Genetic Heterogeneity of MET-Aberrant NSCLC and Its Impact on the Outcome of Immunotherapy.

INTRODUCTION: Robust data on the outcome of MET-aberrant NSCLC with nontargeted therapies are limited, especially in consideration of the heterogeneity of MET-amplified tumors (METamp). METHODS: A total of 337 tumor specimens of patients with MET-altered Union for International Cancer Control stage IIIB/IV NSCLC were analyzed using next-generation sequencing, fluorescence in situ hybridization, and immunohistochemistry. The evaluation focused on the type of MET aberration, co-occurring mutations, programmed death-ligand 1 expression, and overall survival (OS). RESULTS: METamp tumors (n = 278) had a high frequency of co-occurring mutations (>80% for all amplification levels), whereas 57.6% of the 59 patients with MET gene and exon 14 (METex14) tumors had no additional mutations. In the METamp tumors, with increasing gene copy number (GCN), the frequency of inactivating TP53 mutations increased (GCN < 4: 58.2%; GCN ≥ 10: 76.5%), whereas the frequency of KRAS mutations decreased (GCN < 4: 43.2%; GCN ≥ 10: 11.8%). A total of 10.1% of all the METamp tumors with a GCN ≥ 10 had a significant worse OS (4.0 mo; 95% CI: 1.9-6.0) compared with the tumors with GCN < 10 (12.0 mo; 95% confidence interval [CI]: 9.4-14.6). In the METamp NSCLC, OS with immune checkpoint inhibitor (ICI) therapy was significantly better compared with chemotherapy with 19.0 months (95% CI: 15.8-22.2) versus 8.0 months (95% CI: 5.8-10.2, p < 0.0001). No significant difference in median OS was found between ICI therapy and chemotherapy in the patients with METex14 (p = 0.147). CONCLUSIONS: METex14, METamp GCN ≥ 10, and METamp GCN < 10 represent the subgroups of MET-dysregulated NSCLC with distinct molecular and clinical features. The patients with METex14 do not seem to benefit from immunotherapy in contrast to the patients with METamp, which is of particular relevance for the prognostically poor METamp GCN ≥ 10 subgroup.

Multidisciplinary oral rehabilitation of an adolescent suffering from juvenile Gorlin-Goltz syndrome - a case report.

BACKGROUND: The Gorlin-Goltz syndrome is an autosomal dominant disorder characterized by keratocystic odontogenic tumors in the jaws, multiple basal cell carcinomas and skeletal abnormities. Frequently, the manifestation of the syndrome occurs in the adolescent years. CASE PRESENTATION: An 11-year-old boy was referred to our clinic due to the persistence of the lower deciduous molars. The further diagnosis revealed bilateral keratocystic odontogenic tumors in the region of teeth 33 and 45 representing a symptom of a Gorlin-Goltz syndrome. This case of the oral rehabilitation of an adolescent with bilateral keratocystic odontogenic tumors shows the approach of a multidisciplinary treatment concept including the following elements: Enucleation and bone defect augmentation using a prefabricated bone graft; distraction osteogenesis to extend the graft-block vertically after cessation of growth; accompanying orthodontic treatment, guided implant placement and prosthetic rehabilitation. Six months after implant insertion, a new keratocystic odontogenic tumor in the basal part of the left sinus maxillaris had to be removed combined with the closure of the oroantral fistula. During the follow-up period of 18 months in semi-annual intervals, the patient showed no sign of pathology. CONCLUSION: In the presented case could be shown that distraction osteogenesis of prefabricated bone blocks is possible. With a multidisciplinary approach in a long-term treatment a sufficient oral rehabilitation of the patient suffering from extended keratocystic odontogenic tumors was possible.

Childhood supratentorial ependymomas with YAP1-MAMLD1 fusion: an entity with characteristic clinical, radiological, cytogenetic and histopathological features.

Ependymoma with YAP1-MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1-MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1-MAMLD1 fusion was documented by RT-PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo-rosettes, small to medium-sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot-like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1-MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1-11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow-up, 4.84 years). In this to date, largest series of ependymomas with YAP1-MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings.

Atrial fibrosis and atrial fibrillation: the role of the TGF-ß1 signaling pathway.

