Comparison of three different image enhancement systems for detection of laryngeal lesions.

OBJECTIVE: Image enhancement systems are important diagnostic tools in the detection of laryngeal pathologies. This study aimed to compare three different image enhancement systems: professional image enhancement technology, Image1 S and narrow-band imaging. METHOD: Using the three systems, 100 patients with laryngeal lesions were investigated using a flexible and a 30° rigid endoscope. The lesions were diagnosed by three experts and classified using the Ni classification. The findings were compared. RESULTS: Lesions classified as 'benign' were histopathologically confirmed in 50 per cent of patients, malignant lesions were confirmed in 41 per cent and recurrent respiratory papillomatosis were confirmed in 9 per cent. There was no significant difference between the experts' assessments of each image enhancement system. CONCLUSION: The three systems give comparable results in the detection of laryngeal lesions. With two additional systems, more users can perform image-enhanced endoscopy, resulting in a broadly available tool that can help to improve oncological assessment.

Cold traps as reliable devices for quantitative determination of SARS-CoV-2 load in aerosols.

Spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a demanding challenge. This is of particular importance in schools and public areas of unavoidable access. New viral mutations may increase infectivity and require even better methods to identify areas of potential hazards. High-throughput SARS-CoV-2 testing and legal restrictions are not effective in order to get the current outbreak under control. The occurrence of new SARS-CoV-2 variants with a higher transmissibility requires efficient strategies for early detection and surveillance. Until today, testing focuses on nasal or pharyngeal mucosa swabs, neglecting the origin of aerosolic transmission, thus failing to detect the spread by carriers of the virus. Therefore, in this study, SARS-CoV-2 RNA levels were determined by quantitative real time PCR in aerosols collected by non-powered cold traps. SARS-CoV-2 spreading kinetics were recorded in indoor hotspots within a high-endemic area. These hotspots included a SARS-CoV-2 isolation unit, an outpatient endoscopy facility, a concert hall, and a shopping mall. For determination of viral presence aerosols were collected by cold traps positioned at different locations in the area of interest over a period of 4-6 h. Indoor SARS-CoV-2 hotspots were found in non-ventilated areas and in zones that are predisposed to a buoyancy (chimney) effect. SARS-CoV-2 RNA in those aerosols reached concentrations of 105 copies/mL, while extensive outdoor air ventilation reliably eliminated SARS-CoV-2 aerosol contamination. The method presented herein is effective for the identification of SARS-CoV-2 indoor hotspots and may help to characterize the spreading kinetics of SARS-CoV-2. Moreover, it can be used for the surveillance of emerging SARS-CoV-2 variants. Due to low costs and easy handling, the procedure might enable efficient algorithms for COVID-19 screening and prevention.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) - an underestimated condition: ferritin light chain variant spectrum in German families.

Background In hereditary hyperferritinaemia-cataract syndrome (HHCS), single nucleic acid alterations in the ferritin light chain (L-ferritin) iron response element (IRE) constitutively derepress ferritin synthesis, resulting in hyperferritinaemia, L-ferritin deposits in the lens of the eye and early bilateral cataract onset. Methods In this study, six German families with putative HHCS were analysed. Clinical diagnosis of HHCS was based on medical history, evaluation of ferritin serum levels, transferrin saturation and clinical ophthalmological examination. Diagnosis was confirmed by polymerase chain reaction (PCR)-based DNA sequencing of the L-ferritin IRE. Results Genetic analysis of the L-ferritin IRE revealed relevant single nucleic acid alterations in each of the affected families. Variants c.-168G > A, c.-168G > U and c.-167C > U were located in the C-bulge region; and variants c.-161C > U and c.-157G > A were located in the hexanucleotide loop of the L-ferritin IRE. Conclusions Family history of hyperferritinaemia and juvenile cataracts are strong indicators of HHCS. Genetic analysis of the L-ferritin IRE is a straightforward procedure to confirm the diagnosis. Accurate diagnosis of hyperferritinaemia can avoid unnecessary treatment by venesection, and focus attention on early cataract detection in offspring at risk.

The Detergent Effect of Mesalazine Interferes with Phosphatidylcholine Binding to Mucin 2.

