Development and Validation of a Scoring System to Predict Response to Obeticholic Acid in Primary Biliary Cholangitis.

BACKGROUND & AIMS: Obeticholic acid (OCA) is the only licensed second-line therapy for primary biliary cholangitis (PBC). With novel therapeutics in advanced development, clinical tools are needed to tailor the treatment algorithm. We aimed to derive and externally validate the OCA response score (ORS) for predicting the response probability of individuals with PBC to OCA. METHODS: We used data from the Italian RECAPITULATE (N = 441) and the IBER-PBC (N = 244) OCA real-world prospective cohorts to derive/validate a score including widely available variables obtained either pre-treatment (ORS) or also after 6 months of treatment (ORS+). Multivariable Cox regressions with backward selection were applied to obtain parsimonious predictive models. The predicted outcomes were biochemical response according to POISE (alkaline phosphatase [ALP]/upper limit of normal [ULN]<1.67 with a reduction of at least 15%, and normal bilirubin), or ALP/ULN<1.67, or Normal range criteria (NR: normal ALP, alanine aminotransferase [ALT], and bilirubin) up to 24 months. RESULTS: Depending on the response criteria, ORS included age, pruritus, cirrhosis, ALP/ULN, ALT/ULN, GGT/ULN, and bilirubin. ORS+ also included ALP/ULN and bilirubin after 6 months of OCA therapy. Internally validated c-statistics for ORS were 0.75, 0.78, and 0.72 for POISE, ALP/ULN<1.67, and NR response, which raised to 0.83, 0.88, and 0.81 with ORS+, respectively. The respective performances in validation were 0.70, 0.72, and 0.71 for ORS and 0.80, 0.84, and 0.78 for ORS+. Results were consistent across groups with mild/severe disease. CONCLUSIONS: We developed and externally validated a scoring system capable to predict OCA response according to different criteria. This tool will enhance a stratified second-line therapy model to streamline standard care and trial delivery in PBC.

Plasmacytoid dendritic cell-derived IFN-α promotes murine liver ischemia/reperfusion injury by induction of hepatocyte IRF-1.

UNLABELLED: Plasmacytoid dendritic cells (pDC) constitute the body's principal source of type I interferon (IFN) and are comparatively abundant in the liver. Among various cytokines implicated in liver ischemia and reperfusion (I/R) injury, type I IFNs have been described recently as playing an essential role in its pathogenesis. Moreover, type I IFNs have been shown to up-regulate hepatocyte expression of IFN regulatory factor 1 (IRF-1), a key transcription factor that regulates apoptosis and induces liver damage after I/R. Our aim was to ascertain the capacity of IFN-α released by liver pDC to induce liver damage through hepatic IRF-1 up-regulation after I/R injury. Our findings show that liver pDC mature and produce IFN-α in response to liver I/R. Liver pDC isolated after I/R induced elevated levels of IRF-1 production by hepatocytes compared with liver pDC isolated from sham-operated mice. Notably, hepatic IRF-1 expression was reduced significantly by neutralizing IFN-α. In vivo, IFN-α neutralization protected the liver from I/R injury by reducing hepatocyte apoptosis. This was associated with impaired expression of IRF-1 and proapoptotic molecules such as Fas ligand, its receptor (Fas) and death receptor 5, which are regulated by IRF-1. Furthermore, pDC-depleted mice failed to up-regulate hepatic IFN-α and displayed less liver injury associated with reduced levels of hepatic interleukin (IL)-6, tumor necrosis factor-α, and hepatocyte apoptosis after I/R compared with controls. CONCLUSION: these data support the hypothesis that IFN-α derived from liver pDC plays a key role in the pathogenesis of liver I/R injury by enhancing apoptosis as a consequence of induction of hepatocyte IRF-1 expression.

Roles of dendritic cells in murine hepatic warm and liver transplantation-induced cold ischemia/reperfusion injury.

