Mitochondrial respiration controls neoangiogenesis during wound healing and tumour growth.

The vasculature represents a highly plastic compartment, capable of switching from a quiescent to an active proliferative state during angiogenesis. Metabolic reprogramming in endothelial cells (ECs) thereby is crucial to cover the increasing cellular energy demand under growth conditions. Here we assess the impact of mitochondrial bioenergetics on neovascularisation, by deleting cox10 gene encoding an assembly factor of cytochrome c oxidase (COX) specifically in mouse ECs, providing a model for vasculature-restricted respiratory deficiency. We show that EC-specific cox10 ablation results in deficient vascular development causing embryonic lethality. In adult mice induction of EC-specific cox10 gene deletion produces no overt phenotype. However, the angiogenic capacity of COX-deficient ECs is severely compromised under energetically demanding conditions, as revealed by significantly delayed wound-healing and impaired tumour growth. We provide genetic evidence for a requirement of mitochondrial respiration in vascular endothelial cells for neoangiogenesis during development, tissue repair and cancer.

Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features.

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases.

[Drug-induced Pulmonary Hypertension - a Current Review]. / Medikamenten-induzierte pulmonale Hypertonie - eine aktuelle Übersicht.

BACKGROUND: The current classification of pulmonary Hypertension (PH) consists of five clinical groups, and drug-induced pulmonary arterial hypertension (PAH) is classified under Group 1 as a distinct entity. Our present work encompasses the available data concerning the association between the intake of a wide range of drugs and development of PAH. METHODS: A selective literature search was performed in Pubmed to include published work between the years 1960 - 2015. For this search, the terms pulmonary hypertension, pulmonary arterial hypertension, pulmonary veno-occlusive disease, drug induced pulmonary hypertension and chemotherapy induced PVOD were used. Mainly German, English and French publications regarding this topic were considered. RESULTS: An association between drug intake and PH development was described for different medications, among them appetite-suppressant drugs, interferon alpha and beta, but also several chemotherapeutic drugs. CONCLUSIONS: The present literature regarding drug-induced PH mainly comprises case reports and small patient cohorts. Drug-induced PH has become increasingly discussed in recent years and needs further elucidation. A close cooperation between clinicians, PH expert centers and regulatory agencies is mandatory to identify other potential drugs at an early stage that may be linked to PH development. For the clinician, a thorough patient interview including drug history is necessary in the evaluation of a patient with PH.

[Acute jaw-thoracic pain and syncope in a 41-year-old man]. / Akuter Kiefer-Thorax-Schmerz und Synkope bei einem 41-jährigen Patienten.

A 41-year-old physically active man with no significant past medical history presented with sudden thoracic pain. The patient was referred to the next tertiary care hospital. A CT scan showed an ectasia of the ascending aorta with irregularities of the aortic wall without dissection. Despite initial refusal, the patient was referred to a university hospital with experience in aortic surgery. A triphase ECG-synchronized cardiothoracic flash protocol performed on a 256 line CT scanner confirmed an aortic intramural hematoma and a covered aortic perforation. Shortly afterwards the patient collapsed and had to be resuscitated.

Cell type-dependent pathogenic functions of overexpressed human cathepsin B in murine breast cancer progression.

The cysteine protease cathepsin B (CTSB) is frequently overexpressed in human breast cancer and correlated with a poor prognosis. Genetic deficiency or pharmacological inhibition of CTSB attenuates tumor growth, invasion and metastasis in mouse models of human cancers. CTSB is expressed in both cancer cells and cells of the tumor stroma, in particular in tumor-associated macrophages (TAM). In order to evaluate the impact of tumor- or stromal cell-derived CTSB on Polyoma Middle T (PyMT)-induced breast cancer progression, we used in vivo and in vitro approaches to induce human CTSB overexpression in PyMT cancer cells or stromal cells alone or in combination. Orthotopic transplantation experiments revealed that CTSB overexpression in cancer cells rather than in the stroma affects PyMT tumor progression. In 3D cultures, primary PyMT tumor cells showed higher extracellular matrix proteolysis and enhanced collective cell invasion when CTSB was overexpressed and proteolytically active. Coculture of PyMT cells with bone marrow-derived macrophages induced a TAM-like macrophage phenotype in vitro, and the presence of such M2-polarized macrophages in 3D cultures enhanced sprouting of tumor spheroids. We employed a doxycycline (DOX)-inducible CTSB expression system to selectively overexpress human CTSB either in cancer cells or in macrophages in 3D cocultures. Tumor spheroid invasiveness was only enhanced when CTSB was overexpressed in cancer cells, whereas CTSB expression in macrophages alone did not further promote invasiveness of tumor spheroids. We conclude that CTSB overexpression in the PyMT mouse model promotes tumor progression not by a stromal effect, but by a direct, cancer cell-inherent mode of action: CTSB overexpression renders the PyMT cancers more invasive by increasing proteolytic extracellular matrix protein degradation fostering collective cell invasion into adjacent tissue.