OBJECTIVES: Therapeutically applied delayed-release phosphatidylcholine (PC) revealed mucosa protection and clinical improvement of ulcerative colitis. However, a recent trial with simultaneous application of delayed-release PC and mesalazine showed lack of efficacy. It is hypothesized that mesalazine acts as detergent to prohibit PC integration into mucus as target compartment, thus preventing topical mucus protection. METHODS: In vitro PC-binding studies with mucin 2 and intestinally differentiated CaCo2 cells as well as outcome analysis of a therapeutic trial with delayed-release PC and additional mesalazine. RESULTS: Choline-containing phospholipids, in particular PC, bind to mucin 2 as main scaffold protein of intestinal mucus to establish a hydrophobic barrier towards microbiota in the intestinal lumen. PC also binds to the apical surface of polarized CaCo2 cells with membrane-anchored mucin 3. Mesalazine removes mucin-bound PC and, thus, reduces transepithelial resistance. A post hoc analysis of patients from a previous multicenter phase IIB trial with delayed-release PC revealed that those without mesalazine showed a PC dose-dependent outcome with regard to achievement of partial and complete remission (p < 0.05 for 1.6 and 3.2 g PC daily) whereas those treated simultaneously with mesalazine showed no PC dose dependency. CONCLUSION: Mesalazine solubilizes PC and, thus, prevents the protective action of therapeutically applied delayed-release PC within mucus.

First clinical trial of a newly developed capsule endoscope with panoramic side view for small bowel: a pilot study.

BACKGROUND AND STUDY AIMS: Capsule endoscopy is the first-line diagnostic technique for the small bowel. However, the inability to visualize the duodenal papilla is an inherent limitation of this method. In the present study, we evaluated feasibility of a newly developed CapsoCam SV1 capsule. PATIENTS AND METHODS: This is a prospective dual center study of a newly developed video capsule CapsoCam SV1 from Capsovision, CA, providing panoramic 360° imaging. A high frequency of 20 frames occurs per second for the first 2 h and thereafter 12 frames/s, with a battery life of 15 h. We evaluated feasibility and completeness of small bowel examination together with secondary endpoints of duodenal papilla detection in 33 patients. Patients swallowed the capsules following colonoscopy or were prepared with 2 L of polyethylene glycol solution prior to the examination. All patients swallowed 20 mg of metoclopramide and 160 mg of simethicone 30 min before ingestion of the capsule. RESULTS: Thirty-one of the 33 patients' data could be evaluated. Small bowel examination was complete in all procedures. Mean time to pass the small bowel was 258 ± 136 min. Average small bowel cleanliness was 3.3 ± 0.5. In 71% of the patients, we identified the duodenal papilla. No adverse reaction in relation to the capsule examination was observed. CONCLUSIONS: CapsoCam SV1 is a safe and efficient tool in small bowel examination. The duodenal papilla as the only landmark in small bowel is detected in more than 70% of the patients.

Evidence for a critical role of ceruloplasmin oxidase activity in iron metabolism of Wilson disease gene knockout mice.

BACKGROUND AND AIM: Wilson disease is a genetic disorder associated with copper overload due to mutations within the ATP7B gene. Although copper and iron metabolism are closely linked, the influence of mutations of the ATP7B gene on iron homeostasis is unknown. Therefore, the present study was carried out to elucidate iron metabolism in Atp7b(-/-) mice, an animal model of Wilson disease. METHODS: Hepatic iron content, serum iron parameters and blood hemoglobin levels of Atp7b(-/-) mice and wild type mice were studied. Hepatic and duodenal expression of iron metabolism-related genes was measured quantitatively by real-time reverse transcription-polymerase chain reaction and post-translational expression of Dmt1 was analyzed by immunoblot. RESULTS: Atp7b(-/-) mice displayed copper accumulation (P < 0.001), slightly elevated hepatic iron content (P = NS), and a low serum ceruloplasmin oxidase activity (1.5 +/- 1.9 U/L vs 18.9 +/- 4.0 U/L, P < 0.001) when compared with wild type mice. Serum iron, serum transferrin saturation, and blood hemoglobin levels were significantly lower in Atp7b(-/-) mice compared with controls (121.2 +/- 35.3 microg/dL vs 201.8 +/- 34.9 microg/dL (P < 0.001); 44.0 +/- 12.7% vs 68.0 +/- 8.2% (P < 0.001); and 12.7 +/- 0.2 g/dl vs 15.3 +/- 0.1 g/dl (P < 0.001), respectively). Hepatic mRNA expression of hepcidin, TfR-1, TfR-2, hemojuvelin, and Dmt1 + IRE did not differ significantly between Atp7b(-/-) and wild type mice. In the duodenum of Atp7b(-/-) mice Dmt1 + IRE and hephaestin did not show any differences in their mRNA levels compared with wild type mice, while Dcytb mRNA expression was 1.7-fold increased compared with wild type mice (P = 0.01). CONCLUSION: Atp7b(-/-) mice demonstrated decreased serum iron parameters and hemoglobin levels most likely related to a low serum ceruloplasmin oxidase activity and not due to total body iron deficiency.

