Peripheral Donor-specific Antibodies Are Associated With Histology and Cellular Subtypes in Protocol Liver Biopsies of Pediatric Recipients.

BACKGROUND: The cellular infiltrate in protocol liver biopsies (PB) following pediatric liver transplantation remains mostly uncharacterized, yet there is increasing concern about the role of inflammation and fibrosis in long-term liver allografts. We aimed to define cell types in PB and to analyze their relationship with donor-specific antibodies (DSA) and histological phenotype. METHODS: PB were performed at least 1 year after transplantation. We identified 4 phenotypes: normal, fibrosis, inflammation, inflammation with fibrosis. Cell types were counted after immunostaining for CD3, CD4, CD8, CD68, CD20, MUM1, and FoxP3. RESULTS: Forty-four patients underwent 1 PB between 2000 and 2015. Eleven percent (5/44) of PB displayed normal histology, 13.6% (6/44) fibrosis, 34.1% (15/44) inflammation, and 40.9% (18/44) inflammation and fibrosis. The main cell types in the portal tracts and lobules were CD3+ and CD68+ cells. Frequency of de novo DSA was 63% (27/44). The presence of CD8+ cells in the lobules was associated with fibrosis. Inflammation and fibrosis in PB were associated with the presence of circulating de novo DSA, number of de novo DSA, and C1q binding activity when compared to other phenotypes. CONCLUSIONS: T cells (CD3+) and macrophages (CD68+) were the most prevalent cell-types in PB. In the presence of inflammation, portal tracts were enriched in CD3+, CD20+ but displayed fewer CD68+. This coincided with the presence and number of de novo DSA. How these cellular and humoral actors interact is unclear, but peripheral DSA may be a marker of immune cellular activity in the seemingly quiescent allograft.

Portal inflammation predicts renal dysfunction in patients with nonalcoholic fatty liver disease.

BACKGROUND: The association between nonalcoholic fatty liver disease (NAFLD) and renal function changes remains inconclusive. We explored whether the histological severity of NAFLD is associated with early deterioration of renal function. METHODS: Patients with biopsy-proven NAFLD were prospectively followed for renal function monitoring. A renal outcome was defined as a ≥ 50% increase in serum creatinine, a < 30% decrease in the estimated glomerular filtration rate (eGFR) or an eGFR < 45 mL/min/1.73 m2. RESULTS: Among 455 NAFLD patients, 221 (48.6%) had nonalcoholic steatohepatitis (NASH), and no difference in baseline eGFR was found between NASH and NAFL patients. During a median follow-up of 32 months, a renal outcome occurred in 15 patients; the incidence rate was 12.3 per 1,000 person-years. Compared with NAFL, NASH did not increase the risk of renal outcomes. Among the histological components of NAFLD, lobular inflammation (≥ 2), fibrosis (≥ F3), and portal inflammation (≥ 3) significantly increased the risk of renal outcomes in the crude analysis (HR 3.35, 95% CI 1.10-9.11; HR 3.25, 95% CI 1.12-8.84; and HR 7.73, 95% CI 2.86-22.22). After adjustment for risk factors for renal dysfunction, including sex, age, diabetes, hypertension, and chronic kidney disease, only portal inflammation significantly increased the risk of renal outcomes (HR 5.88, 95% CI 1.87-18.42, p = 0.002). CONCLUSIONS: Portal inflammation predicts early deterioration of renal function in patients with biopsy-proven NAFLD. Individualized monitoring of renal function based on the histological severity of NAFLD may be helpful for early identification of long-term renal outcomes.

AAV-IL-22 modifies liver chemokine activity and ameliorates portal inflammation in murine autoimmune cholangitis.

