Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Non-invasive diagnosis of alcohol-related steatohepatitis in patients ongoing alcohol withdrawal based on cytokeratin 18 and transient elastography.

BACKGROUND AND AIMS: The diagnosis of alcoholic steatohepatitis (ASH) is based on liver biopsy, which is costly and invasive with non-negligible morbidity. The aim of this study was to evaluate the accuracy of circulating cytokeratin 18 M65 fragment (K18-M65) alone or in association with other markers for the non-invasive diagnosis of ASH in patients ongoing alcohol withdrawal. METHODS: This study examined the serum level of K18-M65 in a test cohort of 196 patients. All patients underwent liver biopsy, transient elastography (TE) and serum collection. The diagnostic accuracy of K18-M65 alone or combined with clinico-biological data was assessed and the best defined cut-offs were validated in an independent validation cohort of 58 patients. RESULTS: K18-M65 had an area under the curve (AUC) of 0.82 (test cohort) and 0.90 (validation cohort). Using two cut-off decision points, K18-M65 was able to classify 46.9% (test cohort) and 34.5% (validation cohort) of patients with 95% sensitivity or specificity. Combining K18-M65, alpha-2-macroglobulin, TE, body mass index, and age, we created a score allowing accurate diagnosis of ASH with an AUC of 0.93 (test cohort) and 0.94 (validation cohort). This new score was able to rule out or rule in the diagnosis of steatohepatitis for probability ≤0.135 or ≥0.667 respectively in more than two-thirds of patients. CONCLUSIONS: We propose a new validated non-invasive score for the diagnosis of ASH in patients ongoing alcohol withdrawal. This score can help to identify patients that may benefit from potential therapeutics or motivate them to reduce alcohol consumption.

Novel Insights into Alcoholic Liver Disease: Iron Overload, Iron Sensing and Hemolysis.

The liver is the major target organ of continued alcohol consumption at risk and resulting alcoholic liver disease (ALD) is the most common liver disease worldwide. The underlying molecular mechanisms are still poorly understood despite decades of scientific effort limiting our abilities to identify those individuals who are at risk to develop the disease, to develop appropriate screening strategies and, in addition, to develop targeted therapeutic approaches. ALD is predestined for the newly evolving translational medicine, as conventional clinical and health care structures seem to be constrained to fully appreciate this disease. This concept paper aims at summarizing the 15 years translational experience at the Center of Alcohol Research in Heidelberg, namely based on the long-term prospective and detailed characterization of heavy drinkers with mortality data. In addition, novel experimental findings will be presented. A special focus will be the long-known hepatic iron accumulation, the somewhat overlooked role of the hematopoietic system and novel insights into iron sensing and the role of hepcidin. Our preliminary work indicates that enhanced red blood cell (RBC) turnover is critical for survival in ALD patients. RBC turnover is not primarily due to vitamin deficiency but rather to ethanol toxicity directly targeted to erythrocytes but also to the bone marrow stem cell compartment. These novel insights also help to explain long-known aspects of ALD such as mean corpuscular volume of erythrocytes (MCV) and elevated aspartate transaminase (GOT/AST) levels. This work also aims at identifying future projects, naming unresolved observations, and presenting novel hypothetical concepts still requiring future validation.

Bone morphogenetic protein 6-mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro.

Liver sinusoidal endothelial cell-derived bone morphogenetic protein 6 (BMP6) and the BMP6-small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte-endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Non-invasive Biomarkers of Liver Inflammation and Cell Death in Response to Alcohol Detoxification.