Fatal course of an autochthonous hepatitis E virus infection in a patient with leukemia in Germany.

An acute infection with hepatitis E virus (HEV) genotype 3 subtype c was diagnosed in a patient with chronic lymphatic B-cell leukemia 6 weeks after the infusion of donor lymphocytes. Despite intensive care the patient died 39 days after admission due to pericardial effusion that was related to acute liver failure. We suggest that diagnostic procedures for detection of HEV infection should be seriously considered for the immunocompromised patient with elevated liver enzymes in the absence of a travel history to HEV endemic countries.

Dynamics of polyphosphate-accumulating bacteria in wastewater treatment plant microbial communities detected via DAPI (4',6'-diamidino-2-phenylindole) and tetracycline labeling.

Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

Tumor necrosis factor alpha induces the expression of the nuclear protein p8 via a novel NF kappaB binding site within the promoter.

p8 is a widely expressed HMG-I/Y-like transcription factor which is involved in regulating cell proliferation and tissue stress. Several studies describe a strong upregulation of p8 expression during inflammatory processes like pancreatitis and LPS-induced sepsis. Here we demonstrate that TNFalpha, which is an important inducer of innate defence against gram-negative bacteria, significantly stimulates p8 protein production in H4IIE rat hepatoma cells within 2 hours. Since a putative NF kappaB motif has been described, we further tested whether TNFalpha stimulates p8 expression via activation of NF kappaB. We characterized the TNFalpha-induced binding of NF kappaB to this motif. We show that the TNFalpha-induced NF kappaB pathway contributes to the induction of p8 during pancreatitis and LPS-induced inflammation.

Rango homeostático del metabolismo oxidativo: 60 años de fisiometría integrativa / Homeostatic range of the oxidative metabolism: 60 years of integrative fisiometry

The energetic metabolism and its relationship with body weight generated a vivid controversy, since the Rubner's surface law was introduced into biology. Recently, the multifactor theory (Darveau et al) has caused again a revival of this polemic topic. Moreover, the investigations concerning metabolism and body weight include all terrestrial mammals, from the shrew (3 grams) to the elephant (three tons). The corresponding allometric exponent for standard metabolic rate, both theoretical and empirical, fluctuates around an average value of 0.75, in contrast with the surface law, which postulated a value of 0.67. The "metabolic range" (rest vs maximal exercise) does vary from 1 to 10, due to the prevalent influence of the skeletal muscle activity. Recent investigations have emphasized the fact that the allometric exponent is not unique (0.75), but it should be subjected to statistical variability, both in standard and in maximal exercise.

VISION2003: virtual learning units for medical training and education.

The project VISION2003 consists of several partners with different professions ranging from medicine to medical informatics, from computer science to didactics. Its aim is the development, testing, introduction and a long-time maintenance of an open, web-based, intelligent and adaptive teaching and learning system for medical education. The system is expected to enhance the acceptance and efficiency of conventional ways of learning by supplementing and supporting them and creating new methods for imparting knowledge ["VISION2003, Lehr-und Lernsysteme in der Medizin: Intelligente und Multimediale, Internetbasierte adaptive und intelligente Autorensysteme für kooperatives Training in der Medizin", (last valid on 17 January 2003) and Ein offenes sprachkonzept für verteilte wissensverarbeitung in der medizin, Tagungsabstract XVI International Congress of the European Federation for Medical Informatics MIE, September 2000]. This is done exemplarily in the specific fields of oncology, accident-surgery and cardiology in consideration of actual standards and didactical measures. The range of possible applications is wide, from electronically accessible scripts through example cases to complex simulations. The main focus of the project is the creation of an open and flexible internet platform for delivering multimedia-based learning units and the development of adaptive and intelligent authoring systems.

