Rajan Somasundaram

Assessment of liver viscoelasticity using multifrequency MR elastography

MR elastography (MRE) allows the noninvasive assessment of the viscoelastic properties of human organs based on the organ response to oscillatory shear stress. Shear waves of a given frequency are mechanically introduced and the propagation is imaged by applying motion‐sensitive gradients. An experiment was set up that introduces multifrequency shear waves combined with broadband motion sensitization to extend the dynamic range of MRE from one given frequency to, in this study, four different frequencies. With this approach, multiple wave images corresponding to the four driving frequencies are simultaneously acquired and can be evaluated with regard to the dispersion of the complex modulus over the respective frequency. A viscoelastic model based on two shear moduli and one viscosity parameter was used to reproduce the experimental wave speed and wave damping dispersion. The technique was applied in eight healthy volunteers and eight patients with biopsy‐proven high‐grade liver fibrosis (grade 3–4). Fibrotic liver had a significantly higher (P < 0.01) viscosity (14.4 ± 6.6 Pa · s) and elastic moduli (2.91 ± 0.84 kPa; 4.83 ± 1.77 kPa) than the viscosity (7.3 ± 2.3 Pa · s) and elastic moduli (1.16 ± 0.28 kPa; 1.97 ± 0.30 kPa) of normal volunteers. Multifrequency MRE is well suited for the noninvasive differentiation of normal and fibrotic liver as it allows the measurement of rheologic material properties. Magn Reson Med 60:373–379, 2008.

Viscoelasticity-based Staging of Hepatic Fibrosis with Multifrequency MR Elastography

PURPOSE To analyze the dynamics of the shear modulus of the liver to assess the optimal driving frequency and to determine the diagnostic accuracy of generalized frequency-independent elasticity cutoff values for staging hepatic fibrosis. MATERIALS AND METHODS This institutional review board-approved prospective study included 16 healthy volunteers and 72 patients with biopsy-proved liver fibrosis. After obtaining written informed consent, imaging was performed at 1.5-T by using a motion-sensitized echo-planar imaging sequence. Wave excitation was performed by an actuator introducing a superposition of four frequencies (25.0, 37.5, 50.0, 62.5 Hz) of shear waves. The elasticity µ value and the structure geometry parameter α were calculated by using the two-parameter springpot model. The performance of magnetic resonance (MR) elastography in staging liver fibrosis was assessed with area under the receiver operating characteristic curve (AUROC) analysis and Spearman correlation analysis. RESULTS Elasticity increased with stage of fibrosis, with mean values as follows: for volunteers, 2.25 kPa ± 0.43 (standard deviation); stage F1, 2.61 kPa ± 0.43; stage F2, 3.00 kPa ± 0.63; stage F3, 3.86 kPa ± 0.61; and stage F4, 5.86 kPa ± 1.22. Frequency-independent cutoff values derived for fibrosis and AUROC values, respectively, were as follows: stage F1 or higher, 2.84 kPa and 0.9128; stage F2 or higher, 3.18 kPa and 0.9244; stage F3 or higher, 3.32 kPa and 0.9744; and equivalent to stage F4, 4.21 kPa and 0.9931. The geometry of the tissue (α value) did not correlate with fibrosis. Frequencies of 50.0 Hz and 62.5 Hz displayed the highest diagnostic accuracy. CONCLUSION The diagnostic performance of multifrequency MR elastography in determining the degree of hepatic fibrosis increases with stage of fibrosis. Metrics obtained at the higher frequencies provide better diagnostic performance compared with the lower frequencies. Results of the AUROC analysis demonstrate the high accuracy of frequency-independent cutoff values for staging higher grades of hepatic fibrosis.

In vivo determination of hepatic stiffness using steady-state free precession magnetic resonance elastography.

Objective:The objective of this study was to introduce an magnetic resonance elastography (MRE) protocol based on fractional motion encoding and planar wave acquisition for rapid measurements of in vivo human liver stiffness. Materials and Methods:Vibrations of a remote actuator membrane were fed by a rigid rod to the patients surface beneath the right costal arch resulting in axial shear deflections of the liver. Data acquisition was performed using a balanced steady-state free precession (bSSFP) sequence incorporating oscillating gradients for motion sensitization. Tissue vibrations of frequency fv = 51 Hz were tuned by twice the sequence repetition time (1/fv = 2TR). Twenty axial images acquired by time-resolved through-plane wave encoding were used for planar elasticity reconstruction. The MRE data acquisition was achieved within 4 breathholds of 17 seconds each. The method was applied to 12 healthy volunteers and 2 patients with diffuse liver disease (fibrosis grade 3). Results:MRE data acquisition was successful in all volunteers and patients. The elastic moduli were measured with values between 1.99 ± 0.16 and 5.77 ± 0.88 kPa. Follow-up studies demonstrated the reproducibility of the method and revealed a difference of 0.74 ± 0.47 kPa (P < 0.05) between the hepatic stiffness of 2 healthy male volunteers. Conclusion:bSSFP combined with fractional MRE enables rapid measurement of liver stiffness in vivo. The used actuation principle supports a 2-dimensional analysis of the strain wave field captured by axial wave images. The measured data indicate individual variations of hepatic stiffness in healthy volunteers.