There remain significant obstacles in developing biologics to treat primary biliary cholangitis (PBC). Although a number of agents have been studied both in murine models and human patients, the results have been relatively disappointing. IL-22 is a member of the IL-10 family and has multiple theoretical reasons for predicting successful usage in PBC. We have taken advantage of an IL-22 expressing adeno-associated virus (AAV-IL-22) to address the potential role of IL-22 in not only protecting mice from autoimmune cholangitis, but also in treating animals with established portal inflammation. Using our established mouse model of 2-OA-OVA immunization, including α-galactosylceramide (α-GalCer) stimulation, we treated mice both before and after the onset of clinical disease with AAV-IL-22. Firstly, AAV-IL-22 treatment given prior to 2-OA-OVA and α-GalCer exposure, i.e. before the onset of disease, significantly reduces the portal inflammatory response, production of Th1 cytokines and appearance of liver fibrosis. It also reduced the liver lymphotropic chemokines CCL5, CCL19, CXCL9, and CXCL10. Secondly, and more importantly, therapeutic use of AAV-IL-22, administered after the onset of disease, achieved a greater hurdle and significantly improved portal pathology. Further the improvements in inflammation were negatively correlated with levels of CCL5 and CXCL10 and positively correlated with levels of IL-22. In conclusion, we submit that the clinical use of IL-22 has a potential role in modulating the inflammatory portal process in patients with PBC.

Paneth cell-mediated multiorgan dysfunction after acute kidney injury.

Acute kidney injury (AKI) is frequently complicated by extrarenal multiorgan injury, including intestinal and hepatic dysfunction. In this study, we hypothesized that a discrete intestinal source of proinflammatory mediators drives multiorgan injury in response to AKI. After induction of AKI in mice by renal ischemia-reperfusion or bilateral nephrectomy, small intestinal Paneth cells increased the synthesis and release of IL-17A in conjunction with severe intestinal apoptosis and inflammation. We also detected significantly increased IL-17A in portal and systemic circulation after AKI. Intestinal macrophages appear to transport released Paneth cell granule constituents induced by AKI, away from the base of the crypts into the liver. Genetic or pharmacologic depletion of Paneth cells decreased small intestinal IL-17A secretion and plasma IL-17A levels significantly and attenuated intestinal, hepatic, and renal injury after AKI. Similarly, portal delivery of IL-17A in macrophage-depleted mice decreased markedly. In addition, intestinal, hepatic, and renal injury following AKI was attenuated without affecting intestinal IL-17A generation. In conclusion, AKI induces IL-17A synthesis and secretion by Paneth cells to initiate intestinal and hepatic injury by hepatic and systemic delivery of IL-17A by macrophages. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from AKI.

Cytokine levels and histopathology in chronic hepatitis B and chronic hepatitis C.

The changes in balance of cytokine profile may result in either recovery or persistence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. This study aims to reveal a possible correlation between cytokine levels, ie, tumor necrosis factor (TNF)-α; interferon-gamma (IFN-γ); interleukin (IL)-10, IL-18, and transforming growth factor-beta (TGF-ß); and Ishak score or fibrosis in patients with chronic hepatitis B (CHB) or chronic hepatitis C (CHC). Fifty patients with CHB (n=25), CHC (n=25), and the control group of subjects with negative hepatitis B and C serology (n=30) were included in the study. Patients who did not agree to participate in the study were excluded. Serum cytokine levels were measured by ELISA. Liver biopsies from the patients were also taken for pathological analyses by the same pathologist. The serum levels of TNF-α, IL-10, and IL-18 in the hepatitis C group were significantly high compared with those of the control group (P=0.017, P=0.001, and P=0.004 respectively), but, only IL-10 levels in the hepatitis B group were significantly high (P=0.001). These groups did not show any significant difference with respect to IFN-γ or TGF-ß levels. In patients with CHB or CHC, there was a significant correlation (P=0.000) between TNF-α and Ishak score or fibrosis; but no such correlation was found with IFN-γ, IL-10, IL-18, or TGF-ß. Result of the current study indicated that cytokine activities were important indicators of clinical severity and progression of HBV- and HCV infections. Further investigations on possible effects of cytokines on hepatocellular damage and fibrosis should be done to arrange new immunopathological approaches to viral hepatitis.

High prevalence of autoantibodies to C-reactive protein in patients with chronic hepatitis C infection: association with liver fibrosis and portal inflammation.