INTRODUCTION: Alcohol-related liver disease (ALD) represents the most common liver disease worldwide, however, the underlying molecular mechanisms are still poorly understood. Namely centrilobular inflammation and programmed cell death are characteristic to ALD and it remains to be elucidated why they persist despite the absence of alcohol. AIMS: To study the effects of alcohol withdrawal in a cohort of heavy drinkers and the role of cirrhosis by using non-invasive biomarkers such as cytokines, apoptotic and angiogenic markers. METHODS: Caspase 3-cleaved M30, M65, cytokines (IL-6, IL-8), tumor necrosis factor alpha (TNF-α), transforming growth factor (TGF-ß) and vascular endothelial growth factor (VEGF) were measured in 114 heavy drinkers. The role of alcohol detoxification was investigated in 45 patients. The liver histology was available in 23 patients. Fibrosis stage and steatosis were assessed by measuring liver stiffness (LS) and controlled attenuation parameter (CAP) in all patients using transient elastography (FibroScan, Echosens, Paris). Mean observation interval between the measurements was 5.7 ± 1.4 days (mean + -SD). RESULTS: Patients consumed a mean of 204 ± 148 g/day alcohol with a heavy drinking duration of 15.3 ± 11.0 years. Mean LS was 20.7 ± 24.4 kPa and mean CAP was 303 ± 51 dB/m. Fibrosis distribution was F0-38.1%, F1-2-31%, F3-7.1 and F4-23.9%. Apoptotic markers M30 and M65 were almost five times above normal. In contrast, TNF- α a, IL-8 and VEGF were only slightly elevated. Patients with manifest liver cirrhosis (F4) had significantly higher levels of M30, M65, IL-6 and IL-8. Histology features such as hepatocyte ballooning, Mallory-Denk bodies, inflammation and fibrosis were all significantly associated with elevated LS, and serum levels of TNF-alpha, M30 and M65 but not with CAP and other cytokines. During alcohol detoxification, LS, transaminases, TGF- ß, IL-6, IL-8 and VEGF decreased significantly. In contrast, no significant changes were observed for M30, M65 and TNF- α and M30 even increased during detoxification in non-cirrhotic patients. Profibrogenic cytokine TGF-beta and pro-angiogenic cytokine VEGF showed a delayed decrease in patients with manifest cirrhosis. CONCLUSION: Patients with alcohol-related cirrhosis have a pronounced apoptotic activity and a distinct inflammatory response that only partly improves after 1 week of alcohol detoxification. Alcohol withdrawal may represent an important approach to better dissect the underlying mechanisms in the setting of alcohol metabolism.

Susceptibility based multiparametric quantification of liver disease: Non-invasive evaluation of steatosis and iron overload.

PURPOSE: To evaluate if single-voxel MR spectroscopy (MRS) of iron and fat correlates with biopsy results of hepatic steatosis and iron overload, and to compare MR-measurements with room-temperature susceptometer (RTS), ultrasound, controlled attenuation parameter (CAP) and serum ferritin. MATERIAL AND METHODS: In this prospective study, a set of 42 patients out of 47 screened patients with several chronic liver diseases underwent MRI-examination at 1.5 T including R2-measurements by single-voxel high-speed T2-corrected multiecho spectroscopy, additional liver biopsy, abdominal ultrasound, CAP, and RTS. Routine blood and serum parameters were determined, including ferritin. Atomic absorption spectroscopy (AAS) and histologically confirmed extent of hepatic steatosis from liver biopsy were used as reference standard. For correlation of R2, RTS, CAP, ferritin, and ultrasound with results of AAS and histologically determined fat fraction of liver biopsy specimen, Spearman's and Pearson's correlation as well as receiver operating characteristics curve (ROC) analysis with cut-off values determined by maximizing Youden index was used. RESULTS: MRS iron assessment correlated best with AAS, with a Pearson correlation coefficient of 0.715 (p < 0.001), followed by RTS 0.520 (p < 0.001), and serum ferritin 0.213 (p = 0.088, not significant). MRS fat quantification correlated best with the histological confirmed extent of steatosis hepatis with a Spearman correlation coefficient of 0.836 (p < 0.001), followed by CAP 0.604 (p < 0.001) and sonographically diagnosed steatosis 0.358 (p = 0.013). CONCLUSION: MRS by T2-corrected multiecho single-voxel spectroscopy correlated best with histological results of hepatic fat and iron content compared to RTS, CAP, abdominal ultrasound, and ferritin. Non-invasive methods to assess hepatic fat and iron are of clinical interest for follow-up examinations of patients with chronic liver diseases, where repeated biopsy is not indicated.