BACKGROUND: Atrial fibrosis concurs with chronic atrial fibrillation (AF), a phenomenon that contributes to the resistance to restore and maintain sinus rhythm (SR). Fibrogenesis represents a complex process in which the transforming growth factor-ß1 (TGF-ß1) pathway may play a major role, e.g. in the setting of myocardial infarction. The present study addresses the potential contribution of the TGF-ß1 signaling pathway to atrial fibrosis in patients with AF. METHODS AND RESULTS: Right atrial appendages of 163 patients were excised during heart surgery and grouped according to rhythm (SR vs. AF) and AF duration. Five groups were defined: SR, paroxysmal/chronic persistent AF (<6 months), chronic permanent AF (CAF) of 7-24 months, 25-60 months, and >60 months duration. Collagen content of atria, determined morphometrically, revealed a steady and significant increase in patients with SR (14.6±8.9%) up to patients with CAF of >60 months (28.1±7.1%). Likewise, expression of TGF-ß1 mRNA and protein, TGF-ß-receptor-II protein, profibrotic phospho-Smad-2 and -4 proteins increased. However, the TGF-ß(1) effect appeared to decline with increasing AF duration, characterized by a decrease in TGF-ß-receptor-I protein, increases of TGF-ß inhibiting Smad-7 protein and a reduction of ph-Smad-2. CONCLUSIONS: Human atrial fibrogenesis in patients with atrial fibrillation is accompanied by a biphasic response, an early increase and later loss of responsiveness to TGF-ß(1). It appears that fibrosis progresses despite compensatory changes in the TGF-ß-signaling pathway. The sequential changes in the contribution of different profibrotic processes during the establishment of AF may offer the opportunity to selectively interfere with the atrial remodeling process at different stages.

Atrial fibrillation is associated with cardiac hypoxia.

BACKGROUND: Atrial fibrillation (AF), the most common human arrhythmia, is responsible for substantial morbidity and mortality and may be promoted by selective atrial ischemia and atrial fibrosis. Consequently, we investigated markers for hypoxia and angiogenesis in AF. METHODS: Right atrial appendages (n=158) were grouped according to heart rhythm [sinus rhythm (SR) or AF]. The degree of fibrosis and microvessel density of all patients were determined morphometrically using Sirius-Red- and CD34/CD105-stained sections, respectively. Next, sections (n=77) underwent immunostaining to detect hypoxia- and angiogenesis-related proteins [hypoxia-inducible factor (HIF)1 alpha, HIF2 alpha, vascular endothelial growth factor (VEGF), VEGF receptor 2 (KDR), phosphorylated KDR (pKDR), carboanhydrase IX, platelet-derived growth factor] and the apoptosis-related B-cell lymphoma 2 protein. RESULTS: Fibrosis progressed significantly from 14.7+/-0.8% (SR) to 22.3+/-1.4% (AF). While the positive cytoplasmic staining of HIF1 alpha, HIF2 alpha, VEGF, KDR, and pKDR rose significantly from SR to AF, their nuclear fractions fell (only pKDR significantly). The median CD34/CD105-positive microvessel size increased significantly from SR to AF. CONCLUSIONS: AF is closely associated with an atrial up-regulation of hypoxic and angiogenic markers. Whether this is cause, effect, or co-phenomenon of fibrosis remains to be investigated. It is conceivable that fibrosis might lead to an increased O(2) diffusion distance and thus induce ischemic signaling, which, in turn, leads to angiogenesis.

Hypoxia and myocardial remodeling in human cardiac allografts: a time-course study.

