Hepatic Infiltrates in Operational Tolerant Patients After Liver Transplantation Show Enrichment of Regulatory T Cells Before Proinflammatory Genes Are Downregulated.

Immunosuppression can be discontinued from selected and stable patients after liver transplantation resulting in spontaneous operational tolerance (SOT), although the underlying mechanisms remain elusive. Thus, we analyzed serial liver biopsy specimens from adult liver recipients enrolled in a prospective multicenter immunosuppression withdrawal trial that used immunophenotyping and transcriptional profiling. Liver specimens were collected before the initiation of weaning, at the time of rejection, or at 1 and 3 years after complete drug discontinuation. Unexpectedly, the tolerated grafts developed portal tract expansion with increased T cell infiltration after immunosuppression withdrawal. This was associated with transient and preferential accumulation of CD4(+) FOXP3(+) cells and a trend toward upregulation of immune activation and regulatory genes, without signs of rejection. At the same time, no markers of endothelial damage or activation were noted. Portal infiltrates persisted at 3 years but were characterized by decreased expression of genes associated with chronic immunological damage. Further, SOT was not associated with a progressive liver fibrosis up to 5 years. These data suggest that SOT involves several mechanisms: a long-lasting local immune cell persistence with a transient regulatory T cells accumulation followed by a downregulation of immune-activated genes over years. These results have important implications for designs and follow-up of weaning trials.

Molecular Characterization of Acute Cellular Rejection Occurring During Intentional Immunosuppression Withdrawal in Liver Transplantation.

Acute cellular rejection occurs frequently during the first few weeks following liver transplantation. During this period, its molecular phenotype is confounded by peri- and postoperative proinflammatory events. To unambiguously define the molecular profile associated with rejection, we collected sequential biological specimens from 55 patients at least 3 years after liver transplantation who developed rejection during trials of intentional immunosuppression withdrawal. We analyzed liver tissue and blood samples obtained before initiation of drug withdrawal and at rejection, alongside blood samples collected during the weaning process. Gene expression profiling was conducted using whole-genome microarrays and real-time polymerase chain reaction. Rejection resulted in distinct blood and liver tissue transcriptional changes in patients who were either positive or negative for hepatitis C virus (HCV). Gene expression changes were mostly independent from pharmacological immunosuppression, and their magnitude correlated with severity of histological damage. Differential expression of a subset of genes overlapped across all conditions. These were used to define a blood predictive model that accurately identified rejection in HCV-negative, but not HCV-positive, patients. Changes were detectable 1-2 mo before rejection was diagnosed. Our results provide insight into the molecular processes underlying acute cellular rejection in liver transplantation and help clarify the potential utility and limitations of transcriptional biomarkers in this setting.

Early non-invasive selection of patients at high risk of severe hepatitis C recurrence after liver transplantation.

BACKGROUND: The early identification of patients at high risk of severe post liver transplant hepatitis C recurrence is relevant, as these patients may be treated using interferon (IFN)-free regimens. METHODS: In a retrospective study with prospectively collected data, we investigated whether the use of several non-invasive methods (fibrosis 4 index [FIB-4], AST-to-platelets ratio index [APRI], enhanced liver fibrosis test [ELF], IFN-γ-inducible protein 10 [IP-10], and transient elastography by Fibroscan) and their combinations 6 months after transplantation could identify those recipients at higher risk of severe recurrence, defined by the presence of significant fibrosis (F ≥2) and/or portal hypertension (hepatic venous pressure gradient ≥6 mmHg) 12 months after transplant. Seventy-two hepatitis C virus (HCV)-infected liver transplant patients and 10 recipients in whom HCV was eradicated before transplantation were included in the study. RESULTS: The levels of all biomarkers were significantly higher in HCV-infected recipients than in controls. Among HCV recipients, levels of biomarkers were significantly higher in patients with severe recurrence. Although there were no statistically significant differences between biomarkers, APRI, ELF, and FIB-4 obtained the highest area under the ROC curve values. The combination of serum biomarkers with Fibroscan increased the negative and positive predictive values, although diagnostic accuracy of individual tests was not significantly improved. CONCLUSIONS: Patients at higher risk of severe HCV recurrence can be identified early, 6 months after transplantation, using readily available non-invasive methods.