Fibre-years, pulmonary asbestos burden and asbestosis.

The relations between cumulative asbestos fibre doses at the work-places and asbestos burden of the lung evaluated by lung dust analyses have been tested on 3 different groups of patients of the German Mesothelioma Register: 1. total collective (n = 366), 2. collective without elevated asbestos burden of the lungs (n = 193), 3. collective with asbestoses/minimal asbestoses (n = 64). The relations between the above mentioned parameters are in general only weak. The limit value of > 25 fibre-years is found in 19.6% of persons without increased pulmonary asbestos burden. In spite of reaching or exceeding the cumulative doses of 25 fibre-years, 24% of the whole collective also show no elevated asbestos-concentrations in their lung tissues. By contrast, 42% of patients with asbestos-associated lung fibroses do not attain 25 fibre-years at their work-places. Considering our data it is doubtful that the postulated limit value of 25 fibre-years can be an adequate parameter for the evaluation of asbestos-associated lung fibroses.

Antibodies to Lassa virus Z protein and nucleoprotein co-occur in human sera from Lassa fever endemic regions.

It is not known whether the small 11-kDa Z protein of Lassa virus is immunogenic during human Lassa virus infection. To obtain evidence for the existence of an antibody response and to test the suitability of these antibodies for serosurveys, sera from Lassa fever endemic regions (Guinea and Nigeria, n = 75) were tested for co-reactivity to Z protein and nucleoprotein (NP). Sera from a non-endemic region (Uganda, n = 50) served as a specificity control. Z protein and NP were expressed in Escherichia coli, affinity-purified, and used as antigen in Western blot. Indirect immunofluorescence (IIF) with Lassa virus-infected cells was performed for comparison. Due to high unspecific reactivity of the African sera, Western blot testing was performed with a 1:1,000 serum dilution. Under these conditions, none of the control sera but 12% of the sera from endemic regions co-reacted with both Z protein and NP. Reactivity to Z protein was significantly associated with NP reactivity (P < 10(-6)). NP and Z protein-specific antibodies were co-detected in 33% of the IIF-positive sera and in 5% of the IIF-negative sera (P = 0.001). These data provide evidence for appearance of antibodies to Z protein and NP following Lassa virus infection. A recombinant blot for detection of both antibody specificities seems to be specific but less sensitive than IIF.

[Percentage of tumor in prostatic biopsy cylinders as prognosis factor of organ-limited disease in candidates for radical prostatectomy]. / El porcentaje de tumor en cilindros de biopsia prostática como factor pronóstico de enfermedad órganoconfinada en pacientes candidatos a prostatectomía radical.

UNLABELLED: The present study analyzes the prognostic influence of tumor percentage in cylinders of prostatic biopsy in this patient group. MATERIAL AND METHODS: Retrospective study of 68 patients with a diagnosis of adenocarcinoma, clinical stage T1-T2 who had undergone a radical prostatectomy from May 1997 to october 2000. Following preoperative parameters were analyzed: age, PSA, clinical staging, Gleason and six cylinders of ultrasonography-leaded transrectal prostatic biopsy, studying the amount of positive biopsies, the tumor percentage of the total amount of biopsies and the maximum percentage of tumor in one cylinder. Univariate (square-Chi, Student t) and multivariate (multiple logistic regression) analysis are performed in order to study the relationship of these parameters with the presence or not of an organ-located disease. RESULTS: An organ-located disease was shown at 44 patients through the piece of radical prostatectomy. The univariate analysis gave all studied parameters, except age, a prognostic value of the existence or not of an organ-located disease. In the multivariate analysis only the total percentage of biopsy tumors (p = 0.0002) and PSA (p = 0.005) behaved as independent prognostic factors. CONCLUSION: Tumor percentage in prostatic biopsy seems to be a factor with a high predictive value of an organ-located disease, possibly because it is an index of tumoral volume.