The presence of autoantibodies against C-reactive protein (anti-CRP) has been reported in association with autoimmunity and histopathology in chronic hepatitis C virus (HCV) infection. Resistin could play a role in the pathogenesis of hepatitis, although results on HCV infection are ambiguous. Here we retrospectively analyzed anti-CRP and resistin levels in the sera of 38 untreated and well-characterized HCV patients at the time of their first liver biopsy. HCV activity and general health were assessed by a physician at least yearly until follow-up ended. Anti-CRP and resistin were also measured in patients with autoimmune hepatitis (AIH) and nonalcoholic fatty liver disease (NAFLD). Anti-CRP antibodies were registered in all HCV patients, whereas only a few AIH (11%) and NAFLD (12%) sera were positive. Anti-CRP levels were related to histopathological severity and were highest in patients with cirrhosis at baseline. Resistin levels were similar in HCV, AIH, and NAFLD patients, but high levels of resistin were associated with early mortality in HCV patients. Neither anti-CRP nor resistin predicted a response to interferon-based therapy or cirrhosis development or was associated with liver-related mortality. We conclude that anti-CRP antibodies are frequently observed in chronic HCV infection and could be a useful marker of advanced fibrosis and portal inflammation.

Anti-endotoxin hyperimmune globulin attenuates portal cytokinaemia, phagocytic cell priming, and acute lung injury after lower limb ischaemia-reperfusion injury.

OBJECTIVES: Acute limb ischaemia is a common and often lethal clinical event. Reperfusion of an ischaemic limb has been shown to induce a remote gut injury associated with transmigration of endotoxin into the portal and systemic circulation, which in turn has been implicated in the conversion of the sterile inflammatory response to a sepsis syndrome, after lower torso ischaemia-reperfusion injury. This study tests the hypothesis that an anti-endotoxin hyperimmune globulin attenuates ischaemia-reperfusion (I/R) associated sepsis syndrome. DESIGN: Prospective, randomised placebo controlled trial, animal experiment. MATERIALS AND METHODS: Experimental porcine model, bilateral hind limb I/R injury, randomised to receive anti-endotoxin hyperimmune globulin or placebo. RESULTS: Bilateral hind limb I/R injury significantly increased intestinal mucosal acidosis, portal endotoxaemia, plasma cytokine (TNF-alpha, IL-6, IL-8) concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, oedema, and capillary-alveolar protein leak. Conversely, pigs treated with anti-endotoxin hyperimmune globulin (IgG) 20mg/kg at onset of reperfusion had significantly reduced portal endotoxaemia, early circulating phagocytic cell priming, plasma cytokinaemia and attenuation of acute lung injury. CONCLUSIONS: Endotoxin translocation across a hyperpermeable gut barrier, phagocytic cell priming and cytokinaemia are key events of limb I/R injury induced systemic inflammation and acute lung injury. This study shows that an anti-endotoxin hyperimmune globulin attenuates portal endotoxaemia, which may reduce early phagocytic cell activation, cytokinaemia and ultimately acute lung injury.

Undifferentiated murine embryonic stem cells cannot induce portal tolerance but may possess immune privilege secondary to reduced major histocompatibility complex antigen expression.