Liver stiffness 1 year after transplantation predicts clinical outcomes in patients with recurrent hepatitis C.

The value of transient elastography (TE) to assess clinical outcomes in hepatitis C recurrence after liver transplantation (LT) has not been explored so far. We studied 144 hepatitis C-infected and 48 non-hepatitis C virus (HCV)-infected LT recipients and evaluated the prognostic value of TE 1 year after transplantation to predict clinical decompensations and graft and patient survival. In HCV patients, cumulative probabilities of liver decompensation 5 years after LT were 8% for patients with liver stiffness measurement (LSM) <8.7 kilopascals (kPa) versus 47% for patients with LSM ≥ 8.7 kPa (p<0.001). Five-year graft and patient cumulative survival were 90% and 92% in patients with LSM<8.7 kPa (p<0.001) and 63% and 64% in patients with LSM ≥ 8.7 kPa, respectively (p<0.001). Patients with low LSM 1 year after LT had excellent outcomes independently from receiving antiviral treatment or achieving sustained virological response (SVR). In contrast, graft survival significantly improved in patients with LSM ≥ 8.7 kPa who achieved SVR. No association between outcomes and LSM at 12 months was observed in non-HCV patients. In conclusion, LSM 1 year after LT is a valuable tool to predict hepatitis C-related outcomes in recurrent hepatitis C and can be used in clinical practice to identify the best candidates for antiviral therapy.

A need for biomarkers of operational tolerance in liver and kidney transplantation.

Both kidney and particularly liver recipients can occasionally discontinue all immunosuppressive drugs without undergoing rejection. These patients, who maintain stable graft function off immunosuppressive drugs without clinically significant detrimental immune responses and/or immune deficits, are conventionally termed operationally tolerant and offer a unique paradigm of tolerance in humans. The immune characterization of operationally tolerant transplant recipients has recently received substantial attention. Operationally tolerant patients might exhibit a signature of tolerance that could potentially be useful to select recipients amenable to drug minimization or withdrawal. Furthermore, elucidation of the molecular pathways associated with the operational tolerance phenotype could provide novel targets for therapy. Particular emphasis has been placed on the use of blood samples and high-throughput transcriptional profiling techniques. In liver transplantation, natural killer related transcripts seem to be the most robust markers of operational tolerance, whereas enrichment in B cell-related gene expression markers has been consistently found in blood samples from operationally tolerant kidney recipients, suggesting that different mechanisms operate in the two situations. In this minireview, we summarize the main achievements of recently published reports focused on the identification of transcriptional markers of operational tolerance, we highlight their mechanistic and clinical implications and describe their methodological limitations.

Comparison of transcriptional and blood cell-phenotypic markers between operationally tolerant liver and kidney recipients.

A proportion of transplant recipients can spontaneously accept their grafts in the absence of immunosuppression (operational tolerance). Previous studies identified blood transcriptional and cell-phenotypic markers characteristic of either liver or kidney tolerant recipients. However, the small number of patients analyzed and the use of different transcriptional platforms hampered data interpretation. In this study we directly compared samples from kidney and liver tolerant recipients in order to identify potential similarities in immune-related parameters. Liver and kidney tolerant recipients differed in blood expression and B-cell immunophenotypic patterns and no significant overlaps were detectable between them. Whereas some recipients coincided in specific NK-related transcripts, this observation was not reproducible in all cohorts analyzed. Our results reveal that certain immune features, but not others, are consistently present across all cohorts of operationally tolerant recipients. This provides a set of reproducible biomarkers that should be explored in future large-scale immunomonitoring trials.

Comparative transcriptional and phenotypic peripheral blood analysis of kidney recipients under cyclosporin A or sirolimus monotherapy.

