using Ishak’s modified staging system. The evaluation of each specimen was conducted blindly by two pathologists. ImmunohistochemistryFour-micrometer-thick sections, cut from formalin-fixed, paraffin-embedded tissues, were immunohistochemically stained for SMA, CD68, and CD79α. The following primary antibodies and a staining kit MAX-PO (MULTI), Nichirei Corporation, Tokyo, Japan containing peroxidase-labeled -secondary antibodies were used: anti-alpha-SMA (Nichirei; Code 412021), anti-CD68 (Dako, Tokyo, Japan; Code M0814), and anti-CD79α (Dako; Code N162830).
The immunostaining was performed according to the manufacturer’s instructions. LDLT: Living donor liver transplantation; IS: Immunosuppression; α-SMA: Alpha smooth muscle actin; BA: Biliary atresia; M: Male; F: Female.In addition, the number of α-SMA-positive cells was 227.5 ± 99.0 in the tolerant patients with F0 stage fibrosis, which was higher than that in the patients without tolerance.
The α-SMA-positive area ratio was also calculated using the WinROOF software program. The α-SMA-positive area ratio in the patients without tolerance with any fibrotic stage was 2.3% ± 0.46%; it was 2.2% ± 0.47% in cases with fibrotic stage F0 and 0.75% ± 0.53% in the no-tolerance patients (all patients with fibrotic stage F0). Accordingly, even among patients with no findings of fibrosis, the α-SMA area ratio was higher in patients with tolerance than in those without tolerance. Degree of α-SMA staining in LDLT cases with fibrosisThe α-SMA-positive area ratio was calculated for adult patients with fibrosis using the WinROOF software program. Liver specimens obtained from a total of 10 liver biopsies in fibrosis Cases 1 to 3 were subjected to the analysis (Figure ). Figure shows the timing of the biopsies, fibrosis grade, and α-SMA-positive area ratio.
The α-SMA-positive area continued to increase over time in all patients, and the α-SMA-positive area also increased in all patients even when they were in the pre-fibrotic stage (arrowhead). Changes in alpha smooth muscle actin expression in adult patients with fibrosis. A: The α-smooth muscle actin (SMA)-positive area continued to increase over time in all the patients, even when they were in the pre-fibrotic stage (arrow head); B: The α-SMA positive area ratio in the patients with fibrosis was calculated based on the fibrosis stage. The α-SMA area ratio was higher in the patients with F0-1 fibrosis than in those with F4 fibrosis; C: The photograph on the left shows the α-SMA staining in a representative case with F4 fibrosis.
The photograph on the right shows a WinRoof digital image, with green corresponding to the area of α-SMA-positive staining. Α-SMA: Alpha smooth muscle actin; LDLT: Living donor liver transplantation.The α-SMA-positive area ratio in adult patients with fibrosis was also evaluated based on the fibrosis stage. The area ratio was 1.1% ± 0.5% in the F0-1 stage and 4.6% ± 1.2% in the F4 stage. The α-SMA area ratio was higher in the F0-1 stages than in the F4 stage (Figure ).The α-SMA-positive area continued to increase over time in the pediatric patients with tolerance.
Pediatric Case 1 showed F0 fibrosis in the liver at all time points, whereas pediatric Case 2 showed a slight progression of fibrosis (F1) eight years after the cessation of the immunosuppressant treatment (Figure ). However, there were no significant increases in the α-SMA-positive area in the pediatric cases without tolerance (Figure ). Change in alpha smooth muscle actin expression in the two pediatric cases with immune tolerance.
A: The α-SMA-positive area continued to increase over time in both cases. Case 1 showed F0 fibrosis in the liver at all time points, whereas Case 2 showed a slight progression of fibrosis (F1) eight years after the cessation of immunosuppressive treatment; B: The findings of Azan-Mallory staining and the α-SMA-positive area determined by immunohistochemical analysis are shown. Α-SMA: Alpha smooth muscle actin; IS: Immunosuppression. DISCUSSIONImmune tolerance is the ultimate goal of transplantation, and many transplant patients have been reported to have ceased immunosuppressant medication for a long period while maintaining a favorable clinical course in clinical practice,. However, hepatic fibrosis has been reported to have developed in transplant patients who have ceased immunosuppressant medication, and there are some concerns over whether the cessation of immunosuppressive treatment leads to fibrosis. We performed a histological analysis in patients who had ceased immunosuppressant medication and examined the impact of the cessation on the liver graft.As shown by the findings of this and previous studies, the expression of α-SMA increases with the progression of hepatic fibrosis because liver injury that is caused by hepatic viruses or medication allows T cells and Kupffer cells to release PDGF, IGF-I, TGF-β, activated oxygen, lipid peroxide, and α-SMA into the cytoplasm of HSCs. Subsequently, type I collagen is produced and fibrosis develops.