Induction of donor-specific tolerance using embryonic stem (ES) cells followed by transplantation of ES cell-derived tissues from the same allogeneic strain could theoretically engender successful transplantation without immunosuppression. We sought to induce tolerance using bona fide murine ES cells in immunocompetent mice. ES cells were evaluated for the expression of markers restricted to undifferentiated cells [stage-specific embryonic antigen-1 (SSEA-1) and OCT-4] and the ability to form teratomas in immunodeficient mice. BALB/cByJ mice underwent intraportal inoculation with YC5-EYFP ES cells (129 strain; R1-derived) or saline followed by transplantation with 129X1/SvJ, CBA/J, or BALB/cByJ nonvascularized, neonatal cardiac grafts. Mice were sacrificed at graft failure and underwent histologic evaluation of transplanted grafts and lymphoid organs. ES cells and early differentiated progeny underwent real time (RT)-PCR and fluorescence-activated cell sorting (FACS) analysis to detect major histocompatibility complex (MHC) gene transcription and antigen expression. ES cells expressed markers restricted to undifferentiated cells while maintaining the ability to form teratomas in immunodeficient mice. No prolongation of allograft survival or evidence of lymphoid chimerism was observed in immunocompetent recipient mice despite hepatic teratoma formation. MHC class I, class II, and nonclassical antigens were undetectable on ES cells and early differentiated progeny despite the presence of mRNA transcripts. Class I expression was strongly upregulated upon exposure to gamma-interferon. Intraportal inoculation with murine ES cells does not produce lymphoid chimerism or induce donor-specific unresponsiveness to neonatal cardiac grafts in unmanipulated immunocompetent hosts. However, specific differentiated cell types such as ES cellderived dendritic cells, or alternate routes of ES cell administration, may be effective. ES cells appear to have immune privilege, allowing them to form teratomas in immunocompetent mice.

Cytokine production associated with periportal fibrosis during chronic schistosomiasis mansoni in humans.

Volunteers living in an area where schistosomiasis mansoni is endemic were subjected to ultrasound examination and classified into groups according to the levels of fibrosis diagnosed, namely, absence of indications of fibrosis (group 0), incipient fibrosis (group 1), and moderate/severe fibrosis (group 2). Peripheral blood mononuclear cells (PBMC) collected from the volunteers were stimulated with soluble antigens from adult schistosomes or from schistosome eggs, and the production of the cytokines gamma interferon, tumor necrosis factor alpha, transforming growth factor beta (TGF-beta), interleukin-4 (IL-4), IL-10, and IL-13 was determined. Potential associations of the level of fibrosis with age, sex, intensity of infection, and cytokine production were investigated between the three groups. Univariate analysis identified associations of age (>50), gender (male), and absence of eggs/g of feces with moderate/severe fibrosis and an association of intensity of infection (>100 eggs) with incipient fibrosis. When cytokine production in PBMC cultures stimulated by soluble egg antigens was categorized as low or high, significant differences in the distribution of IL-13 levels were established between groups 0 and 2. No significant differences were detected between the groups in the cytokines produced by PBMC cultures stimulated with soluble antigens from adult schistosomes. When all variables were tested in multivariate analyses, only IL-13 was strongly associated with fibrosis (odds ratio = 5.8; 95% confidence interval [CI] = 1.1 to 30.5). While high levels of TGF-beta appeared to be associated with protection against fibrosis, the strength of the association was low.

Immune-mediated liver injury: prognostic value of CD4+, CD8+, and CD68+ in infants with extrahepatic biliary atresia.

BACKGROUND: Hepatic fibrosis and cirrhosis develop progressively in extrahepatic biliary atresia (EHBA) despite timely surgical intervention. PURPOSE: The aim of the study was to define CD4+ helper T lymphocytes, cytotoxic CD8+ T lymphocytes, and CD68+ (macrophages) infiltration of portal tracts and lobules and hepatic fibrosis as possible predictive measures of outcome of infants having EHBA. METHODS: The outcome of 32 infants with EHBA was correlated to their percutaneous biopsy and postportoenterostomy core liver tissue infiltration by CD4+, CD68+, and CD8+ cells and to the degree of detected fibrosis. RESULTS: Portoenterostomy cores were heavily infiltrated by CD4+, CD8+, and CD68+, compared with the preoperative liver biopsy (P = .008, .004, and .017, respectively). Infants having favorable outcome had more macrophage infiltration in portoenterostomy core compared with those having an unfavorable outcome (25.66 +/- 29.77 per HPF compared with 11.62 +/- 4.58, P = .000). Mean CD4+/CD8+ ratio was 1.54 +/- 1.37 in those who died within 18 months postoperatively and 0.733 +/- 0.48 in others (P = .021). CONCLUSION: Immune-mediated destruction of portal tracts is an integral part of pathogenesis of EHBA.