UNLABELLED: Dendritic cells (DCs) induce and regulate both innate and adaptive immune responses; however, their in vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by using DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) knockout (KO) mice in hepatic warm (70% partial clamping for 60 minutes) and cold IR injury (liver transplant [LTx] with 24-hour cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c+ F4/80- DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in wildtype (WT) mice with lower alanine aminotransferase (ALT) levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic messenger RNA (mRNA) and protein levels for inflammatory cytokines (tumor necrosis factor alpha [TNFα], interleukin [IL]-6) and chemokines (CCL2, CXCL2) were also significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3L KO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. CONCLUSION: Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury.

Antiviral activity and safety profile of silibinin in HCV patients with advanced fibrosis after liver transplantation: a randomized clinical trial.

Response to interferon-based therapies in HCV recurrence after liver transplantation (LT) is unsatisfactory, and major safety issues aroused in preliminary experience with boceprevir and telaprevir. As transplant community identified HCV viral clearance as a critical matter, efficacious and safe anti-HCV therapies are awaited. The aim of this study was to assess efficacy and safety of intravenous silibinin monotherapy in patients with established HCV recurrence after LT, nonresponders to pegylated interferon and ribavirin. This is a single center, prospective, randomized, parallel-group, double-blind, placebo-controlled, phase 2 trial including 20 patients randomly assigned (3:1) to receive daily 20 mg/kg of intravenous silibinin or saline as placebo, for 14 consecutive days. On day 14 of treatment, viral load decreased by 2.30 ± 1.32 in silibinin group versus no change in the placebo group (P = 0.0002). Sixteen days after the end of the treatment, viral load mean values were similar to baseline. Treatment resulted well tolerated apart from a transient and reversible increase in bilirubin. Neither changes in immunosuppressant through levels nor dosage adjustments were necessary. Silibinin monotherapy has a significant antiviral activity in patients with established HCV recurrence on the graft not responding to standard therapy and confirms safety and tolerability without interaction with immunosuppressive drugs (ClinicalTrials.gov number: NCT01518933).

Comparison of Transient Elastography and Point Shear Wave Elastography for Analysis of Liver Stiffness: A Prospective Study.

Liver stiffness measurement (LSM) by Fibroscan is the most used non-invasive method to assess liver fibrosis. Recently, point-shear wave elastography (pSWE) has been introduced as a simple alternative non-invasive test. Therefore, we aimed to compare the results of these two techniques. One hundred and eighty-four consecutive patients attending our outpatient ultrasound clinic were recruited. LSM was performed by both Fibroscan and pSWE. Statistical analysis was conducted by Spearman's test for correlation and linear regression. Bland-Altman graphs and ROC curves were drawn with area under the curve (AUC). Overall, the correlation of LS between Fibroscan and pSWE was substantial (r = 0.68, p < 0.001). Linear regression showed a coefficient b= 0.94 ± 0.02. The Bland-Altman plot found a bias of -0.10, with only 11 values exceeding the 95% confidence interval. When only considering patients with a LSM of > 10 kPa (n = 31), we found an excellent r = 0.79 (0.60-0.90, p < 0.001). A cutoff of 12.15 kPa for pSWE had sensitivity = 74.2% and specificity = 99.3% to detect relevant fibrosis, with an AUC = 0.98. The highest correlation was observed for hepatitis C (r = 0.91) and alcoholic liver disease (ALD)(r = 0.99). In conclusion, pSWE shows LSM estimation in agreement with Fibroscan in most cases, and the best concordance was observed for hepatitis C and ALD, and for higher ranges of LS.

DAP12 promotes IRAK-M expression and IL-10 production by liver myeloid dendritic cells and restrains their T cell allostimulatory ability.