Due to its low level of nephrotoxicity and capacity to harness tolerogenic pathways, sirolimus (SRL) has been proposed as an alternative to calcineurin inhibitors in transplantation. The exact mechanisms underlying its unique immunosuppressive profile in humans, however, are still not well understood. In the current study, we aimed to depict the in vivo effects of SRL in comparison with cyclosporin A (CSA) by employing gene expression profiling and multiparameter flow cytometry on blood cells collected from stable kidney recipients under immunosuppressant monotherapy. SRL recipients displayed an increased frequency of CD4 + CD25highFoxp3 + T cells. However, this was accompanied by an increased number of effector memory T cells and by enrichment in NFkB-related pro-inflammatory expression pathways and monocyte and NK cell lineage-specific transcripts. Furthermore, measurement of a transcriptional signature characteristic of operationally tolerant kidney recipients failed to detect differences between SRL and CSA-treated recipients. In conclusion, we show here that the blood transcriptional profile induced by SRL monotherapy in vivo does not resemble that of operationally tolerant recipients and is dominated by innate immune cells and NFkB-related pro-inflammatory events. These data provide novel insights on the complex effects of SLR on the immune system in clinical transplantation.

ATG-Fresenius treatment and low-dose tacrolimus: results of a randomized controlled trial in liver transplantation.

We report the results of a prospective randomized controlled trial in liver transplantation assessing the efficacy and safety of antithymocyte globulin (ATG-Fresenius) plus tacrolimus monotherapy at gradually decreasing doses. Patients were randomized to either: (a) standard-dose tacrolimus plus steroids;or (b) peritransplant ATG-Fresenius plus reduced-dose tacrolimus monotherapy followed by weaning of tacrolimus starting 3 months after transplantation. The primary end-point was the achievement of very low-dose tacrolimus (every-other-day or once daily dose with <5 ng/mL trough levels) at 12 months after transplantation. Acute rejection occurring during the first 3 months after transplantation was more frequent in the ATG group (52.4% vs. 25%). Moreover, late acute rejection episodes occurred in all recipients in whom weaning was attempted and no recipients reached the primary end-point. This motivated the premature termination of the trial. Tacrolimus trough levels were lower in the ATG-Fresenius group but no benefits in terms of improved renal function, lower metabolic complications or increased prevalence of tolerance-related biomarkers were observed. In conclusion, the use of ATG-Fresenius and tacrolimus at gradually decreasing doses was associated with a high rate of rejection, did not allow for the administration of very low doses of tacrolimus and failed to provide detectable clinical benefits. ClinicalTrials.gov identifier: NCT00436722.

Anti-viral therapy can be delayed or avoided in a significant proportion of HBeAg-negative Caucasian patients in the Grey Zone.

BACKGROUND: Grey Zone (GZ) is an ill-defined situation including patients falling between inactive carrier (IC) state and HBeAg-negative chronic hepatitis B (HBeAg-negative CHB). AIMS: To assess the long-term outcomes of GZ patients compared to IC in the absence of treatment. METHODS: Retrospective analysis of 287 IC and GZ HBeAg-negative patients. Patients were classified into 4 groups at baseline: HBV-DNA <2000 IU/mL and ALT <40 U/L (IC), HBV-DNA <2000 IU/mL and ALT 40-80 U/L (GZ-1), HBV-DNA 2000-20 000 IU/mL and ALT <40 U/L (GZ-2) or ALT 40-80 U/L (GZ-3). Data were also analysed using AASLD ALT criteria. RESULTS: After a median follow-up of 8.2 (5-19) years, HBsAg loss occurred in about 15% ICs or GZ patients. Transition into IC state occurred in 40% of GZ patients. DNA fluctuations >2000 IU/mL correlated inversely with transition into IC and HBsAg loss. HBsAg levels were significantly lower in ICs than in GZ patients (338 IU/mL [20-3269] vs 5763 IU/mL [2172-17 754]; P < 0.05). Among the latter group, there was an increasing gradient of HBsAg levels from GZ-1 to GZ-3 patients (P < 0.05). HBeAg-negative CHB occurred in only 18 (6.3%) GZ patients. No patient developed cirrhosis nor advanced fibrosis. ALT/HBV-DNA fluctuations and HBeAg-negative CHB development were more frequent in genotype B/C patients, whereas HBsAg loss occurred only in genotype A/D patients. CONCLUSIONS: Most Caucasian GZ patients present excellent long-term outcomes in the absence of treatment, with a high rate of HBsAg loss and low rate of progression to HBeAg-negative CHB. HBV-genotyping and HBsAg levels could help to predict outcomes and better classify GZ patients.

Hepatitis B reactivation in patients with chronic hepatitis C undergoing anti-viral therapy with an interferon-free regimen.

