Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

UNLABELLED: Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1ß for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. CONCLUSION: Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here.

Preformed antibodies detected by cytotoxic assay or multibead array decrease liver allograft survival: role of human leukocyte antigen compatibility.

The significance of human leukocyte antigen (HLA) compatibility and preformed antibodies in liver transplantation remains unclear. The objectives of this study were to evaluate, in a single-center cohort comprising 896 liver transplants, whether the degree of donor-recipient compatibility and preformed antibodies modified graft survival. Univariate Kaplan-Meier analysis demonstrated that donor-recipient HLA compatibility had a marginal impact on allograft survival. As for compatibility at individual antigen loci, 2 mismatches at HLA-A conferred a survival advantage in retransplanted allografts (P = 0.011). HLA-B and HLA-DR loci did not play a significant role in outcome in any pathology. The concordance of results on preformed antibodies detected by complement-dependent cytotoxicity (CDC) and a multiple bead assay (Luminex xMAP) showed a strong correlation between both techniques (P < 0.0001). Both CDC-detected and Luminex-detected antibodies were associated with shorter graft survival within the first year post-transplant (P = 0.01 and P = 0.016, respectively). Positive CDC T crossmatches and Luminex-detected HLA class II antibodies played a significant role in decreasing graft survival (P = 0.043 and P = 0.0019 at 1 year, respectively, and P = 0.005 and P = 0.038 at 5 years, respectively). A correlation was also observed between the presence of preformed Luminex-detected class II or Luminex I and II antibodies and allograft rejection (P = 0.001 and P = 0.042, respectively). In conclusion, although HLA typing is not a prerequisite for transplantation, screening of HLA antibodies with Luminex techniques and CDC crossmatch may be useful in the detection of at-risk patients that could benefit from increased surveillance and tailored therapy following transplantation.

DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs.

Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8-/- mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2-driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8-/- Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.

Harmful effect of preformed anti-MICA antibodies on renal allograft evolution in early posttransplantation period.

BACKGROUND: Pretransplantation anti-major histocompatibility complex class I chain-related molecule A (MICA) sensitization is an uncommon event and its role on kidney graft evolution is not completely defined. METHODS: A retrospective study of patients transplanted between 2005 and 2011 in our center (n=727) was performed. Recipients were classified in four groups, according either to multiplexed flow cytometry-recorded anti-human leukocyte antigen (HLA) and anti-MICA antibodies or to percent panel-reactive antibody (PRA; by complement-dependent cytotoxicity) and anti-MICA antibodies. RESULTS: In the total cohort, 52 (7.15%) patients had preformed anti-MICA antibodies, and these were not related with anti-HLA, previous transplantations, or recipient female sex (potential pregnancies). Kaplan-Meier curves showed global allograft survival differences (P=0.042) mostly due to pronounced decrease in PRA+MICA+ group early after transplantation. Biopsy-proven allograft rejection rate increased after month 12 in PRA+MICA- group and was higher early after transplantation in PRA+MICA+ group (P=0.033). In paired comparisons, rejection incidence was superior in PRA+MICA- versus PRA-MICA- patients (17% vs. 7%; P=0.007) at 24 months, confirming the widely reported deleterious effect of PRA+ status, but at 3 months rejection was higher in PRA+MICA+ versus PRA-MICA- patients (14% vs. 2%; P=0.009). Among patients categorized according anti-HLA and anti-MICA antibodies, the most striking difference in rejection was observed at 3 months (8% in HLA-MICA+ vs. 2% in HLA-MICA- patients; P=0.032). In the multivariate analysis, HLA-MICA+ status at 3 months independently conferred the highest risk for rejection (odds ratio, 5.07; P=0.049). CONCLUSIONS: Pretransplantation sensitization against MICA and HLA are independent events. Preformed anti-MICA antibodies independently increase risk for kidney rejection and enhance the deleterious effect of PRA+ status early after transplantation.

Histologic findings predictive of a diagnosis of de novo autoimmune hepatitis after liver transplantation in adults.

