Jürgen Graf

Activation of hepatic stellate cells is associated with cytokine expression in thioacetamide-induced hepatic fibrosis in mice

The pathophysiological mechanisms of thioacetamide (TAA)-induced hepatic fibrogenesis are not yet fully understood. In particular, the role of hepatic stellate cells (HSCs) remains unclear. We therefore examined proliferation and transdifferentiation of HSC as well as the underlying molecular mechanisms in TAA-induced fibrosis. Hepatic fibrogenesis was induced in mice by addition of TAA to drinking water. Liver damage was determined by assessment of alanine aminotransferase and aspartate aminotransferase levels, and measurement of collagen deposition. Additionally, expression patterns of α-smooth muscle actin, glial fibrillary acidic protein (GFAP, specific hepatic biomarker for HSC), cysteine- and glycine-rich protein 2 (CRP2, specific marker of HSC transdifferentiation), tissue inhibitor of metalloproteinases-1, matrix metalloproteinase-9 (MMP-9), interleukins (IL-1β, IL-6), platelet-derived growth factors (PDGF-B, PDGF-D) , tumor necrosis factor (TNF)-α, and (transforming growth factor (TGF)-β1 were assessed by real-time PCR. Transcription of GFAP and CRP2 were transiently upregulated during TAA-induced fibrogenesis (punctum maxima (p.m.) week 10 for GFAP and week 14 for CRP2). Similar transient expression patterns were demonstrated for IL-1β, IL-6, TGF-β1, and PDGF-B (p.m. week 12) whereas TNF-α and PDGF-D continuously increased with ongoing liver injury. In particular, not only neutrophil granulocytes, but also macrophages and leukocytes served as a major source for MMP-9 expression. GFAP and CRP2 expression patterns demonstrated transiently increased HSC-activation during TAA-induced hepatic fibrogenesis. The rate of increase of transcription of GFAP correlated best with PDGF-B, whereas CRP2 levels correlated with PDGF-B, PDGF-D, and IL-1β expression. This study demonstrates for the first time that transiently increased activation patterns of HSC are observed in toxically induced hepatic fibrosis. Thus, TAA in drinking water is an effective and elegant model to induce reproducible states of liver fibrosis without parenchymal damage in mice.

Health care costs, long-term survival, and quality of life following intensive care unit admission after cardiac arrest

IntroductionThe purpose of this study was to investigate the costs and health status outcomes of intensive care unit (ICU) admission in patients who present after sudden cardiac arrest with in-hospital or out-of-hospital cardiopulmonary resuscitation.MethodsFive-year survival, health-related quality of life (Medical Outcome Survey Short Form-36 questionnaire, SF-36), ICU costs, hospital costs and post-hospital health care costs per survivor, costs per life year gained, and costs per quality-adjusted life year gained of patients admitted to a single ICU were assessed.ResultsOne hundred ten of 354 patients (31%) were alive 5 years after hospital discharge. The mean health status index of 5-year survivors was 0.77 (95% confidence interval 0.70 to 0.85). Women rated their health-related quality of life significantly better than men did (0.87 versus 0.74; P < 0.05). Costs per hospital discharge survivor were 49,952 €. Including the costs of post-hospital discharge health care incurred during their remaining life span, the total costs per life year gained were 10,107 €. Considering 5-year survivors only, the costs per life year gained were calculated as 9,816 € or 14,487 € per quality-adjusted life year gained. Including seven patients with severe neurological sequelae, costs per life year gained in 5-year survivors increased by 18% to 11,566 €.ConclusionPatients who leave the hospital following cardiac arrest without severe neurological disabilities may expect a reasonable quality of life compared with age- and gender-matched controls. Quality-adjusted costs for this patient group appear to be within ranges considered reasonable for other groups of patients.