Freshly isolated hepatic dendritic cells (DC) are comparatively immature, relatively resistant to maturation, and can downmodulate effector T cell responses. Molecular mechanisms that underlie these properties are ill defined. DNAX-activating protein of 12 kDa (DAP12) is an ITAM-bearing transmembrane adaptor protein that integrates signals through several receptors, including triggering receptor expressed on myeloid cells-1, -2, and CD200R. Notably, DC propagated from DAP12-deficient mice exhibit enhanced maturation in response to TLR ligation. Given the constitutive exposure of liver DC to endotoxin draining from the gut, we hypothesized that DAP12 might regulate liver DC maturation. We show that DAP12 is expressed by freshly isolated liver, spleen, kidney, and lung myeloid DC. Moreover, inhibition of DAP12 expression by liver DC using small interfering RNA promotes their phenotypic and functional maturation, resulting in enhanced TNF-α, IL-6, and IL-12p70 production, reduced secretion of IL-10, and enhanced CD4(+) and CD8(+) T cell proliferation. Furthermore, DAP12 silencing correlates with decreased STAT3 phosphorylation in mature liver DC and with diminished expression of the IL-1R-associated kinase-M, a negative regulator of TLR signaling. These findings highlight a regulatory role for DAP12 in hepatic DC maturation, and suggest a mechanism whereby this function may be induced/maintained.

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.

High Extra Virgin Olive Oil Consumption Is Linked to a Lower Prevalence of NAFLD with a Prominent Effect in Obese Subjects: Results from the MICOL Study.

Extra virgin olive oil (EVOO) has healthy benefits for noncommunicable diseases (NCDs). However, limited evidence is available about the effects of liver disease and non-alcoholic fatty liver disease (NAFLD). We evaluate whether dose-increased consumption of EVOO is associated with a lower prevalence of NAFLD and if these effects vary based on body weight. The study included 2436 subjects with a 33% NAFLD prevalence. Daily EVOO was categorized into tertiles: low (0-24 g/day), moderate (25-37 g/day), and high consumption (>37 g/day). Subjects were also classified by body mass index (BMI) as normo-weight (18.5-24.9), overweight (25-29.9), and obese (≥30). Logistic regression analysis was applied to calculate odds ratios (ORs) for NAFLD, considering a 20-gram increment in EVOO intake and accounting for EVOO categories combined with BMI classes. The ORs were 0.83 (0.74;0.93) C.I. p = 0.0018 for continuous EVOO, 0.89 (0.69;1.15) C.I. p = 0.37, and 0.73 (0.55;0.97) C.I. p = 0.03 for moderate and high consumption, respectively, when compared to low consumption. Overall, the percent relative risk reductions (RRR) for NAFLD from low to high EVOO consumption were 18% (16.4%;19.2%) C.I. and 26% (25%;27.4%) C.I. in overweight and obese subjects. High EVOO consumption is associated with a reduced risk of NAFLD. This effect is amplified in overweight subjects and even more in obese subjects.

Age-Related Dietary Habits and Blood Biochemical Parameters in Patients with and without Steatosis-MICOL Cohort.

BACKGROUND: Steatosis is now the most common liver disease in the world, present in approximately 25% of the global population. The aim of this study was to study the association between food intake and liver disease and evaluate the differences in blood parameters in age classes and steatosic condition. METHODS: The present study included 1483 participants assessed in the fourth recall of the MICOL study. Patients were subdivided by age (65 years) and administered a validated food frequency questionnaire (FFQ) with 28 food groups. RESULTS: The prevalence of steatosis was 55.92% in the adult group and 55.88% in the elderly group. Overall, the results indicated many statistically significant blood parameters and dietary habits. Analysis of food choices with a machine learning algorithm revealed that in the adult group, olive oil, grains, processed meat, and sweets were associated with steatosis, while the elderly group preferred red meat, dairy, seafood, and fruiting vegetables. Furthermore, the latter ate less as compared with the adult group. CONCLUSIONS: Many differences were found between the two age groups, both in blood parameters and food intake. The random forest also revealed different foods predicted steatosis in the two groups. Future analysis will be useful to understand the molecular basis of these differences and how different food intake causes steatosis in people of different ages.

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation.

UNLABELLED: Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7-H1), a recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7-H1 plays crucial roles during innate immune responses induced by hepatic I/R injury, and using B7-H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)-to-WT LT enhanced constitutive B7-H1 expression on dendritic cells and sinusoidal endothelial cells and promptly induced B7-H1 on hepatocytes. When B7-H1 KO liver grafts were transplanted into WT recipients, serum alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT-to-WT LT. Augmented tissue injury in B7-H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3(+) T cells (particularly CD8(+) T cells). B7-H1 KO grafts had significantly fewer annexin V(+) CD8(+) T cells, and this indicated a failure to delete infiltrating CD8(+) T cells. To evaluate the relative contributions of parenchymal cell and bone marrow-derived cell (BMDC) B7-H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7-H1 on parenchymal cells or BMDCs. A selective B7-H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7-H1 expression was involved in liver damage control. Human livers up-regulated B7-H1 expression after LT. CONCLUSION: The study demonstrates that graft tissue expression of B7-H1 plays a critical role in regulating inflammatory responses during LT-induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses.