Controlled attenuation parameter and alcoholic hepatic steatosis: Diagnostic accuracy and role of alcohol detoxification.

BACKGROUND & AIMS: Controlled attenuation parameter (CAP) is a novel non-invasive measure of hepatic steatosis, but it has not been evaluated in alcoholic liver disease. Therefore, we aimed to validate CAP for the assessment of biopsy-verified alcoholic steatosis and to study the effect of alcohol detoxification on CAP. METHODS: This was a cross-sectional biopsy-controlled diagnostic study in four European liver centres. Consecutive alcohol-overusing patients underwent concomitant CAP, regular ultrasound, and liver biopsy. In addition, we measured CAP before and after admission for detoxification in a separate single-centre cohort. RESULTS: A total of 562 patients were included in the study: 269 patients in the diagnostic cohort with steatosis scores S0, S1, S2, and S3 = 77 (28%), 94 (35%), 64 (24%), and 34 (13%), respectively. CAP diagnosed any steatosis and moderate steatosis with fair accuracy (area under the receiver operating characteristic curve [AUC] ≥S1 = 0.77; 0.71-0.83 and AUC ≥S2 = 0.78; 0.72-0.83), and severe steatosis with good accuracy (AUC S3 = 0.82; 0.75-0.88). CAP was superior to bright liver echo pattern by regular ultrasound. CAP above 290 dB/m ruled in any steatosis with 88% specificity and 92% positive predictive value, while CAP below 220 dB/m ruled out steatosis with 90% sensitivity, but 62% negative predictive value. In the 293 patients who were admitted 6.3 days (interquartile range 4-6) for detoxification, CAP decreased by 32 ±â€¯47 dB/m (p <0.001). Body mass index predicted higher CAP in both cohorts, irrespective of drinking pattern. Obese patients with body mass index ≥30 kg/m2 had a significantly higher CAP, which did not decrease significantly during detoxification. CONCLUSIONS: CAP has a good diagnostic accuracy for diagnosing severe alcoholic liver steatosis and can be used to rule in any steatosis. In non-obese but not in obese, patients, CAP rapidly declines after alcohol withdrawal. LAY SUMMARY: CAP is a new ultrasound-based technique for measuring fat content in the liver, but has never been tested for fatty liver caused by alcohol. Herein, we examined 562 patients in a multicentre setting. We show that CAP highly correlates with liver fat, and patients with a CAP value above 290 dB/m were highly likely to have more than 5% fat in their livers, determined by liver biopsy. CAP was also better than regular ultrasound for determining the severity of alcoholic fatty-liver disease. Finally, we show that three in four (non-obese) patients rapidly decrease in CAP after short-term alcohol withdrawal. In contrast, obese alcohol-overusing patients were more likely to have higher CAP values than lean patients, irrespective of drinking.

Sensitive and non-invasive assessment of hepatocellular iron using a novel room-temperature susceptometer.