SLA/LP/tRNP((Ser)Sec) antigen in autoimmune hepatitis: identification of the native protein in human hepatic cell extract.

A diagnostic subgroup of AIH type 1 is characterized by specific serum antibodies against soluble liver protein. The respective autoantigen was named SLA/LP/tRNP((Ser)Sec), after three homologous recombinant polypeptides were isolated from expression gene libraries. We analyzed human cultured liver cells for the human homologue of recombinant SLA/LP/tRNP((Ser)Sec) by antigen purification. In addition, a monoclonal antibody was generated against recombinant SLA-p35, a truncated recombinant SLA-reactive polypeptide. With a positive patient serum, immune affinity chromatography was performed on the 52 kD-SLA main antigenic determinant pre-enriched by ion exchange chromatography. By mass spectrometry, the 52 kD-SLA/LP/tRNP ((Ser)Sec) autoantigen was unambiguously identified in the purification product. The identity of the recombinant SLA-p35 and its human homologue was further confirmed by a specific signal of the anti SLA-p35 monoclonal antibody with purified human SLA/LP/tRNP((Ser)Sec). The 48 kD-SLA species frequently comigrating in SLA-immunoblotting however was not identified by either approach. We conclude that the native counterpart of recombinant tRNP((Ser)(Sec)) indeed is detectable with a molecular weight of 52 kD in soluble liver extract of human cells as the major antigenic component of SLA/LP/tRNP((Ser)Sec).

The iron regulatory peptide hepcidin is expressed in the heart and regulated by hypoxia and inflammation.

The peptide hormone hepcidin plays a central role in iron homeostasis. It is predominantly expressed in the liver and regulated by iron, hypoxia, and inflammation. Although it has been shown that iron plays a key pathophysiological role in cardiac diseases, including iron-overload cardiomyopathy, myocardial ischemia-reperfusion injury, and atherosclerosis, very little is known about the putative expression and the role of hepcidin in the heart. In the present study, expression and regulation of hepcidin in rat heart were analyzed. Basal cardiac expression of hepcidin was demonstrated on mRNA and protein level in vivo in a rat model and compared with its regulation in the liver. The cellular localization was analyzed by immunofluorescence microscopy. Sixteen hours after a single injection of turpentine, a more than 2-fold increase of cardiac hepcidin mRNA and a more than 3-fold increase of hepatic hepcidin mRNA was observed. In response to hypoxia, expression of hepcidin in the liver decreased. In contrast, hypoxia resulted in a strong up-regulation of hepcidin expression on mRNA and protein level in the heart, accompanied by an increased immunoreactivity of hepcidin pronounced at the myocardial intercalated disc area. The finding of a regulated expression of the iron-regulatory peptide hormone hepcidin in the heart suggests that hepcidin may have an important role in cardiac diseases.

Localization of the iron-regulatory proteins hemojuvelin and transferrin receptor 2 to the basolateral membrane domain of hepatocytes.