HCV-positive kidney transplant patients treated with direct-acting antivirals maintain stable medium-term graft function despite persistent reduction in tacrolimus trough levels.

Background/aim: Direct-acting antivirals (DAAs) have improved the treatment of HCV-positive kidney transplant recipients (KTRs). However, their medium-term follow-up effects on graft function are conflicting. This study aimed to analyze how the interplay between DAAs, calcineurin inhibitors (CNI), and HCV eradication impacts 12-month kidney graft function. Methods: This double-center retrospective study with a prospective follow-up enrolled 35 KTRs with HCV treated with DAAs for 12 weeks. We compared three parameters: estimated glomerular filtration rate (eGFR), 24-h proteinuria, and CNI trough levels at three time points: baseline, end of treatment (EOT), and 12 months later. Results: Kidney allograft function remained stable when comparing baseline and 12-month post-treatment values of eGFR (60.7 versus 57.8 ml/min; p = 0.28) and 24-h proteinuria (0.3 versus 0.2 g/24 h; p = 0.15), while tacrolimus (Tac) trough levels underwent a statistically significant decline (6.9 versus 5.4 ng/ml; p = 0.004). Using an ongoing triple Tac-based maintenance therapy as a conservative measure, a dose escalation of Tac was applied only in seven patients. No variation in CyA and mTOR levels was detected. Conclusion: DAA therapy is safe and effective in HCV-positive KTRs. It also produces a persistent significant reduction in Tac trough levels that does not influence graft function at 12 months.

Tolerogenic plasmacytoid DC.

Plasmacytoid DC (pDC) are type-I IFN-producing cells known for their capacity to promote anti-viral innate and adaptive immune responses. Despite their potent anti-viral function, when compared with conventional DC, pDC exhibit poor immunostimulatory ability and their interaction with T cells often favors the generation of Treg. pDC are activated primarily in response to ssRNA and ssDNA through TLR7 and TLR9, respectively, but also through TLR-independent mechanisms. Non-lymphoid tissue pDC, such as those residing in the airways, gut, and liver, play a significant role in regulating mucosal immunity and are critical for the development of tolerance to inhaled or ingested antigens. Herein we discuss properties that define tolerogenic pDC and how their unique characteristics translate into an ability to regulate immunity and promote the development of tolerance. We cover the importance of pDC during intrathymic Treg development and the maintenance of peripheral tolerance, as well as their regulatory role in transplantation, autoimmunity, and cancer. We highlight recent findings regarding danger-associated molecular pattern and PAMP signaling in the regulation of pDC function, and how the ability of pDC to promote tolerance translates into the potential clinical applications of these cells as therapeutic targets to regulate immune reactivity.

NOD2 ligation subverts IFN-alpha production by liver plasmacytoid dendritic cells and inhibits their T cell allostimulatory activity via B7-H1 up-regulation.