Portal capillary C4d deposits and increased infiltration by macrophages indicate humorally mediated mechanisms in acute cellular liver allograft rejection.

Almost no data exist concerning the role of antibody-mediated mechanisms in human acute cellular liver allograft rejection (ACR). Therefore, the aim of this study was to determine whether ACR is associated with depositions of complement split products and increased infiltration by B-lymphocytes, plasma cells and macrophages. A total of 35 liver biopsy specimens (ACR n=22, controls n=13) were analyzed by immunohistochemical single and double staining. The average numbers of CD 20(+), CD 38(+) and CD 68(+) cells per portal tract were established while the presence of C4d and C3d deposits was evaluated semiquantitatively. Significantly greater numbers of CD 20(+) (P=0.029) and CD 38(+) (P=0.014) cells were found in the ACR specimens than in the control specimens. Additionally, 50% of patients diagnosed with ACR showed C4d deposits along portal capillaries, which was associated with a significantly increased portal infiltration by macrophages (P=0.007). Taken together these results support the involvement of humorally mediated mechanisms in some cases of ACR.

Biliary atresia is associated with CD4+ Th1 cell-mediated portal tract inflammation.

A proposed mechanism in the pathogenesis of biliary atresia involves an initial virus-induced, progressive T cell-mediated inflammatory obliteration of bile ducts. The aim of this study was to characterize the inflammatory environment present within the liver of infants with biliary atresia to gain insight into the role of a primary immune-mediated process versus a nonspecific secondary response to biliary obstruction. Frozen liver tissue obtained from patients with biliary atresia, neonatal giant cell hepatitis, total parenteral nutrition (TPN)-related cholestasis, choledochal cysts, and normal control subjects was used for fluorescent immunohistochemistry studies of cellular infiltrates, cytokine mRNA expression, and in situ hybridization for localization of cytokine-producing cells. Immunohistochemistry revealed increases in CD8(+) and CD4(+) T cells and Kupffer cells (CD68(+)) in the portal tracts of biliary atresia. Reverse transcription-PCR analysis of biliary atresia tissue showed a Th1-type cytokine profile with expression of IL-2, interferon-gamma, tumor necrosis factor-alpha, and IL-12. This profile was not seen in normal, neonatal hepatitis or choledochal cyst livers but was present in TPN-related cholestasis. In situ hybridization revealed that the Th1 cytokine-producing cells were located in the portal tracts in biliary atresia and in the parenchyma of TPN-related cholestasis. A distinctive portal tract inflammatory environment is present in biliary atresia, involving CD4(+) Th1 cell-mediated immunity. The absence of similar inflammation in other pediatric cholestatic conditions suggests that the portal tract inflammation in biliary atresia is not a secondary response to cholestasis but rather indicates a specific immune response involved in the pathogenesis of biliary atresia.

The normal and metastases-bearing livers retain various specific subsets of live lymphocytes from portal circulation.

The liver is among the organs that trap lymphocytes flowing through their blood vasculature. These cells, marginated in sinusoids, participate in the liver's anti-viral and anti-tumor processes. The molecular mechanism of this lymphocyte margination and cooperation with resident sinusoidal cells remains obscure and inadequately studied due to the difficulties in obtaining samples of sinusoidal blood from a living animal. To overcome these shortcomings, we have worked out an in situ rat liver perfusion model in exsanguinated animals that enables quantitative observations of blood lymphocyte trapping in sinusoids. The cell populations trapped by the liver and retained in the perfusing blood were characterized with respect to their phenotypes and cytotoxicity. Perfused livers, previously washed out of sinusoidal lymphocytes, halted leukocytes from normal perfusing blood. The numbers of halted post-perfusion CD5+, CD4+, CD8+, CD56+ (ED1) and MHC class II+ (OX6) subsets did not differ statistically from the pre-perfusion population, which suggests active extraction of leukocytes during perfusion. Moreover, cytotoxicity of post- and preperfusion populations against CC531 and K562 remained at a similar level. The perfused livers with CC531 colon adenocarcinoma metastases halted higher numbers of the CD14 and MHC class II+ and fewer of CD11b+ and CD54+ normal blood leukocytes than normal livers. The phenotypes of cells retrieved from sinusoids after perfusion were almost identical to those obtained prior to perfusion. Interestingly, the post-perfusion populations displayed higher cytotoxic capacity than before perfusion. Taken together, the in situ liver perfusion method allows the study of the specificity and kinetics of recruitment of specific populations of host leukocytes in metastatic tumor tissue and evaluation of their cytotoxicity levels.

Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients.

PCR DNA amplification of IgH genes was performed on liver biopsy samples of 42 unselected hepatitis C virus (HCV)-positive patients. Genotypic analysis and signal amplification by branched DNA were used to characterize and quantitate HCV RNA genomic sequences. Intraportal lymphoid follicle-like structures were isolated from surrounding hepatocytes by microdissection technique. IgH VDJ PCR products were cloned and sequenced. IgH VDJ gene rearrangements were detected in the liver of 26 (62%) patients. Unequivocal monoclonal or oligoclonal patterns of B cell expansions were found in 14 (33.3%) and 12 (28.6%) patients, respectively. Patients with intrahepatic B cell monoclonal expansions showed liver HCV RNA levels higher than those with oligoclonal or polyclonal features (1106.4 +/- 593.5 vs 677.3 +/- 424.3 vs 406.2 +/- 354.3 pg HCV RNA/g tissue; p = 0.048 and p = 0.001, respectively). Although a single dominant band was obtained with total DNA, characterization of DNA recovered from intraportal inflammatory aggregates resulted in the detection of multiple IgH VDJ gene rearrangements, pointing to an oligoclonal pattern of lymphoproliferation. Cloning and sequence analyses showed that B cell clonalities were differently distributed in adjacent portal tracts of the same liver area. In addition, HCV RNA genomic sequences could be consistently amplified from each of the portal inflammatory aggregates examined. These data support the concept that in chronic HCV infection the intrahepatic B cell repertoire is frequently clonally restricted and that HCV may have a direct role in sustaining in situ B cell proliferation.

Soluble IL-6 receptor leads to a paracrine modulation of the IL-6-induced hepatic acute phase response in double transgenic mice.

There is a growing number of soluble agonistic (IL-6, ciliary neurotropic factor, IL-11, and glia-derived neurotropic factor receptors) and antagonistic (IL-1 and TNF receptors) receptor proteins, modulating the biological functions of their cognate ligands. The physiologic role of these receptor molecules in vivo is unclear. In particular, it is not known how the specificity of function of soluble receptors after release into the blood stream is maintained. We addressed this question by studying the function of the soluble IL-6R (sIL-6R) at the cellular level in the liver. We have generated double transgenic mice coexpressing human sIL-6R and human IL-6 in the liver and have analyzed the expression patterns by in situ hybridization. The expression of the human sIL-6R, driven by the phosphoenolpyruvate carboxykinase promoter, is located mainly in periportal areas, whereas human IL-6 under the control of the metallothionein promoter is uniformly expressed throughout the liver. We show here by in situ hybridization that acute phase protein gene expression induced by human IL-6 and human sIL-6R correlated with the periportal expression of sIL-6R, indicating that sIL-6R acts mainly in an area where it is generated. We conclude that in a concentration-dependent manner, at low concentrations of sIL-6R, there is a predominantly paracrine action at the site of its generation, whereas at higher concentrations of the sIL-6R there are both local and systemic effects.

Immunohistochemical features of the portal tract mononuclear cell infiltrate in chronic aggressive hepatitis.