BACKGROUND: A few cases of hepatitis B virus (HBV) reactivation during anti-viral therapy against hepatitis C (HCV) have been reported. However, the information regarding the real impact of this phenomenon is scarce. AIM: To evaluate the risk of HBV reactivation during anti-viral therapy against HCV with an interferon-free regimen with direct-acting anti-virals (DAAs). METHODS: Observational and prospective study of 352 patients receiving DAAs therapy between September 2015 and May 2016. HBV-DNA and ALT levels were monitored at baseline, at week 4 of anti-viral therapy, at end of treatment and 12 weeks after treatment discontinuation in patients with HBV surface antigen (HBsAg) positive or HBV core antibody (anti-HBc) positive before starting anti-viral therapy. RESULTS: Ten (2.8%) and 64 (18%) patients were HBsAg and anti-HBc positive at baseline, respectively. Five (50%) of 10 HBsAg positive and one (1.6%) of 64 anti-HBc positive patients presented HBV virological reactivation (>1log increase in HBV-DNA levels). None of these patients presented clinical reactivation (increase in ALT levels). CONCLUSIONS: HBV virological reactivation is frequent in HBsAg+ patients receiving anti-viral therapy against HCV. However, HBV-DNA elevations were modest (<20 000 IU/mL) and without clinical impact (no ALT elevation).

Predicting the development of liver cirrhosis by simple modelling in patients with chronic hepatitis C.

BACKGROUND: Data are scarce on the natural history of chronic hepatitis C (CHC) in patients with mild hepatitis C who did not respond to anti-viral therapy. AIM: To predict the risk of progression to cirrhosis, identifying patients with the more urgent need for therapy with effective anti-virals. METHODS: A cohort of 1289 noncirrhotic CHC patients treated with interferon-based therapy between 1990 and 2004 in two referral hospitals were followed up for a median of 12 years. RESULTS: Overall, SVR was achieved in 46.6% of patients. Data from a randomly split sample (n = 832) was used to estimate a model to predict outcomes. Among nonresponders (n = 444), cirrhosis developed in 123 (28%) patients. In this group, the 3, 5 and 10-year cumulative probabilities of cirrhosis were 4%, 7% and 22%, respectively, compared to <1% in the SVR-group (P < 0.05). Baseline factors independently associated with progression to cirrhosis in nonresponders were: fibrosis ≥F2, age >40 years, AST >100 IU/L, GGT >40 IU/L. Three logistic regression models that combined these simple variables were highly accurate in predicting the individual risk of developing cirrhosis with areas under the receiving operating characteristic curves (AUC) at 5, 7 and 10 years of ~0.80. The reproducibility of the models in the validation cohort (n = 457, nonresponders = 244), was consistently high. CONCLUSIONS: Modelling based on simple laboratory and clinical data can accurately identify the individual risk of progression to cirrhosis in nonresponder patients with chronic hepatitis C, becoming a very helpful tool to prioritise the start of oral anti-viral therapy in clinical practice.

Five-year follow-up of a trial comparing Tacrolimus and cyclosporine microemulsion in liver transplantation.

We evaluate 5-year results of a prospective randomized trial that compared cyclosporine microemulsion (CsA-me) and Tacrolimus (Tac) for primary immunosuppression. One hundred one adult patients undergoing liver transplantation were randomized to receive Tac (n = 50) or CsA-me (n = 51). The most frequent indication for the procedure was cirrhosis due to virus C followed by alcoholism. Survival rates at 1, 3, and 5 years were 86%, 75%, and 72%, respectively; there was no significant difference between CsA-me versus Tac arms. Acute rejection occurred in 30 cases (30%), independent of the type of primary immunosuppression. Serious adverse events were reported significantly more among patients under CsA-me (48 episodes) than under Tac (32 episodes). Nineteen patients were switched to the other calcineurin inhibitor. The switch was much more frequent from CsA-me to Tac (n = 15; 29.4%), mainly because of lack of efficacy (n = 10; 19.6%). There were no cases of chronic rejections in the Tac arm. Four patients were switched from Tac to CsA-me for side effects; only 1 remains alive, after treatment was changed from CsA-me to an antimetabolite. There were no statistical differences in renal dysfunction, diabetes, hypertension, neurologic disorders, new-onset malignancies, or infections. There were no differences in survival or rejection among the intention-to-treat groups. Serious adverse events, total patients with switch of calcineurin inhibitor, as well as switches due to lack of efficacy, were statistically more frequent under CsA-me. Tacrolimus seems to be a more appropriate drug to be used for primary immunosuppression in liver transplantation.