The nucleotide-binding oligomerization domain (NOD)2/CARD15 protein, which senses muramyl dipeptide (MDP), a product of bacterial peptidoglycan, appears to play an important role in regulating intestinal immunity. Although the liver is exposed to gut-derived MDP, the influence of NOD2 ligation on hepatic APC, in particular dendritic cells (DC), is unknown. Freshly isolated mouse liver and spleen plasmacytoid (p)DC expressed higher levels of NOD2 message than conventional myeloid (m)DC. Following MDP stimulation in vivo, liver pDC, but not mDC, up-regulated expression of IFN regulatory factor 4 (IRF-4), a negative regulator of TLR signaling, and induced less allogeneic T cell proliferation and IFN-gamma production. The adoptive transfer of liver pDC from MDP-treated mice failed to prime allogeneic T cells in vivo. By contrast, splenic DC IRF-4 levels and T cell stimulatory activity remained unchanged. Liver pDC from MDP-stimulated mice also displayed greater IkappaBalpha, cell surface B7-H1, and B7-H1 relative to CD86 than control liver pDC. No similar effects were observed for liver mDC or spleen DC. Absence of B7-H1 on liver pDC reversed the inhibitory effect of MDP. After ex vivo stimulation with LPS or CpG, liver pDC but not mDC from MDP-treated animals secreted less IL-12p70, IL-6, and TNF-alpha and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN-alpha than their splenic counterparts, and systemic levels of IFN-alpha were reduced in MDP-treated animals after CpG administration. These findings suggest that differential effects of NOD2 ligation on liver pDC may play a role in regulating hepatic innate and adaptive immunity.

Real-world experience with obeticholic acid in patients with primary biliary cholangitis.

BACKGROUND & AIMS: Obeticholic acid (OCA) is the second-line treatment approved for patients with primary biliary cholangitis (PBC) and an inadequate response or intolerance to ursodeoxycholic acid. We aimed to evaluate the effectiveness and safety of OCA under real-world conditions. METHODS: Patients were recruited into the Italian PBC Registry, a multicentre, observational cohort study that monitors patients with PBC at national level. The primary endpoint was the biochemical response according to Poise criteria; the secondary endpoint was the biochemical response according to normal range criteria, defined as normal levels of bilirubin, alkaline phosphatase (ALP), and alanine aminotransferase (ALT) at 12 months. Safety and tolerability were also assessed. RESULTS: We analysed 191 patients until at least 12 months of follow-up. Median age was 57 years, 94% female, 61 (32%) had cirrhosis, 28 (15%) had histologically proven overlap with autoimmune hepatitis (PBC-AIH). At 12 months, significant median reductions of ALP (-32.3%), ALT (-31.4%), and bilirubin (-11.2%) were observed. Response rates were 42.9% according to Poise criteria, and 11% by normal range criteria. Patients with cirrhosis had lower response than patients without cirrhosis (29.5% vs. 49.2%, p = 0.01), owing to a higher rate of OCA discontinuation (30% vs. 12%, p = 0.004), although with similar ALP reduction (29.4% vs. 34%, p = 0.53). Overlap PBC-AIH had a similar response to pure PBC (46.4% vs. 42.3%, p = 0.68), with higher ALT reduction at 6 months (-38% vs. -29%, p = 0.04). Thirty-three patients (17%) prematurely discontinued OCA because of adverse events, of whom 11 experienced serious adverse events. Treatment-induced pruritus was the leading cause of OCA discontinuation (67%). CONCLUSIONS: Effectiveness and safety of OCA under real-world conditions mirror those in the Poise trial. Patients with cirrhosis had lower tolerability. Overlap PBC-AIH showed higher ALT reduction at 6 months compared with patients with pure PBC. LAY SUMMARY: Obeticholic acid (OCA) was shown to be effective in more than one-third of patients not responding to ursodeoxycholic acid in a real-world context in Italy. Patients with cirrhosis had more side effects with OCA, and this led to suspension of the drug in one-third of patients. OCA was also effective in patients who had overlap between autoimmune hepatitis and primary biliary cholangitis.

Monitoring the operationally tolerant liver allograft recipient.

PURPOSE OF REVIEW: A fundamental goal in transplantation is the establishment of allograft function without ongoing immunosuppression. Robust allograft tolerance has been established in experimental transplantation models, whereas clinical operational tolerance has been described most frequently following human liver transplantation. RECENT FINDINGS: Clinical assessment of tolerance has been limited to laboratory evaluation of organ function. Additional tools include graft monitoring through biopsy and blood sampling for biomarker analysis. Current biomarkers under assessment in recent years include dendritic cell subsets, regulatory T cells, antidonor antibodies, and gene polymorphisms. Emerging microarray analysis that is being prospectively validated will also be reviewed. A further tool in the characterization of the tolerant patient will be the accurate enrollment of such patients into a multicenter registry that will prospectively follow the natural history of the patient withdrawn from immunosuppression and help facilitate the entry of interested patients to mechanistic and immune monitoring trials. The International Solid Organ Transplant Tolerance Registry (www.transplant-tolerance.org) will be briefly described. SUMMARY: Effective biomarker characterization of the operationally tolerant liver allograft recipient would allow earlier, well tolerated, prospective drug withdrawal with the goal of extending the potential benefits of drug minimization to an increasing number of patients in a more predictable fashion.