BACKGROUND: Cardiac allografts are known to develop myocardial fibrosis, which may be a cause of progressive cardiac dysfunction. Apart from the renin-angiotensin and transforming growth factor-beta system, hypoxia has been proposed as an important player in the pathogenesis of fibrosis, but its significance remains unclear. This study examines the degree of myocardial fibrosis, cellular remodeling and hypoxic signaling over a time-course of 10 years after human cardiac allograft transplantation. METHODS: Serial right ventricular biopsies of 57 patients were collected in 6-month intervals after cardiac transplant surgery for a total of 10 years to allow a retrospective longitudinal analysis. Over this period, tissue remodeling, including interstitial fibrosis and cellular changes, were determined morphometrically. Immunohistochemistry (IHC) was used to analyze expression of the following hypoxia-related proteins: hypoxia-induced factor 1-alpha (HIF1alpha); the oxygen sensor prolyl hydroxylase 3 (PHD3); and vascular endothelial growth factor (VEGF). RESULTS: Fibrosis increased significantly from 12.6 +/- 6.5% at the point of transplantation throughout follow-up to 28.8 +/- 7.7% at 10 years. The DNA content and number of nuclei changed over the period of follow-up, displaying signs of cellular hypertrophy and a loss of myocytes. Whereas HIF1alpha expression revealed a U-shaped pattern with both early and late elevation during fibrogenesis, PHD3 and VEGF expression patterns showed a gradual increase with PHD3 decreasing again in later fibrogenesis. CONCLUSIONS: In cardiac allografts, extensive and progressive tissue remodeling is present. Hypoxia may play a role in this process by up-regulating HIF1alpha and leading to differential regulation of pro-angiogenic signals.

Age-related atrial fibrosis.

Many age-related diseases are associated with, and may be promoted by, cardiac fibrosis. Transforming growth factor (TGF)-beta, hypoxia-induced factor (HIF), and the matrix metalloproteinase (MMP) system have been implicated in fibrogenesis. Thus, we investigated whether age is related to these systems and to atrial fibrosis. Right atrial appendages (RAA) obtained during heart surgery (n = 115) were grouped according to patients' age (<50 years, 51-60 years, 61-70 years, or >70 years). Echocardiographic ejection fractions (EF) and fibrosis using Sirius-red-stained histological sections were determined. TGF-beta was determined by quantitative RT-PCR and hypoxia-related factors [HIF1 alpha, the vascular endothelial growth factor (VEGF)-receptor, CD34 (a surrogate marker for microvessel density), the factor inhibiting HIF (FIH), and prolyl hydroxylase 3 (PHD 3)] were detected by immunostaining. MMP-2 and -9 activity were determined zymographically, and mRNA levels of their common tissue inhibitor TIMP-1 were determined by RT-PCR. Younger patients (<50 years) had significantly less fibrosis (10.1% +/- 4.4% vs 16.6% +/- 8.3%) than older individuals (>70 years). While HIF1 alpha, FIH, the VEGF-receptor, and CD34 were significantly elevated in the young, TGF-beta and PHD3 were suppressed in these patients. MMP-2 and -9 activity was found to be higher while TIMP-1 levels were lower in older patients. Statistical analysis proved age to be the only factor influencing fibrogenesis. With increasing age, RAAs develop significantly more fibrosis. An increase of fibrotic and decrease of hypoxic signalling and microvessel density, coupled with differential expression of MMPs and TIMP-1 favouring fibrosis may have helped promote atrial fibrogenesis.

Association of echocardiographic atrial size and atrial fibrosis in a sequential model of congestive heart failure and atrial fibrillation.

BACKGROUND: Cardioversion (CV) success of atrial fibrillation (AF) inversely correlates to the size of the left atrium (LA). Atrial fibrillation and its most important risk factor, congestive heart failure (CHF), both induce atrial structural enlargement and fibrosis. To investigate the effect of AF and CHF on atrial dilatation and fibrosis, and to estimate whether echocardiographically determined atrial size may be used as a marker for atrial fibrosis. METHODS: In six dogs, pacemakers were implanted followed by HIS bundle ablation. After 4 weeks of rapid ventricular stimulation (185 bpm) for CHF induction, additional rapid atrial stimulation (500 bpm) was maintained for 7 weeks to induce AF. Serial determinations of echocardiographic atrial size were performed. Seven dogs with sinus rhythm served as histological controls. Postmortem tissue was obtained to determine the degree and composition of atrial fibrosis. RESULTS: While the ejection fraction of the AF/CHF dogs decreased significantly from 57+/-5% to 19+/-7% (P<.01), an increased degree of atrial fibrosis was found (right atrium [RA], 4.9+/-2.0% to 19.9+/-5.4%; LA, 4.4+/-1.6% to 22.2+/-3.2%; P<.01), accompanied by a significant increase of atrial volumes (LA: 21+/-4 to 44+/-4 mm3; P<.01; RA: 10+/-3 to 18+/-6 mm3; P<.05) and LA diameters (34+/-4 to 43+/-2 mm, P<.05). Atrial fibrosis and size significantly correlated. CONCLUSIONS: Atrial fibrillation/CHF leads to a significant atrial fibrosis and dilation. The increased echocardiographic size correlates to the degree of atrial fibrosis and may be used as clinical marker for atrial fibrosis. The fibrosis accompanying atrial dilatation may also explain why LA size, as determined by echocardiography, is a strong predictor of CV success.