Therefore, it is supposed that the α-SMA level is increase in the pre-fibrotic stage, although there have been no reports on the correlation between the α-SMA expression level and fibrotic changes.Here, we investigated the association between α-SMA expression and the fibrotic stage in patients with hepatic fibrosis. An increase in α-SMA over time was observed in all patients with fibrosis, suggesting that there is a correlation between the progression of fibrosis and the increase in α-SMA. Furthermore, all patients with fibrosis had increased α-SMA expression at the pre-fibrotic stage. In the current study, in two cases of tolerance, the progression of fibrosis remained low, although the ratio of α-SMA-positive areas remained high in both cases. However, Yoshitomi et al reported that liver fibrosis progression in tolerant patients was higher than that in non-tolerant patients.
Therefore, we propose that α-SMA may be a potential marker of the progression of liver fibrosis. Thus, we should continue to follow these two cases carefully to ensure that the progression of fibrosis is not missed. Because this study was a retrospective analysis and the time of biopsy varied for the individual patients, periodic follow-up examination will be required to evaluate and support this hypothesis. These findings suggest that a routine protocol biopsy could be an important tool to understand the dynamic state of α-SMA in detail.These assumptions suggest that the possibility of developing fibrosis is higher in tolerant patients than in patients who continue immunosuppression, because α-SMA expression was consistently high in the tolerant patients in this study.
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In addition, the expression of α-SMA gradually increased in the tolerant patients, indicating that care should be taken in future follow-ups for these patients to ensure that their liver function does not deteriorate.The liver is known to be the organ most susceptible to immune tolerance compared with other organs, and phenomena in different animal species have been observed that demonstrate that the major histocompatibility complex was successfully engrafted without immunosuppressant medication after an allogeneic liver transplant,. The effects of immunosuppressive factors produced in the liver and the correlations among antigen-presenting cells in the transplanted liver, including Kupffer cells, sinusoidal endothelial cells, and recipient-derived T cells, are believed to be involved in this immune tolerance. In addition, there have been some studies in mice showing that T cells became unresponsive to the antigen presented from sinusoidal endothelial cells of the specific donor type,.
A treatment strategy leading to the acquisition of immune tolerance is considered to be important for human liver transplantation to prevent damage to hepatic sinusoidal endothelial cells.CD68 (KCs) and CD79α (T cells) were immunostained to search for factors related to fibrosis in patients with and without immune tolerance, but there were no significant differences in either KCs or T cells. The major factor determining the progression of fibrosis in patients with immune tolerance still remains unknown, and predictors for the development of tolerance are also unknown. Therefore, we confirm that liver fibrosis staging assessed by biopsy is the main parameter influencing the treatment course.A previous study indicated that calcineurin inhibitors (CNIs) may inhibit the activity of HSCs and the progression of fibrosis, but convincing evidence has not yet been provided. However, there is a good possibility that the cessation of immunosuppressant medication may cause a certain degree of rejection without abnormal hepatic function or histological rejection. There were also no differences in α-SMA expression between hepatitis C virus-infected patients with and without liver transplants. In addition, CNIs administration may not always inhibit α-SMA expression if there is an infectious background.
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In addition, the infiltration of inflammatory cells stimulates the expression of pre-fibrotic growth factors, and inflammatory cells and activated HSCs are actually mixed in patients with chronic hepatic dysfunction. Therefore, controlling inflammation is considered to inhibit the progression of fibrosis regardless of the use of immunosuppressant medication.When deciding whether to resume immunosuppressant medication, it is important to determining whether the progression of fibrosis is due to an antigen response is important. However, the factors associated with an increase in α-SMA were not determined in the present study in the two pediatric patients, who had most likely acquired immune tolerance. Immunosuppressant medication has not been resumed in these two pediatric patients because they have not shown clear abnormalities in liver function. However, we are performing a strict follow-up regime for both patients to determine whether they will continue to have a good long-term prognosis.