BACKGROUND & AIMS: Liver iron accumulates in various chronic liver diseases where it is an independent factor for survival and carcinogenesis. We tested a novel room-temperature susceptometer (RTS) to non-invasively assess liver iron concentration (LIC). METHODS: Two hundred and sixty-four patients with or without signs of iron overload or liver disease were prospectively enrolled. Thirty-five patients underwent liver biopsy with semiquantitative iron determination (Prussian Blue staining), atomic absorption spectroscopy (AAS, n=33), or magnetic resonance imaging (MRI, n=15). RESULTS: In vitro studies demonstrated a highly linear (r2=0.998) association between RTS-signal and iron concentration, with a detection limit of 0.3mM. Using an optimized algorithm, accounting for the skin-to-liver capsule distance, valid measurements could be obtained in 84% of cases. LIC-RTS showed a significant correlation with LIC-AAS (r=0.74, p<0.001), LIC-MRI (r=0.64, p<0.001) and hepatocellular iron (r=0.58, p<0.01), but not with macrophage iron (r=0.32, p=0.30). Normal LIC-RTS was 1.4mg/g dry weight. Besides hereditary and transfusional iron overload, LIC-RTS was also significantly elevated in patients with alcoholic liver disease. The areas under the receiver operating characteristic curve (AUROC) for grade 1, 2 and 3 hepatocellular iron overload were 0.72, 0.89 and 0.97, respectively, with cut-off values of 2.0, 4.0 and 5.0mg/g dry weight. Notably, the positive and negative predictive values, sensitivity, specificity and accuracy of severe hepatic iron overload (HIO) (grade ≥2) detection, were equal to AAS and superior to all serum iron markers. Depletion of hepatic iron could be efficiently monitored upon phlebotomy. CONCLUSIONS: RTS allows for the rapid and non-invasive measurement of LIC. In comparison to MRI, it could be a cost-effective bedside method for LIC screening. Lay summary: Novel room-temperature susceptometer (RTS) allows for the rapid, sensitive, and non-invasive measurement of liver iron concentration. In comparison to MRI, it could be a cost-effective bedside method for liver iron concentration screening.

Occupational consequences after isolated reconstruction of the insufficient posterior cruciate ligament.

BACKGROUND: With incorrect or even without treatment, acute injuries of the posterior cruciate ligament (PCL) can lead to chronic instability of the knee joint. After delayed treatment, negative occupational changes and reduced quality of life can occur. These aspects have not yet been investigated. The purpose of this study was to evaluate occupational consequences after isolated reconstruction in cases of chronic PCL insufficiency. FINDINGS: 12 patients treated with PCL reconstruction in a single bundle technique, using hamstring tendon grafts, were evaluated. All patients were operated upon at least 3 months after injury. Mean time of follow-up was 51 ± 18.2 months (14-75). Radiological assessment (Telos stress device) showed a side comparison of total translation of 4.5 ± 2.6 mm. Occupational consequences have been evaluated by the classification system "REFA". Median time incapacity for work was 8 weeks. Nearly all patients achieved the mental status of the normal population (SF-36), but physical status was still restricted. A pre- to postoperative improvement of the clinical scores could be seen: Lysholm-Score: 46.4 ± 17.3 to 84.7 ± 14.1, HSS-Score: 74.3 ± 10.5 to 88.3 ± 10.7. Postoperative evaluated scores were: Tegner score: 4.8 ± 1.2, IKDC score: 80.0 ± 16.2, VPS: 3.4 ± 2.7. Patients with low physical load in their workplace described significantly better clinical results in every clinical score (p < .05) and less pain than patients with high physical load prior to the accident (VPS: REFA < 2: 2.4 ± 2.6, REFA ≥ 2: 5.5 ± 1.7; p < 0.05). CONCLUSIONS: Operative treated patients with a chronic PCL insufficiency achieve an improvement of the clinical result. Patients with low physical load at their workplace achieve less restrictions.

Return to work and clinical outcome after open wedge HTO.