The portal tract mononuclear cell infiltrate has been characterised in 28 liver biopsy samples showing features of chronic aggressive hepatitis from 12 patients with autoimmune chronic active hepatitis, 12 with primary sclerosing cholangitis, and four with other chronic liver diseases (two with alpha 1-antitrypsin deficiency, one with Wilson's disease, and one with chronic hepatitis B infection). In all patients liver disease had started in childhood. The mononuclear cell infiltrate was investigated by a two step immunoperoxidase technique using monoclonal antibodies to: total, alpha/beta T cell receptor positive, helper/inducer, suppressor/cytotoxic T lymphocytes; B lymphocytes; killer/natural killer cells; monocyte/macrophages; and to the activation markers HLA-DR antigens, interleukin 2 receptor (IL-2R), transferrin receptor, and 4F2Ag. In all samples the infiltrate consisted of mainly alpha/beta T cell receptor T lymphocytes. Although T helper/inducer cells predominated in patients with autoimmune chronic active hepatitis, T suppressor/cytotoxic lymphocytes were preponderant in patients with primary sclerosing cholangitis and the other chronic liver diseases. Killer/natural killer cells accounted for up to 25% of the mononuclear cell infiltrate in patients with autoimmune chronic active hepatitis, being rare or absent in the other diseases. Monocytes/macrophages were always found, but they were more numerous in primary sclerosing cholangitis than in the other chronic liver diseases. B lymphocytes were rare or absent in all subjects. Activated mononuclear cells were present in all subjects, but although in patients with autoimmune chronic active hepatitis and primary sclerosing cholangitis most cells of the infiltrate expressed HLA-DR antigens and up to 75% IL-2R, in other forms of chronic liver diseases HLA-DR positive cells were less common and IL-2R positive cells ere rare or absent. These results show that the cells responsible for the histological characteristics of chronic aggressive hepatitis vary in their functional phenotype and state of activation according to the type of underlying liver disorder, confirming the involvement of different pathogenetic mechanisms.

Changes of IgG-bearing cell populations in the portal tracts of patients with chronic liver disease of viral etiology: an evaluation by immunoperoxidase method and computerized image analysis.

Little is known about the distribution of IgG-bearing cell subpopulations in normal liver and their possible changes in disease conditions. We developed an immunohistochemical method that proved suitable and accurate for the identification and characterization of IgG-bearing cells and their subpopulations in liver specimens. The method uses specific monoclonal antibodies on serial mirror liver sections. We applied this method to four normal liver tissue specimens and 25 liver biopsy samples of chronic hepatitis of viral etiology. Only rare IgG-bearing cells could be observed in the portal tracts of normal liver specimens. In contrast, a dense infiltrate of such cells was seen in liver specimens from patients with chronic viral hepatitis. The density of IgG-bearing cells in such patients ranged from 6 to 20 cells x 10(-4) micron2 in the different specimens (mean = 11 x 10(-4) micron2). The increase in IgG-bearing cells did not appear to be related to the histological diagnosis, to the degree of histological inflammatory activity or to the type of viral infection. The major population of IgG-bearing cells consisted of IgG1-positive cells (68%); IgG2- (17%), IgG3- (8%) and IgG4 (7%)-bearing cells represented only minor fractions. The increased prevalence of IgG1-bearing cells observed in chronic hepatitis but not in normal liver specimens suggests that these findings may reflect an activation of antibody production directed toward viral antigens or antigenic structures of self. The identification of the antigenic specificities of the antibodies produced by IgG-bearing cells might provide important clues in understanding the pathogenesis of chronic viral hepatitis.

HLA-DR expression on the microvasculature of portal tracts in idiopathic portal hypertension. Immunohistochemical characteristics and relation to portal phlebosclerosis.