Telomerase in nasal polyps.

BACKGROUND: The aim of this study was to determine the presence and location of telomerase activities and the possible influence of elevated human telomerase reverse transcriptase (hTERT) mRNA levels on the outcome after surgical treatment in nasal polyposis. METHODS: Telomerase activity in nasal polyps of 21 patients was quantified by measuring the hTERT mRNA contents with one-step real-time polymerase chain reaction. Inferior turbinates of 12 patients served as controls. Immunohistochemistry with specific antibodies was performed against hTERT. The number of hTERT marked cells was determined in 15 randomly selected fields. All patients were followed up after surgery for 60 months. RESULTS: Elevated hTERT mRNA expression and number of hTERT+ cells was detected in nasal polyps in comparison with inferior turbinates (p < 0.001). hTERT+ cells were detected in the basal layer of the epithelia, the endothelia, and in some seromucous glands. During follow-up, it was discovered that tissue samples of five patients with recurrent polyposis did not have higher amounts of hTERT when compared with patients without relapse. CONCLUSION: Telomerase activity is elevated in nasal polyps. Elevated hTERT expression does not predict the recurrence of nasal polyposis after surgical treatment.

Decreased plasminogen activator inhibitor and tissue metalloproteinase inhibitor expression may promote increased metalloproteinase activity with increasing duration of human atrial fibrillation.

INTRODUCTION: Atrial fibrosis has been shown to concur with the persistence of atrial fibrillation (AF) and is only incompletely reversible, thus counteracting attempts to restore and maintain sinus rhythm (SR). Besides the angiotensin system, the matrix metalloproteinases (MMP) play a major role in the pathogenesis of fibrosis. Thus, the present study investigated changes of the MMP system during the development of human AF. METHODS AND RESULTS: Right atrial appendages of 146 patients were excised during heart surgery and grouped according to rhythm (SR vs AF) and AF duration. Hydroxyproline as a surrogate for collagen content and morphometrically determined collagen content increased significantly from SR (14.3 +/- 7.7%) to chronic permanent AF (CAF) of 6-24 months (21.2 +/- 9.2%, P = 0.02), and CAF of > 60 months (25.3 +/- 4.7%, P < 0.01). From SR to paroxysmal and chronic persistent AF (CPAF) and to CAF MMP-2 and MMP-9 activity rose, while their mRNA and protein levels were not altered significantly. Plasminogen activator inhibitor (PAI), an inhibitor of a potent activator of many MMPs, was significantly decreased with increasing duration of AF. In parallel, the mRNA levels of the tissue inhibitors of MMPs TIMP-1 and -2 decreased significantly. CONCLUSION: Human atrial fibrogenesis is enhanced with increasing duration of AF: a longer AF duration is associated with elevated atrial interstitial MMP activity, but decreased PAI and TIMP expression.

Morphological effects of nanosecond- and femtosecond-pulsed laser ablation on human middle ear ossicles.

We evaluate the feasibility of nanosecond-pulsed and femtosecond-pulsed lasers for otologic surgery. The outcome parameters are cutting precision (in micrometers), ablation rate (in micrometers per second), scanning speed (in millimeters per second), and morphological effects on human middle ear ossicles. We examine single-spot ablations by a nanosecond-pulsed, frequency-tripled Nd:YAG laser (355 nm, beam diameter 10 microm, pulse rate 2 kHz, power 250 mW) on isolated human mallei. A similar system (355 nm, beam diameter 20 microm, pulse rate 10 kHz, power 160-1500 mW) and a femtosecond-pulsed CrLi:SAF-Laser (850 nm, pulse duration 100 fs, pulse energy 40 microJ, beam diameter 36 microm, pulse rate 1 kHz) are coupled to a scanner to perform bone surface ablation over a defined area. In our setups 1 and 2, marginal carbonization is visible in all single-spot ablations of 1-s exposures and longer: With an exposure time of 0.5 s, precise cutting margins without carbonization are observed. Cooling with saline solution result is in no carbonization at 1500 mW and a scan speed of 500 mms. Our third setup shows no carbonization but greater cutting precision, although the ablation volume is lower. Nanosecond- and femtosecond-pulsed laser systems bear the potential to increase cutting precision in otologic surgery.