PURPOSE: The purpose of this study was to examine the incapacity of work related to work load according to the classification that has been introduced by the REFA Association and the clinical outcome after open wedge HTO with autologous bone graft from the iliac crest. METHODS: A total of 32 patients who were employed and able to work at the time of the surgery as well as treated with an open wedge HTO with the LC-DCP and autologous bone wedges from the iliac crest could be included in the radiological and clinical examination (77 months, SD ± 19). Postoperative duration of the incapacity of work and subjective ratings were based on the information provided by the patients themselves. The German classification that has been established by the REFA Association was used to classify the work load. Several clinical scores were used for clinical assessment. RESULTS: The duration of incapacity of work (median, 87 days; range, 14-450) demonstrated a relation to work load according to REFA. The Lysholm score, the HSS score and the score according to Lequesne augmented by 19.2 ± 16.8 (p < 0.0001), 15.6 ± 13.2 (p < 0.0001) and -6.0 ± 5.1 (p < 0.0001), respectively. The Tegner score gained from median 3 (range, 1-5) to 4 (range, 1-8). CONCLUSION: In this study, a relation was found between work load divided into different categories according to the classification established by the REFA Association and the duration of incapacity of work after open wedge HTO. An improvement of all clinical scores was observed. Typical neurological complications after autologous bone transplantation from the iliac crest were observed in 19% of our patient population. LEVEL OF EVIDENCE: IV.

Digital planning of high tibial osteotomy. Interrater reliability by using two different software.

PURPOSE: The purpose of the study was to determine the interrater reliability as well as the correlation of mediCAD(®) and PreOPlan(®) in deformity analysis and digital planning of osteotomies. METHODS: Digital radiographs were obtained from 81 patients planned to undergo an open wedge high tibial osteotomy. The JPEG files of the radiographs were imported to landmark-based software. Deformity analysis and planning of correction were performed by 1 experienced and 2 unexperienced observers. Osteotomy planning was aimed at correction to the predefined mechanical tibiofemoral angle of 3° valgus leg alignment. The interrater reliability of measurements was assessed using intraclass correlation coefficients (ICCs) and the confidence interval. RESULTS: The ICC of PreOPlan(®) was from 0.841 (mechanical lateral distal femur angle) to 0.993 (wedge-angle) and from 0.896 (joint line convergence angle) to 0.995 (mechanical tibiofemoral angle) of mediCAD(®). The ICC of height of wedge-base was 0.979 with PreOPlan(®) and 0.969 with mediCAD(®). Comparing PreOPlan(®) and mediCAD(®), the ICC of the height of wedge-base of the observers was 0.966, 0.956 and 0.969, respectively. CONCLUSIONS: The results show a high interrater reliability of digital planning software. Experience of the observer had no influence on results. Furthermore, a high interrater reliability and correlation of digital planning specific parameters was found. Surgeons need to master limb geometry measurements and osteotomy planning on digital radiographs as digital planning reports are used for intercolleagual correspondence, teaching purposes and as medicolegal documents. The digital planning software tested agrees with the actual demands and could be recommended for deformity analysis and planning of osteotomies. LEVEL OF EVIDENCE: Diagnostic studies, Level I.

The retrourethral transobturator sling suspension in the treatment of male urinary stress incontinence: results of a single institution experience.

Objective. To evaluate functional outcome of the retrourethral transobturator sling suspension (RTS) in the treatment of stress urinary incontinence (SUI) caused by prior prostate surgery. Methods. The RTS (AdVance male sling) was implanted in 32 patients who suffered from mild to severe postsurgical-treatment incontinence at the University Hospital Ulm from September 2010 to September 2011 including 10 patients with prior radiation therapy. Functional data (uroflowmetry, daily pad use, and postvoid residual urine) as well as quality of life with impact of urinary problems (ICIQ-UI SF) were prospectively assessed at baseline and during followup. Results. After a median followup of 9 months (range, 3-14) the incontinence cure rate (no pad usage) was 56.2% and the improvement rate (1-2 pads/day or ≥50% reduction) was 21.9%. No improvement was observed in 21.9%. Daily pad use and ICIQ-UI SF score improved significantly. No major perioperative complications occurred. Postoperatively, 15.6% of the patients exhibited transient acute urinary retention which resolved without further treatment after a maximum of 3 weeks. One patient underwent sling explantation due to dislocation and persistent perineal pain. Conclusions. The implantation of the RTS is a safe and effective procedure in selected patients with SUI resulting from prostate surgery.