Professional recognition of female and male doctors.

PRECEDENTS AND AIM: The awards for the excellence of the Official College of Physicians of Barcelona (COMB) were instituted in 2004 to recognize the excellence of the professional exercise. The winners are yearly chosen by juries appointed by the board of government, whose members propose for the award doctors who, in their opinion, have an exemplary professional and human behaviour. SUBJECTS AND METHODS: The number of male and female doctors who have obtained this recognition has been analysed in relation with the sex distribution in the juries. Likewise it has been compared the ratios men-to-women of those who have been rewarded and this ratio among physicians of more than 45 years. RESULTS: Between the awarded physicians the ratio men-to-women was of 2.7/1 (range, from 1.2/1 for awardees in primary care to 6/1 in research). The men-to-women ratio among those who were awarded was in parallel to the man-to-women ratios of the juries. The ratio between men and women among members of the COMB of more than 45 years was 1.4/1, whereas in those who were awarded it was of 2.7/1. The increase in the proportion of women in the juries in the last four years has been followed by an increase in the number of female physicians awarded. CONCLUSION: This data demonstrates that the predominance of male doctors among those who were awarded does not depend so much on the age factor, but basically on the proportion of male and female doctors in the juries.

Reconocimiento profesional a médicos y médicas / Professional recognition of female and male doctors

Antecedentes y objetivo: Los premios a la excelencia del Colegio Oficial de Médicos de Barcelona (COMB) fueron instituidos en 2004 para reconocer la excelencia en el ejercicio profesional. Los premiados son elegidos anualmente por jurados nombrados por la junta de gobierno, cuyos miembros proponen médicos que en su opinión tienen un comportamiento profesional y humano ejemplar. Sujetos y métodos: Hemos analizado el número de médicos y médicas que han obtenido este reconocimiento y la relación entre el número de varones y mujeres premiados con la distribución por géneros en los jurados. Asimismo se han comparado las edades de médicos y médicas premiados y la de colegiados y colegiadas de más de 45 años. Resultados: La relación de varones y mujeres entre los premiados fue de 2,7/1 (rango: 1,2/1 para los premiados en Atención Primaria y 6/1 en el apartado Investigación), con una correlación directa no estadísticamente significativa entre la relación varones/mujeres premiados y jurados. La relación entre varones y mujeres colegiados con más de 45 años fue de 1,4/1, mientras que en los premiados fue de 2,7/1. En los últimos 4 años ha incrementado la proporción de mujeres en los jurados y de forma paralela ha aumentado el número de médicas premiadas. Conclusión: El predomino de varones entre los premiados parece relacionarse con la mayor proporción de varones en la composición de los jurados (AU)

Precedents and aim: The awards for the excellence of the Official College of Physicians of Barcelona (COMB) were instituted in 2004 to recognize the excellence of the professional exercise. The winners are yearly chosen by juries appointed by the board of government, whose members propose for the award doctors who, in their opinion, have an exemplary professional and human behaviour. Subjects and methods: The number of male and female doctors who have obtained this recognition has been analysed in relation with the sex distribution in the juries. Likewise it has been compared the ratios men-to-women of those who have been rewarded and this ratio among physicians of more than 45 years. Results: Between the awarded physicians the ratio men-to-women was of 2.7/1 (range, from 1.2/1 for awardees in primary care to 6/1 in research). The men-to-women ratio among those who were awarded was in parallel to the man-to-women ratios of the juries. The ratio between men and women among members of the COMB of more than 45 years was 1.4/1, whereas in those who were awarded it was of 2.7/1. The increase in the proportion of women in the juries in the last four years has been followed by an increase in the number of female physicians awarded. Conclusion: This data demonstrates that the predominance of male doctors among those who were awarded does not depend so much on the age factor, but basically on the proportion of male and female doctors in the juries (AU)