Chief complaints in medical emergencies: do they relate to underlying disease and outcome? The Charité Emergency Medicine Study (CHARITEM)

Objectives To evaluate the relationship between chief complaints and their underlying diseases and outcome in medical emergency departments (EDs). Methods All 34 333 patients who attended two of the EDs of the Charité Berlin over a 1-year period were included in the analysis. Data were retrieved from the hospital information system. For study purposes, the chief complaint (chest pain, dyspnoea, abdominal pain, headache or ‘none of these symptoms’) was prospectively documented in an electronic file by the ED-physician. Documentation was mandatory. Results The majority of patients (66%) presented with ‘none of these symptoms’, 11.5% with chest pain, 11.1% with abdominal pain and 7.4% with dyspnoea. In total, 39.4% of all patients were admitted to the hospital. The leading diagnosis was acute coronary syndrome (50.7%) for chest pain in-patients and chronic obstructive pulmonary disease (16.5%) and heart failure (16.1%) for in-patients with dyspnoea. The causes of abdominal pain in in-patients were of diverse gastrointestinal origin (47.2%). In-hospital mortality of in-patients was 4.7%. Patients with chest pain had significantly lower in-hospital mortality (0.9%) than patients with dyspnoea (9.4%) and abdominal pain (5.1%). Conclusion The majority of emergency patients lack diagnosis-specific symptoms. Chief complaints help preselect patients but must not be mistaken as disease specific. Mortality largely differs depending on the chief complaint. In chest pain patients, standardized processes may be one factor that explains the low mortality in this group.

Role of IL28B polymorphism in the development of hepatitis C virus-induced hepatocellular carcinoma, graft fibrosis, and posttransplant antiviral therapy.

Background. The development of liver graft disease is partially determined by individual genetic background. Interleukin 28B (IL28B) is strongly suspected to be involved in susceptibility for hepatitis C virus (HCV) infection, inflammation, and antiviral treatment response before and after liver transplantation (LT). Currently, the role of IL28B polymorphism (rs12979860) in the development of hepatocellular carcinoma (HCC) is unclear, and only limited data are available on the course of HCV recurrence. Methods. One hundred sixty-seven HCV-positive patients after LT were genotyped for IL28B (C→T; rs12979860). Sixty-one patients with histologically confirmed HCC in the explanted liver were compared with 106 patients without HCC regarding IL28B genotypes. Among patients with HCC, IL28B genotypes were correlated with tumor histology and pretransplant &agr;-fetoprotein (AFP) levels. Furthermore, the role of IL28B polymorphism was evaluated regarding interferon-based treatment success and fibrosis progression after LT. Results. The prevalence of HCC in explanted livers was significantly higher among patients with TT genotype, suggesting a protective role of the C allele in HCC development (P=0.041). Median AFP level was closely to significance higher in the presence of T allele (P=0.052). Significant differences in IL28B genotype distribution were detected between AFP-negative and AFP-positive HCCs (<15 &mgr;g/L vs. >15 &mgr;g/L; P=0.008). Although no impact could be observed regarding acute cellular rejection (P=0.940), T allele was significantly associated with antiviral therapy failure (P=0.028) and faster development of advanced fibrosis (P=0.017) after LT. Conclusion. IL28B polymorphism seems to be involved in the development of HCV-induced HCC and in the course of HCV recurrence after LT. T allele may be regarded as a genetic risk factor for HCV-related carcinogenesis, posttransplant fibrosis progression, and antiviral therapy failure.

The alpha 2 chain of collagen type VI sequesters latent proforms of matrix-metalloproteinases and modulates their activation and activity.

The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified alpha-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the alpha2 chain of microfilamentous CVI alpha2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that alpha2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the alpha2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and alpha2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the alpha2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its alpha2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.

Hydroxyproline-containing collagen analogs trigger the release and activation of collagen-sequestered proMMP-2 by competition with prodomain-derived peptide P33-42

BackgroundFibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated.ResultsIn situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity.ConclusionsThese findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.

Impact of neoadjuvant transarterial chemoembolization on tumor recurrence and patient survival after liver transplantation for hepatocellular carcinoma: a retrospective analysis

Transarterial chemoembolization (TACE) has gained wide acceptance as a bridge to liver transplantation (LT) in patients with hepatocellular carcinoma (HCC). Aim of this analysis was to compare long‐term results with and without neoadjuvant TACE and to identify subgroups, which particularly benefit from TACE.

An active extract of Lindera obtusiloba inhibits adipogenesis via sustained Wnt signaling and exerts anti-inflammatory effects in the 3T3-L1 preadipocytes

Obesity, the related metabolic syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea polyphenols influencing adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and differentiation of (im)mature adipocytes using 3T3-L1, an established cell line for studying adipogenesis. L. obtusiloba extract reduced the de novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC(50) of ∼135 μg/ml paralleled by induction of caspase 3/7 mediated apoptosis. Hormone-induced 3T3 L1 differentiation in the presence of L. obtusiloba extract resulted in a reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the adipogenesis-associated proteins glucose transporter-4 and vascular endothelial growth factor, in reduced secretion of the proadipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein β-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-γ. Treatment of mature adipocytes with L. obtusiloba extract also significantly reduced intracellular lipid droplets. In addition to this strong interference of L. obtusiloba extract with adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor α-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for obesity associated with the metabolic syndrome.

The Elongated First Fibronectin Type III Domain of Collagen XIV Is an Inducer of Quiescence and Differentiation in Fibroblasts and Preadipocytes

Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln29-Pro154 that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.