Complement factor 5 is a quantitative trait gene that modifies liver fibrogenesis in mice and humans.

Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.

Expression of interleukin-5, interleukin-8, and interleukin-10 mRNA in the osteomeatal complex in nasal polyposis.

BACKGROUND: Several cytokines are expressed in chronic sinusitis with and without underlying allergy. Their local production and regulation in the osteomeatal complex, the key area of paranasal sinuses, still is not fully understood. This study was performed to investigate differences of cytokine messenger RNA (mRNA) expression between the medial and the lateral part of the middle turbinate and anterior ethmoid mucosa of allergic and nonallergic patients. METHODS: Using the LightCycler system for real-time reverse-transcription polymerase chain reaction, we investigated the content of interleukin (IL)-5, IL-8, and IL-10 mRNA in tissue samples from middle turbinates and anterior ethmoids of 18 patients with chronic sinusitis and nasal polyps. Inferior turbinate mucosa of six control subjects without sinusitis and allergy served as control. RESULTS: IL-5 mRNA was detectable in 32 (60%) of 54 samples (two of six controls) in significant different amounts between the various locations (p < or = 0.001). Anterior ethmoid mucosa (0.96+/-0.99) expressed the highest amount of IL-5 mRNA followed by the lateral (0.37+/-0.54) and the medial portion of the middle turbinate (0.12+/-0.29) with no difference between allergic and nonallergic subgroups. IL-8 was detected in significant higher amounts in all three origins with no significant difference in concentrations between the examined locations as compared with controls. Patients expressed either IL-5 or IL-8 or both cytokine mRNA. IL-10 was expressed in all three specimens from five of eight allergic patients. All five individuals with clinical symptoms of allergy at the time of operation expressed IL-10 in at least one specimen. CONCLUSION: IL-5 cytokine expression in the osteomeatal complex is linked to the presence of nasal polyps, whereas IL-8 is up-regulated without distinct correlation to nasal polyps. IL-10 expression was detectable in five of eight allergic patients.

Enlarged cervical lymph nodes and elevated liver chemistry tests: a therapeutic dilemma.

We describe the case of a 36-years-old male patient, originating from India, who presented with enlarged cervical lymph nodes and elevated liver chemistry tests. Histologically necrosing granulomas were observed in the lymph nodes, and PCR revealed DNA from mycobacterium tuberculosis. However, in the liver biopsy granulomatous hepatitis without central necrosis was seen. With a positive PCR for mycobacteria from liver tissue and no evidence for other hepatic diseases we started drug treatment with standard quadruple regimen consisting of isoniazid, rifampicin, ethambutol, and pyrazinamide. Five days after onset of therapy, liver chemistry tests rose 10-fold, forcing us to interrupt treatment. Gradual step-wise re-exposition with the same medication after return of liver chemistry tests to baseline was well tolerated without any further side effects. Liver involvement of tuberculosis can have many facets and may be treated by gradual dosing of standard drugs.

Haplotype-tagging RANTES gene variants influence response to antiviral therapy in chronic hepatitis C.

The response to antiviral therapy for chronic hepatitis C virus (HCV) is complex and is determined by both environmental and genetic factors. Recently, interacting gene polymorphisms of the chemokine RANTES have been shown to affect HIV disease progression. Our aim was to assess if these RANTES variants are associated with response to anti-HCV therapy. Three linked RANTES single nucleotide polymorphisms (403 G/A, Int1.1 T/C, and 3' 222 T/C) were determined in 297 Caucasian patients who were treated for chronic HCV infection and 152 control subjects. Characteristic nucleotide combinations on single chromosomes (haplotypes) were reconstructed and tested for disease association. Four common RANTES haplotypes (prevalence > 3%) were identified in patients and controls [corrected]. There was a strong association of RANTES haplotype distribution with outcome of antiviral combination therapy (P = .007). Specifically, RANTES haplotypes carrying Int1.1 C and 3' 222 C alleles were more frequent in nonresponders than in patients with a sustained response to antiviral therapy (odds ratio 1.9, P = .01). The influence of these RANTES haplotypes on the outcome of therapy was more pronounced in patients infected with HCV genotypes 1 and 4 (odds ratio 2.3, P = .02). Because RANTES haplotypes carrying Int1.1 C are known to down-regulate RANTES transcriptional activity in vitro, the haplotype analysis fits the hypothesis of a diminished T helper 1 lymphocyte response in patients with a negative response to antiviral therapy. In conclusion, RANTES haplotypes might contribute to the polygenic interaction between HCV and the host immune system and could help to risk stratify patients prior to antiviral therapy.

Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C.

BACKGROUND: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients. METHODS: Liver biopsies from 166 patients were scored for inflammatory activity (A0-4) and hepatic fibrosis (F0-4). Gene mutations were determined by LightCycler, restriction fragment length polymorphism analysis, or direct sequencing. RESULTS: The frequencies of common HFE mutations C282Y and H63D are 4.2% and 21.3%, whereas the recently described S65C substitution and the Y250X mutation in the transferrin receptor 2 gene are very rare. In regression analysis, heterozygous carriers of C282Y or H63D mutations display significantly (P < 0.05) higher inflammatory activities and more advanced fibrosis than patients without mutations. For C282Y heterozygous patients, the odds ratios for marked inflammatory activity (A2-4) and advanced liver fibrosis or cirrhosis (F2-4) are 4.9 and 4.6, respectively, compared with patients carrying homozygous wild-type alleles. C282Y mutations are associated with significantly (P < 0.05) increased serum iron and aminotransferase levels, whereas H63D heterozygotes display higher transferrin saturation, serum iron, and ferritin concentrations compared to wild-type (P < 0.01). CONCLUSIONS: Common heterozygous hemochromatosis mutations are associated with higher grades of inflammation and more severe hepatic fibrosis. Our findings support a role of HFE mutations as primary risk factors for fibrogenesis and disease progression in chronic hepatitis C.

Mixed chimerism of cardiomyocytes and vessels after allogeneic bone marrow and stem-cell transplantation in comparison with cardiac allografts.

Controversy persists about mixed chimerism (mCh) occurring in the hearts of patients after orthotopic cardiac transplantation in comparison with allogeneic bone marrow (BM) and peripheral blood stem-cell (PBSC) transplants. Cadaver hearts were examined after sex-mismatched transplantation by immunophenotyping combined with dual color fluorescence in situ hybridization (X and Y chromosome-specific probes). A striking disparity in the extent of mCh depending on the different transplantation procedures was recognizable. After allografting with PBSCs, 1.7% chimeric cardiomyocytes were detectable contrasting 5.4% of donor cells after full BM transplantation. In cardiac transplants, host-type endothelial cells (16.2%) and myocytes (14.3%) of the vessel walls were more often encountered than after BM and PBSC allografting. A sprouting of vascular structures into the donor heart after orthotopic cardiac transplantation has to be assumed, as does a pivotal role of the mesenchymal stem cells of the BM in the development of mCh.

The presence of non-organ-specific autoantibodies is associated with a negative response to combination therapy with interferon and ribavirin for chronic hepatitis C.

BACKGROUND: Non-organ-specific autoantibodies are found in a considerable number of anti-HCV positive patients. Previous studies investigated the clinical relevance of these antibodies in patients treated with interferon monotherapy, but not combination therapies. METHODS: Anti-nuclear, anti-smooth muscle, anti-mitochondrial, anti-neutrophil-cytoplasmatic and anti-liver/kidney microsomal antibodies were determined in 78 consecutive anti-HCV positive patients by indirect immunofluorescence. The presence of these antibodies was related to demographic variables and to the outcome of antiviral combination therapy with interferon-alpha and ribavirin in 65 patients. RESULTS: In our study, positivity for autoantibodies was associated with higher alanine aminotransferase levels and higher mean values for HCV-RNA (p < 0.01). Furthermore, negativity for non-organ-specific autoantibodies was associated with a favourable treatment outcome of combination therapy with at least one negative RT-PCR for HCV-RNA during treatment (OR 4.65, 95% CI 1.31 to 16.48, p = 0.02). ANA and SMA staining patterns and titers were not correlated to treatment response. With multiple logistic regression analysis, positivity for autoantibodies and HCV genotype were independently associated with outcome of antiviral combination therapy (p = 0.02). CONCLUSIONS: The absence of non-organ-specific autoantibodies might indicate a significantly higher chance for viral clearance in response to combination therapy for chronic hepatitis C infection. Therefore, despite of an overall higher treatment response, the addition of the immunomodulatory drug ribavirin could accentuate immunological differences that affect treatment outcome and might have been less obvious in earlier studies analysing interferon monotherapy.