Bone marrow transplantation demonstrates medullar origin of CD34+ fibrocytes and ameliorates hepatic fibrosis in Abcb4−/− mice

Bone marrow (BM)‐derived stem cells and CD34+ fibrocytes are associated with fibrogenesis in several organs. In an Abcb4−/− mouse model for sclerosing cholangitis alpha‐smooth muscle actin‐positive (α‐SMA+) myofibroblasts are thought to play a pivotal role in hepatic fibrogenesis. The aim of this study was 2‐fold: (1) to demonstrate that the origin of an important fibrogenetic cell population is the BM; and (2) to investigate whether transplantation of BM (BM‐Tx) affects liver function, staging, and grading. Surrogate markers for fibrogenesis and regulation of hepatic stellate cells (HSC) as well as progenitor‐cell‐derived fibrocytes in liver tissue were analyzed by quantitative real‐time polymerase chain reaction (PCR) and immunohistology. After lethal irradiation of recipient mice, BM‐Tx was carried out by way of tail vein injection of BM cells from marker protein donors (green fluorescent protein, GFP+) or Abcb4−/− mice as control (syngeneic Tx). Parameters of liver function were assessed serologically and histologically. Activated HSC of α‐SMA+/CRP2+ phenotype were expressed in ≈50% of proliferating bile ducts, whereas fibrotic liver parenchyma showed no expression thereof. Epithelial mesenchymal transfer (EMT) was visualized in the areas of proliferating bile ducts. The hematopoietic origin of CD34+ fibrocytes was demonstrated immunohistologically in livers of BM chimeric mice. These CD34+ cells infiltrated hepatic lobules from portal fields and developed a desmin+ phenotype expressing collagen type I in fibrotic parenchyma as well as in vitro after isolation by magnetic cell separation. Transplantation of GFP+/Abcb4+ BM improved liver function and staging compared with sham transplantation, but no significant differences were noticed among allogeneic and syngeneic Tx. Conclusion: The present study is the first to identify that both BM‐derived fibrocytes and HSC are involved in biliary fibrogenesis in Abcb4−/− mice. Our data suggest that changes in immunity subsequent to BM‐Tx may alter hepatic fibrosis. (HEPATOLOGY 2009.)

Serum matrix metalloproteinases in adult CF patients: Relation to pulmonary exacerbation

BACKGROUNDnMatrix metalloproteinases (MMPs) virtually degrade all components of the extracellular matrix and are mainly expressed in tissues following inflammation or remodeling. Increased expression of certain MMPs in sputum and bronchoalveolar lavage of patients with cystic fibrosis (CF) is related to impaired lung function. We investigated whether a panel of serum MMPs is associated with both, impairment of pulmonary function and occurrence of pulmonary exacerbations (PEx) in adult patients with CF.nnnMETHODSnSerum concentrations of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-12, MMP-13, and TIMP-1 were determined by ELISA in 54 adult CF patients. PEx was defined based on a score established by Rosenfeld in 2001. MMP-1, MMP-8, MMP-9 and TIMP-1 were assessed in 7 CF patients with PEx before and after systemic antibiotic therapy.nnnRESULTSnOf the 54 CF patients, PEx was diagnosed in 16 different CF patients. Compared to healthy controls, MMP-1, MMP-8, and MMP-9, serum levels were elevated in CF patients and correlated with PEx. MMP-8 expression was associated with impaired lung function. For MMP-2, we observed a decreased expression and an association of MMP-2 decline with PEx. Antibiotic treatment of CF patients with PEx led to a decrease of MMP-1, MMP-8 and active MMP-9 protein concentration.nnnCONCLUSIONSnIncreased serum expression of certain MMPs is associated with occurrence of PEx and impaired lung function in CF. Hence, these MMPs might serve as surrogate markers for PEx.

Cellular sources of MMP-7, MMP-13 and MMP-28 in ulcerative colitis.