BACKGROUND: Autoimmune hepatitis (AIH) after liver transplantation has been defined histologically as a "hepatitic" pattern of injury, characterized by lymphoplasmacytic inflammation with necroinflammatory activity (NIA), comparable with findings seen in native livers. This definition, however, is difficult to apply in practice because specific histologic criteria are not clearly delineated. This study aimed to determine which histologic features correlated best with clinical and serologic features of dAIH. METHODS: Index liver biopsies from patients with autoimmune-like hepatitis transplanted for non-AIH in two centers (n=35 and 20) were reviewed. Histologic features were correlated with the clinical diagnosis of AIH based on a retrospective review of clinical and serologic data, including therapeutic response. RESULTS: A clinical diagnosis of AIH was retrospectively assigned to 24 of 35 (68%) and 18 of 20 (90%) patients, respectively (P=0.10). In multivariate analysis, centrilobular NIA and centrilobular plasma cell (PC) ratio of 30% to 50% were independently discriminating for a clinical diagnosis of AIH (P=0.04 and 0.05, respectively). The best level of predictability (99.6%) was mathematically achieved when severe centrilobular NIA and centrilobular PC ratio of 30% to 50% were both present. CONCLUSION: A histologic pattern of centrilobular injury including increased NIA and increased PC infiltration correlates with measurements of autoimmunity in liver recipients. It could be used to segregate cases for further study and introduced into the AIH scoring systems when applied in the context of liver transplantation.

Intravenous immunoglobulin treatment increased live birth rate in a Spanish cohort of women with recurrent reproductive failure and expanded CD56(+) cells.

PROBLEM: Natural killer (NK, CD3(-)CD56(+)/CD16(+)) and NKT-like cells (CD3(+)CD56(+)/CD16(+)) activity is considered among the key factors for reproductive success. In the absence of immunological screening, beneficial effects of intravenous immunoglobulin (IVIG) in preventing recurrent reproductive failure (RRF) have not been reported. Here, we analyse the IVIG influence on pregnancy success in women with RRF and circulating NK or/and NKT-like cells expansion. METHOD OF STUDY: One hundred fifty-seven women with previous recurrent miscarriage and/or recurrent implantation failure after in vitro fertilization were consecutively studied. Sixty-four patients with CD56(+) cell expansion, no apparent underlying disease and who maintained their desire to conceive were selected. Forty of them received IVIG during pregnancy. RESULTS: Overall, the clinical pregnancy rate for the women under IVIG therapy was 92.5% and the live birth rate was 82.5%. Significantly lower pregnancy and live birth rates (25% and 12.5%, respectively) were observed for the patients with recurrent pregnancy loss and NK/NKT-like cells expansion without IVIG. After three cycles of IVIG, NK cell percentages decreased significantly and these values persisted throughout gestation. CONCLUSION: Intravenous immunoglobulin therapy for women with RRF and NK or NKT-like cell expansion was a safe and beneficial therapeutic strategy that associated with high clinical pregnancy and live birth rates.

Peripheral blood lymphocyte populations in end-stage liver diseases.

GOALS/BACKGROUND: The aim of this study was to decipher whether end-stage liver failure modifies peripheral blood lymphocytes (PBL) in a homogeneous manner, independently of the base pathology, or, if on the contrary, PBL subsets show a different profile in each hepatic disease. METHODS: We studied PBL subsets in 71 patients with end-stage liver disease, before liver transplant, and 74 healthy controls by flow cytometry. The results were statistically compared between patients and controls, and cohorts of patients classified according to their base pathology. RESULTS: We observed lower absolute numbers in all lymphocyte populations in patients compared with controls. We found an increment of CD3+ activated cells (P<10) and CD45RO+CD4+ (P<10) in chronic hepatitis C virus versus controls; hepatitis B virus showed high TCRgammadelta+ and CD8+ T cells with respect to controls (P=0.008 and P=0.029, respectively); alcoholic cirrhotic patients showed low CD8+, mainly CD45RA+CD8+ (P=0.007) and high CD45RO+CD4+ (P<10) compared with the normal population; autoimmune diseases showed lower CD3+ and TCRalphabeta+ (P=0.002 and P=0.0001) than controls. CONCLUSIONS: Regardless of the base pathology, patients with end-stage liver disease show a low absolute number of lymphocyte populations compared with controls. However, PBL profiles are different, characteristic, and specific of every disease causing chronic liver failure.