CC chemokine receptor 5 delta32 polymorphism in two independent cohorts of hepatitis C virus infected patients without hemophilia.

Recently CC chemokine receptor 5 (CCR5) related immune mechanisms and a functional mutation of the CCR5 gene have been implicated in hepatitis C virus (HCV) infection in a cohort of predominantly hemophiliac patients. The present study investigated the frequency and clinical consequences of the CCR5 Delta32 mutation in two genetically homogeneous populations of HCV infected patients with a different risk profile for infection. Genomic DNA samples from 333 German patients with chronic HCV infection were screened by PCR for the presence of the CCR5 Delta32 polymorphism. In-hospital patients admitted for other diseases than viral hepatitis but with a comparable risk for HCV exposure were used as control population ( n=125). Allele frequencies of CCR5 Delta32 polymorphism did not differ significantly between the two groups (7.6% and 9.5%, respectively) and control subjects (10.4%), and did not deviate from Hardy-Weinberg equilibrium in any group. Furthermore, there were no major differences between patients with respect to HCV genotypes, viral loads, liver enzymes, or fibrosis scores in relation to the presence or absence of the heterozygous CCR5 Delta32 mutation. Differences in inflammatory scores in liver biopsy samples and response to antiviral therapy in CCR5 Delta32 heterozygotes in one cohort could neither be reproduced in the other group of patients nor when both cohorts were pooled. These results argue against a strong effect of the CCR5 Delta32 deletion regarding these phenotypes. In conclusion, we found no increased frequency of the CCR5 Delta32 polymorphism in two independent cohorts of patients with HCV infection but without hemophilia as the main risk factor for infection. As the major difference to investigations demonstrating an association between CCR5 Delta32 and HCV infection is the selection of cases and controls, our study emphasizes the importance of epidemiological criteria for association studies of HCV infection.

Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats.

BACKGROUND & AIMS: Numerous studies implicate transforming growth factor (TGF)-beta signaling in liver fibrogenesis. To perturb the TGF-beta pathway during this process, we overexpressed Smad7, an intracellular antagonist of TGF-beta signaling, in vivo and in primary-cultured hepatic stellate cells (HSCs). METHODS: Ligation of the common bile duct (BDL) was used to induce liver fibrosis in rats. Animals received injections of an adenovirus carrying Smad7 cDNA into the portal vein during surgery and via the tail vein at later stages. The effect of Smad7 on TGF-beta signaling and activation of HSC was further analyzed in primary-cultured cells. RESULTS: Smad7-overexpressing BDL rats displayed reduced collagen and alpha-SMA expression and reduced hydroxyproline content in the liver, when compared with animals administered AdLacZ. Such a beneficial effect was also observed when Smad7 was expressed in animals with established fibrosis. Accordingly, Smad7 arrested transdifferentiation of primary-cultured HSCs. AdSmad7 infected cells remained in a quiescent stage and retained storage of vitamin A droplets. Smad7 expression totally blocked TGF-beta signal transduction, shown by inhibiting Smad2/3 phosphorylation, nuclear translocation of activated Smad complexes, and activation of (CAGA)(9)-MLP-Luc, resulting in decreased collagen I expression. Smad7 also abrogated TGF-beta-dependent proliferation inhibition of HSC. Smad7 did not decrease expression of alpha-SMA, but immunofluorescent staining with anti alpha-SMA antibodies displayed destruction of the fibrillar organization of the actin cytoskeleton. CONCLUSIONS: In summary, gene transfer of Smad7 inhibits experimental fibrogenesis in vivo. Studies with isolated HSC suggest that the underlying mechanisms involve inhibition of TGF-beta signaling and HSC transdifferentiation.