Abstract Objective. Matrix metalloproteinases (MMPs) are considered the predominant proteases in the pathogenesis of mucosal ulcerations associated with inflammatory bowel disease (IBD). Whether the malignancy associated MMP-7 and MMP-13 or the recently cloned MMP-28 convey a certain meaning for intestinal homeostasis and pathogenesis of IBD is currently unknown. We therefore set off to analyze regulation patterns and cellular origins of these MMPs in mucosal tissues of patients with ulcerative colitis (UC). Material and methods. Biopsy samples of affected and healthy tissues were obtained from 35 Norwegian patients with UC. RNA was quantified by quantitative real-time polymerase chain reaction to study MMP gene expression in both pathological and healthy mucosal specimens. Cellular origins were determined by immunohistology using surrogate markers for inflammation, neovascularization, and epithelial structures. Protein expression of MMP-7 and MMP-13 was quantified using enzyme-linked immunosorbent assay. Results. MMP-7 and MMP-13 gene expression was significantly increased in UC affected colonic mucosa whereas MMP-28 showed a decreased expression in inflamed mucosa. Endothelial cells and infiltrating leukocytes were identified as the major cellular sources of MMP-7 and MMP-13 in UC. Enterocytes represented the major cellular source of MMP-28 in healthy and inflamed mucosa. Conclusions. MMP-7 and MMP-13 expression in inflammatory and endothelial cells indicate a role of these MMPs for both colitis associated neoangiogenesis and inflammatory changes. Decreased MMP-28 expression in UC is most likely the result of colitis associated epithelial destruction and loss of cryptal architecture.

Latent MMP-9 is bound to TIMP-1 before secretion

Abstract Expression patterns of matrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), are closely correlated with physiological and pathological processes characterized by the degradation and accumulation of the extracellular matrix (ECM). Both, activated MMP-9 and pro-MMP-9 can bind to TIMP-1, and most cell types secrete MMP-9 in complex with TIMP-1. Utilizing immunofluorescence, we observed intracellular co-localization of MMP-9 and TIMP-1 in stimulated human fibrosarcoma cells. In the present study we searched for the origin of the complex formation between the latent enzyme and its specific inhibitor on a subcellular level. Fluorescence resonance energy transfer (FRET) between the fluorescently labeled enzyme and its inhibitor in co-transfected cells were measured. MMP-9 and TIMP-1 were fused to cyan (CFP) and yellow (YFP) variants of the green fluorescent protein and transiently expressed in human hepatoma cells. The intracellular distribution of fluorescently labeled TIMP-1 and MMP-9 was analyzed by confocal laser scanning microscopy. Intracellular complex formation in the Golgi apparatus was verified, demonstrating FRET between MMP-9-CFP and TIMP-1-YFP. Our data provide evidence that the proMMP-9-TIMP-1 complex is already present in the Golgi apparatus. This may be of significance for a number of intracellular and extracellular biochemical processes involving proMMP-9. However, the magnitude and functional relevance of this finding remain unknown.

In-flight medical emergencies.

BACKGROUNDnOne in every 10 000 to 40 000 passengers on commercial aircraft will have a medical incident while on board. Many physicians are unaware of the special features of the cabin atmosphere, the medical equipment available on airplanes, and the resulting opportunities for medical intervention.nnnMETHODSnA selective literature search was performed and supplemented with international recommendations and guidelines and with data from the Lufthansa registry.nnnRESULTSnData on in-flight medical emergencies have been collected in various ways, with varying results; it is generally agreed, however, that the more common incidents include gastrointestinal conditions (diarrhea, nausea, vomiting), circulatory collapse, hypertension, stroke, and headache (including migraine). Data from the Lufthansa registry for the years 2010 and 2011 reveal the rarity of cardiopulmonary resuscitation (mean: 8 cases per year), death (12 cases per year), childbirth (1 case per year), and psychiatric incidents (81 cases per year). If one assumes that one medical incident arises for every 10 000 passengers, and that there are 400 passengers on board each flight, then one can calculate that the probability of experiencing at least one medical incident reaches 95% after 24 intercontinental flights.nnnCONCLUSIONnAn in-flight medical emergency is an exceptional event for the physician and all other persons involved. Physician passengers can act more effectively if they are aware of the framework conditions, the available medical equipment, and the commonly encountered medical conditions.