The newly discovered proteins hemojuvelin (Hjv) and transferrin receptor type 2 (TfR2) are involved in iron metabolism. Mutations in the Hjv and TfR2 gene cause hemochromatosis. We investigated the expression and cellular localization of Hjv and TfR2 in rat and human liver. The expression of Hjv and TfR2 was shown on mRNA and protein level by RT-PCR and immunoblot experiments. Their cellular localization was studied by immunofluorescence with antibodies raised against Hjv and TfR2. Hjv and TfR2 are present in human and rat liver and in primary human hepatocytes. Antisera raised against Hjv identified immunoreactive proteins with an apparent size of 44 and 46 kDa in immunoblot experiments of rat and human liver extracts, which are in accordance with the putative membrane-bound and cleaved soluble forms of this protein, respectively. TfR2 was detected as a 105 kDa protein corresponding to the predicted size of glycosylated TfR2 monomers. In immunofluorescence experiments, Hjv and TfR2 were found in rat liver only in hepatocytes. At the subcellular level, both proteins were predominantly localized to the basolateral membrane domain of hepatocytes. The localization of Hjv and TfR2 at the same membrane domain renders a functional interaction of these two proteins in iron homeostasis possible.

Iron stores modulate hepatic hepcidin expression by an HFE-independent pathway.

BACKGROUND/AIMS: In HFE-related hereditary hemochromatosis an inappropriately low hepatic expression of the iron-regulatory peptide hepcidin (encoded by HAMP) has been suggested to cause iron overload. The aim of the present study was to evaluate whether the hepatic expression of HAMP in relation to iron stores requires HFE or might involve other important iron-related genes including HJV (encoding hemojuvelin) and TFR2 (encoding transferrin receptor-2). METHODS: Using quantitative RT-PCR, the iron-dependent hepatic expression patterns of HAMP, HJV, and TFR2 were evaluated in human and murine HFE-related hemochromatosis. RESULTS: The overall level of hepatic HAMP expression in human and murine HFE-related hemochromatosis is impaired but can still be modulated by iron stores. Moreover, we demonstrate an HFE-independent correlation between the expression of HAMP and TFR2 in mouse and human livers. On the other hand, a strong correlation between the hepatic expression of HAMP and HJV was only found in hemochromatosis patients and Hfe-deficient mice. CONCLUSION: The central pathogenetic step in HFE-related hemochromatosis is an impaired basal expression of HAMP rather than a lack of HAMP upregulation in response to iron stores. An HFE-independent pathway that seems to involve TFR2 and HJV can regulate HAMP expression under conditions of iron overload.

Angiotensin II stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner.

The renin-angiotensin system contributes to atherogenesis. Matrix metalloproteinases (MMP) are thought to participate in plaque destabilization through degradation of extracellular matrix. This study tested whether angiotensin II (ANG II) induces MMP in human vascular smooth muscle cells (SMC). ANG II induced expression of MMP-1, -3, and -9, but not of MMP-2 in SMC. The expression of MMP-1, a key enzyme for collagen degradation, was studied in detail. SMC stimulated with ANG II concentration-dependently released enzymatically active MMP-1. The ANG II type 1 receptor antagonists losartan and candesartan blocked ANG-II-induced MMP-1 release. Inhibition experiments with actinomycin D suggest ANG-II-induced MMP-1 mRNA regulation at the transcriptional level. Decoy oligodeoxynucleotides against nuclear factor-kappaB and activator protein 1 inhibited MMP-1 secretion, demonstrating participation of these transcription factors in MMP-1 transcription. Stimulation of MMP-1 by ANG II depended on cyclooxygenase 2. The antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine, the flavin protein inhibitor diphenylene iodonium, and the NADP(H) oxidase inhibitor apocynin blocked MMP-1 release, suggesting a redox-sensitive mechanism involving NADP(H) oxidase. The reactive oxygen species (ROS) donor 2,3-dimethoxy-1,4-naphthoquinone induced MMP-1 secretion and enhanced ANG-II-stimulated MMP-1 expression. These findings indicate that ROS may increase their own production by activation of NADP(H) oxidase. The capability of ANG II to induce functionally active MMP in human SMC may contribute to the altered plaque composition seen in complicated stages of atherosclerosis.

Iron, the HFE gene, and hepatitis C.

Intrahepatic iron overload is commonly seen in chronic hepatitis C infection. High levels of intrahepatic iron may lead to accelerated liver injury and development of fibrosis and cirrhosis. This is frequently seen in hereditary hemochromatosis, which in most of the cases is caused by homozygous mutations in the HFE gene. In patients suffering from chronic hepatitis C, the presence of heterozygous HFE mutations associates with higher hepatic iron scores and advanced stages of fibrosis. HFE mutations must therefore be considered as important comorbidity factors in chronic hepatitis C infection.

Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin.

Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe(-/-) mice, an Hfe(-/-) mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe(-/-) mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe(-/-) mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.

Hemochromatosis and transferrin receptor gene polymorphisms in chronic hepatitis C: impact on iron status, liver injury and HCV genotype.

Mild iron overload in chronic hepatitis C is associated with liver fibrosis, hepatitis C virus (HCV) genotype 1b infection, and an impaired response to interferon therapy. In this study we evaluated whether polymorphisms in the hemochromatosis gene HFE and the transferrin receptor gene TFR1 are associated with these typical findings. The study considered 246 HCV-infected patients and 200 blood donors as controls, in which C282Y, H63D, and S65C mutations ( HFE) and the S142G polymorphism ( TFR1) were detected. HCV genotype, serum ferritin levels, stainable intrahepatic iron, and grade of fibrosis according to the METAVIR score (F0-F4) were determined. In HCV-infected patients, heterozygosity for the C282Y mutation in HFE was significantly associated with elevated serum ferritin levels, stainable liver iron, and advanced fibrosis or cirrhosis (F2-F4). By multivariate logistic regression analysis the odds ratio for the development of advanced fibrosis or cirrhosis (F2-F4) was 2.5 for HCV-infected patients carrying a heterozygous C282Y mutation and 4.8 for HCV-infected patients with C282Y/H63D and C282Y/S65C compound heterozygosity. Heterozygosity for the C282Y mutation in HFE contributes to iron accumulation and fibrosis progression in chronic hepatitis C.

Expression of hepcidin in hereditary hemochromatosis: evidence for a regulation in response to the serum transferrin saturation and to non-transferrin-bound iron.

Experimental data suggest the antimicrobial peptide hepcidin as a central regulator in iron homeostasis. In this study, we characterized the expression of human hepcidin in experimental and clinical iron overload conditions, including hereditary hemochromatosis. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we determined expression of hepcidin and the most relevant iron-related genes in liver biopsies from patients with hemochromatosis and iron-stain-negative control subjects. Regulation of hepcidin mRNA expression in response to transferrin-bound iron, non-transferrin-bound iron, and deferoxamine was analyzed in HepG2 cells. Hepcidin expression correlated significantly with serum ferritin levels in controls, whereas no significant up-regulation was observed in patients with hemochromatosis despite iron-overload conditions and high serum ferritin levels. However, patients with hemochromatosis showed an inverse correlation between hepcidin transcript levels and the serum transferrin saturation. Moreover, we found a significant correlation between hepatic transcript levels of hepcidin and transferrin receptor-2 irrespective of the iron status. In vitro data indicated that hepcidin expression is down-regulated in response to non-transferrin-bound iron. In conclusion, the presented data suggest a close relationship between the transferrin saturation and hepatic hepcidin expression in hereditary hemochromatosis. Although the causality is not yet clear, this interaction might result from a down-regulation of hepcidin expression in response to significant levels of non-transferrin-bound iron.

UbcH5A, a member of human E2 ubiquitin-conjugating enzymes, is closely related to SFT, a stimulator of iron transport, and is up-regulated in hereditary hemochromatosis.

SFT, a stimulator of iron (Fe) transport, has been described as a transmembrane protein that facilitates the uptake of ferrous and ferric iron in mammalian cells. This study was initiated to investigate the 5' regulatory region of SFT and its role in the etiology of hereditary hemochromatosis. Sequence analyses of the putative 5' regulatory region revealed that the SFT cDNA sequence corresponds to intron 6/exon 7 of UbcH5A, a member of E2 ubiquitin-conjugating enzymes, which is involved in the iron-dependent ubiquitination of the hypoxia-inducible factor (HIF) by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. Further mRNA expression studies using a sequence-specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that UbcH5A is significantly up-regulated in the liver of iron-overloaded patients with hereditary hemochromatosis, as previously published for SFT. However, in vitro studies on HepG2 cells failed to demonstrate any significant UbcH5A regulation in response to iron loading or iron chelation. In conclusion, in vivo mRNA expression data previously obtained for SFT might be attributed to UbcH5A. The role of UbcH5A and the ubiquitination pathway in the etiology of hereditary hemochromatosis remains to be elucidated further.