Immunomodulating effects of the anti-viral agent Silibinin in liver transplant patients with HCV recurrence.

BACKGROUND: Silibinin has been shown to have anti-HCV activity and immune-modulating properties by regulating dendritic cell (DC) function. DCs are antigen-presenting cells that, together with regulatory T cells (Treg), play a pivotal role in controlling alloimmune, as well as anti-HCV immune responses. METHODS: Twelve liver transplant patients with HCV recurrence received iv infusion of Silibinin (iv-SIL) for 14 consecutive days. Using flow cytometry, before and at the end of treatment, we determined the frequencies of circulating myeloid (m) and plasmacytoid (p) DC and Treg and the expression of costimulatory/coregulatory molecules by the DC subsets and Treg. Statistical analysis was performed using the paired Student's t test and Pearson correlation test. RESULTS: After iv-SIL treatment, we observed an elevated plasmacytoid dendritic cell (pDC)/myeloid dendritic cell (mDC) ratio, while pDC displayed lower HLA-DR and higher immunoglobulin-like transcript 4 (ILT4), CD39, and HLA-G expression compared to the pretreatment baseline. In addition, after iv-SIL, mDC showed increased inducible costimulator ligand (ICOSL) expression. No changes were detected in Treg frequency or programed death (PD)-1 expression by these cells. Moreover, several correlations between DC/Treg markers and clinical parameters were detected. CONCLUSIONS: This descriptive study, in liver transplant patients with HCV recurrence, reveals the impact of iv-SIL on DC and Treg. The changes observed in circulating pDC and mDC that have previously been associated with tolerogenic conditions shed new light on how iv-SIL may regulate anti-viral and alloimmunity. We have also observed multiple clinical correlations that could improve the clinical management of liver transplant patients and that deserve further analysis.

Identification and characterization of intestinal Peyer's patch interferon-alpha producing (plasmacytoid) dendritic cells.

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-alpha (IFN-alpha) producing cells in blood. In this study, C57BL/10 (B10;H2b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 microg/d; i.p.; 10 days) that expands DC selectively in vivo. Putative pDC (CD11c+B220+) were identified in the subepithelial dome and in interfollicular regions of intestinal Peyer's patches (PP) from both normal and Flt3L-treated animals. Freshly-isolated, immunobead-purified CD11c+ DC from PP were flow-sorted to obtain lineage- (CD11b-CD19-) CD11c+ B220+ DC (purity>96%). Flow cytometric analysis revealed that these sorted PPpDC were negative for surface markers associated with myeloid DC (CD11b) and expressed only low levels of the "lymphoid-related" DC marker CD8alphaalpha+. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of naïve allogeneic (C3H; H2k) T-cell proliferation. Cytospin preparations of sorted CD11c+B220+ cells revealed plasmacytoid morphology similar to that of human pDC. Immunocytochemistry and enzyme immunoassay revealed that, within 24-hour culture with Herpes simplex virus (10 p.f.u./cell), a subpopulation of stimulated (but not unstimulated) CD11c+B220+ DC produced and secreted IFN-alpha. This novel DC subset may play important roles in innate and adaptive immune responses of the gut and in the regulation of mucosal immune reactions.

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin-modifying HDACi exhibit anti-inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM-CSF-stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b(+)Ly6C(+)F4/80(int)CD115(+)), which suppressed allogeneic T cell proliferation in a NOS- and HO-1-dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin(-)Sca-1(+)c-Kit(+)). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM-CSF administration and increased the number of CD11b(+)Gr1(+) cells in BM and spleen. Increased numbers of Gr1(+) cells, which suppressed T cell proliferation, were recovered from spleens of TSA-treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti-inflammatory action of HDACi.