We recently reported that HLA-DR antigen was expressed on the microvasculature of portal tracts more frequently in idiopathic portal hypertension (IPH) than in normal livers or in other hepatic diseases, and that this HLA-DR expression may be involved in the development of the portal venopathy characteristic of IPH. The present study was performed to evaluate the relationship between the HLA-DR expression and portal tract lesions, as well as to investigate the immunohistochemical characteristics of the HLA-DR-positive microvasculature using liver wedge biopsy specimens obtained from 32 patients with IPH. According to the degree of phlebosclerosis of the portal veins, the portal tracts were divided into three groups: mild, moderate, and severe. The microvasculature in portal tracts was positive for HLA-DR in 21 (66%) of the 32 specimens and in 133 (44%) of 302 portal tracts. In the 21 specimens, there was no significant difference in the prevalence of HLA-DR-positive microvasculature among the three groups: it occurred in 57 (66%) of 86 portal tracts in the mild group, 53 (61%) of 87 portal tracts in the moderate group, and 23 (49%) of 47 portal tracts in the severe group. The HLA-DR-positive microvasculature was positive for type IV collagen and receptors of Ulex europaeus lectin I, suggesting that HLA-DR-positive microvessels are blood vessels. These findings suggest that HLA-DR antigen is already expressed on portal microvessels in the incipient stage of IPH, and that HLA-DR expression persists during the progression of portal phlebosclerosis. The HLA-DR expression may be an initiating factor leading to immunologic assault on portal microvessels in IPH.

Immunohistochemical studies on the main entrance-route of CA19-9 into the peripheral venous blood of gastric cancer patients. Correlation with CA19-9 levels in peripheral and portal blood.

The correlation between CA19-9 levels of portal and peripheral venous blood, and immunohistochemical variables of cancer lesions was examined in 53 gastric cancer patients and eight patients with benign diseases. Immunohistochemically, CA19-9 was found in 33 (62.5%) of 53 primary lesions. The antigen was found in the cancer cells of invasive lymphatics and node metastases of every CA19-9 localized cancer, although the cancer cells in veins showed little or no CA19-9. There was little or no antigen in the cancer cells in veins, lymphatics, or metastases of 20 CA19-9 nonlocalized primary lesions. Patients with CA19-9 nonlocalized cancer or with benign diseases showed no elevation of the antigen levels in peripheral or portal blood. CA19-9 levels of portal blood (mean, 76.4 U/ml; positive rate, 33.3%) were not different from those of peripheral blood (mean, 91.5 U/ml; positive rate, 33.3%). Additionally, the antigen levels of the blood in patients with lymphatic invasion or node metastases were significantly higher than those in patients without the invasion or the metastases, and every patient without the invasion showed no elevation of the antigen. These results suggest that production of the antigen in cancer cells may be a premise of CA19-9 elevation in peripheral blood and that CA19-9 may be drained by the thoracic duct of the lymphatic system via node metastases or invasive lymphatics, but not by the hematogenous portal system.

Immunohistologic pattern of the portal T-lymphocyte infiltration in hepatic allograft rejection.

Monoclonal antibodies were used to identify helper T cells (TH) and suppressor/cytotoxic T cells (TS/C) in liver allograft biopsy specimens obtained 7,21,90,180, and 365 days postoperatively and then annually or during episodes of graft dysfunction and after treatment of rejection episodes. Biopsy specimens were obtained from 70 hepatic allografts from patients treated with cyclosporine and corticosteroids. Rejection was diagnosed by the presence of appropriate laboratory and light microscopic findings and at least 16 weeks of follow-up to exclude other causes of graft dysfunction. Three immunohistologic patterns were noted: no or only a trace of T lymphocytes, predominantly TH infiltrate with or without a small amount of TS/C cells (portal TH), and a mixture of TH with an equal or greater number of TS/C infiltrate (portal mix). Of 68 biopsy specimens obtained during quiescent periods, only 3 had a portal tract T-lymphocyte infiltrate. Of 30 protocol biopsy specimens, 24 contained such an infiltrate a mean of 12.4 days before biochemical and routine histologic indications of rejection in the allograft. At the time of the rejection episode, 33 biopsy specimens were immunohistologically labeled; portal tract T-lymphocyte infiltrate was predominantly TH in 8 and a mixture of TH and TS/C in 25. All rejection episodes with a predominantly TH pattern responded to methylprednisolone. Of the 25 rejection episodes with a portal mix pattern, only 3 responded to methylprednisolone. Eighty-seven biopsy specimens were obtained more than 10 days after treatment of rejection.(ABSTRACT TRUNCATED AT 250 WORDS)