Innovative immunohistochemistry identifies MMP-9 expressing macrophages at the invasive front of murine HCC

AIMnTo investigate the proteolytic contribution of tumor-associated macrophages (TAM) in tumor invasion, we analyzed whether TAM at the invasive front of small HCC in Abcb4(-/-)-mice show an enhanced expression of MMP-9.nnnMETHODSnLiver cryosections of the hepatocellular carcinoma (HCC) invasive front from 12 mo old Abcb4(-/-)-mice were stained for collagen type I and MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies. Afterwards, the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies. Finally, photographs of the invasive tumor front were digitally overlaid and analyzed.nnnRESULTSnAfter complete bleaching of the primary dye, specific fluorescence staining of a third antigen, here F4/80, was successfully performed on the same histological section. With this method, we were able to identify conglomerates of matrix metalloproteinase (MMP-9) expressing macrophages within the tumor capsule of HCC.nnnCONCLUSIONnMMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC. The described staining protocol provides a simple yet powerful extension of conventional immuno-histochemistry, facilitating visualization of at least three different antigens plus nuclei in one single histological section.

YKL-40 and transient elastography, a powerful team to assess hepatic fibrosis

Abstract Objective. Transient elastography (TE) is a non-invasive and accurate method for the diagnosis of severe hepatic fibrosis and cirrhosis (F = 3 and F = 4). However, the assessment of significant fibrosis (F = 2) by TE is impaired due to a high variation in the diagnostic accuracy. Within this study, we aim to compare the diagnostic value of TE and experimental biomarkers of liver fibrosis. Material and methods. A total of 55 patients with chronic liver disease of different etiologies were included in the study. Among them, patients with HCV infection represented the largest cohort (n = 25). Liver fibrosis was evaluated according to the Desmet/Scheuer score. All patients received TE. Serum concentrations of YKL-40, hyaluronic acid (HA), Laminin, C-terminal procollagen I peptide, MMP-9, TIMP-1, TIMP-2 and MMP-9/TIMP-1 complex were determined by ELISA. Results. In the total patient population, areas under the receiver operator characteristic curve (AUROC) for TE were 0.798 (F ≥ 2), 0.880 (F ≥ 3) and 1 (F = 4). Among the serum markers, highest diagnostic accuracies were calculated for YKL-40 for F ≥ 2 (0.792) and F ≥ 3 (0.914) and for YKL-40 and HA for F = 4 (both 0.936). In the subgroup of HCV patients, the following AUROCs for TE were calculated: 0.802 (F ≥ 2), 0.798 (F ≥ 3) and 0.998 (F = 4). YKL-40 exhibited the highest diagnostic accuracy of all biomarkers in the HCV population (0.880, 0.854 and 0.986, respectively). Conclusions. YKL-40 is a powerful fibrosis marker with high diagnostic accuracy, in particular in HCV-associated liver disease. Its determination may confirm and improve the diagnostic accuracy of TE especially in early stages of liver fibrosis.

Endothelial NOS (NOS3) impairs myocardial function in developing sepsis.

Endothelial nitric oxide synthase (NOS)3-derived nitric oxide (NO) modulates inotropic response and diastolic interval for optimal cardiac performance under non-inflammatory conditions. In sepsis, excessive NO production plays a key role in severe hypotension and myocardial dysfunction. We aimed to determine the role of NOS3 on myocardial performance, NO production, and time course of sepsis development. NOS3−/− and C57BL/6 wildtype mice were rendered septic by cecum ligation and puncture (CLP). Cardiac function was analyzed by serial echocardiography, in vivo pressure and isolated heart measurements. Cardiac output (CO) increased to 160xa0% of baseline at 10xa0h after sepsis induction followed by a decline to 63xa0% of baseline after 18xa0h in wildtype mice. CO was unaltered in septic NOS3−/− mice. Despite the hyperdynamic state, cardiac function and mean arterial pressure were impaired in septic wildtype as early as 6xa0h post CLP. At 12xa0h, cardiac function in septic wildtype was refractory to catecholamines in vivo and respective isolated hearts showed impaired pressure development and limited coronary flow reserve. Hemodynamics remained stable in NOS3−/− mice leading to significant survival benefit. Unselective NOS inhibition in septic NOS3−/− mice diminished this survival benefit. Plasma NOx- and local myocardial NOx- and NO levels (via NO spin trapping) demonstrated enhanced NOx- and bioactive NO levels in septic wildtype as compared to NOS3−/− mice. Significant contribution by inducible NOS (NOS2) during this early phase of sepsis was excluded. Our data suggest that NOS3 relevantly contributes to bioactive NO pool in developing sepsis resulting in impaired cardiac contractility.