The ileal neobladder in female patients with bladder cancer: long-term clinical, functional, and oncological outcome.

PURPOSE: To assess long-term clinical and oncological outcome in women undergoing radical cystectomy and ileal neobladder reconstruction for invasive bladder cancer. METHODS: From 1995 to 2010, a total of 121 women with clinically organ-confined urothelial carcinoma underwent radical cystectomy with an orthotopic ileal neobladder. Median follow-up was 56 months. Clinical course, functional, pathological, and oncological outcome of these women were analyzed. RESULTS: Seventy-six patients (62.8%) experienced a complication of some type within 90 days of the procedure. 56 patients (46.3%) experienced minor complications, whereas 20 patients (16.5%) experienced major complications. Pathological subgroups included 70 patients (57.9%) with organ confined, lymph node-negative tumors, 24 (19.8%) with extravesical, lymph node-negative disease and 27 (22.3%) patients with lymph node-positive disease. The 5-year overall survival rate in patients with organ-confined (≤pT2, pN0), locally advanced (≥pT3, pN0), and metastatic disease was 80.2%, 81.9%, and 45.1%, respectively. 4 women (3.3%) experienced a local (pelvic) recurrence. One patient presented with a urethral recurrence (0.8%). Daytime and nighttime urinary continence (0-1 pad) was reported by 82.4 and 76.5%, respectively. Clean intermittent self-catheterization was required by 58.0%. The retrospective study design was the major limitation of the study. CONCLUSIONS: Despite a considerable complication rate, radical cystectomy with orthotopic diversion in female patients with bladder cancer may be considered a standard therapeutic option for selected patients with excellent oncological outcome including a low incidence of local and urethral recurrence.

Detection of disseminated tumor cells in mediastinoscopic lymph node biopsies and lymphadenectomy specimens of patients with NSCLC by quantitative RT-PCR.

OBJECTIVE: Detection of disseminated tumor cells in mediastinoscopic biopsies could improve staging and might be helpful concerning indications for neoadjuvant therapy regimens. This prospective study was performed to evaluate a simple and observer-independent polymerase chain reaction (PCR)-based method for the detection of disseminated tumor cells in regional lymph nodes. METHODS: Lymph nodes of 32 consecutive patients without neoadjuvant therapy were removed by systematic lymphadenectomy during resection of primary NSCLC. One hundred of these lymph nodes were cut into two equal halves which were examined using either routine histopathology or quantitative reverse transcriptase PCR (qRT-PCR). qRT-PCR amplification of cytokeratin 19 (CK19) transcripts was applied for the detection of tumor cell-specific RNA. We differentiated between illegitimate marker gene transcription and cancer-specific expression by using a cut-off value that was obtained from the analysis of 18 lymph nodes of patients with benign lung diseases. Subsequent to the evaluation of qRT-PCR, a pilot project with five additional patients was conducted to examine 19 mediastinoscopic biopsies, which were cut into two equal halves and proceeded as described above. RESULTS: Ninety-four (94%) lymph nodes were tumor-free by histopathology. qRT-PCR detected disseminated tumor cells in 26 (28%) of these lymph nodes. All of the remaining six lymph nodes that were judged by the pathologist to contain tumor cells exhibited CK19 transcripts. Twenty-three patients had a pN0 status. qRT-PCR detected disseminated tumor cells in 13 (56%) of these pN0 patients. The mediastinoscopic biopsies showed disseminated tumor cells in four (21%) out of 19 histopathologically tumor-free samples. CONCLUSIONS: CK19 qRT-PCR is a sensitive and specific tools for the detection of disseminated tumor cells in regional lymph nodes of patients with operable NSCLC. Further studies are required to asses if this molecular method might improve mediastinoscopic staging.