Biomarkers and imaging in non-malignant and malignant osteomalacia.

Deoxypyridinium (DPD) cross-links are a specific parameter for collagen type I degradation. We report the longitudinal tracking of DPD in relation to other bone markers and imaging techniques in a patient with osteomalacia and secondary hyperparathyroidism from reduced light exposure due to attire. This patient was first admitted for diffuse skeletal pain. X-rays showed general demineralization and Looser's transformation zones in the neck of the left femur. MRI examinations of the pelvis and the proximal femora demonstrated bilateral signs of acute sacroiliitis, as well as edema-like lesions in the femoral heads and necks bilaterally. The baseline parathyroid hormone level was 8 times higher than the normal upper limit, whereas 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D levels were significantly reduced. A 7-fold increase in free urinary DPD and a 17-fold increase in bone-specific alkaline phosphatase (bone-AP) were also measured. Percutaneous transiliac bone biopsy revealed markedly increased osteoidosis. Osteomalacia was diagnosed due to chronically reduced sun exposure caused by restrictive attire, and cholecalciferol substitution therapy was begun. After a follow-up of 28 weeks, non-specific parameters of bone turnover (parathyroid hormone, total alkaline phosphatase, serum calcium and serum phosphate) had normalized, while DPD, as a specific bone degradation marker, and bone-AP, as a bone formation parameter, both remained elevated. This example underlines the validity of DPD and bone-AP as indicators of increased bone metabolism: not only were they the parameters with the highest baseline deviation, but they were also the last to normalize.

Nucleotide binding of an ADP analog to cooperating sites of chloroplast F1-ATPase (CF1).

Pre-steady state nucleotide binding to the chloroplast F1-ATPase (CF1) was measured in a stopped-flow apparatus by monitoring the change of fluorescence intensity of TNP-ADP upon binding. The analysis of the time courses led to the proposal of a mechanism of nucleotide binding with the following characteristics. (a) It involves three sites binding nucleotides in a concerted manner. (b) Each binding site is able to undergo a conformational change from a loose binding state into a state refraining from any direct release of the bound nucleotide into the medium. Only the reverse reaction via the loose binding state enables release out of the tight binding state. (c) Due to very strong negative cooperativity, a maximum of two of the three sites can be found in the state of tight binding. (d) Cooperativity between the three sites leads to a slower nucleotide binding of the second nucleotide compared to the first nucleotide. Furthermore, the conformational change from the loose binding state to the tight binding state is slowed down if one of the other sites already is in the tight binding state. Three-sites mechanisms in which rotation leads to an exchange of the properties of the binding sites failed to simulate the observed time courses of nucleotide binding. However, as the experimental set up was designed to prevent catalysis taking place, our results entirely agree with the current finding that rotation requires catalytic turnover of the enzyme.

A comparison of bone-related biomarkers and CA27.29 to assess response to treatment of osseous metastatic breast cancer.

BACKGROUND: The assessment of bone metastases by clinical examination or imaging techniques is still considered unreliable. We compared a specific marker of bone resorption, urinary deoxypyridinoline (DPD)-crosslinks, with serum calcium (Ca), alkaline phosphatase (AP) and CA27.29, to evaluate the status of bone metastases in patients with breast cancer. MATERIALS AND METHODS: Second morning voided urine was collected from 2 groups of patient (pts), those without evidence of disease (n = 118), and those with bone metastases (n = 85) under specific therapy plus pamidronate. DPD and CA27.29 were measured on the automated ACS180 system (Bayer Diagnostics, Tarrytown, NY, USA). Receiver operating characteristics (ROC) curves were established for each of the 4 biomarkers to determine whether they could distinguish the 2 subsets of pts with clinically sufficient validity, and to establish the corresponding cut-off values. RESULTS: Neither Ca nor AP was useful in discriminating the 2 subgroups. At a DPD cut-off of 13 nmol/mmol, we found a specificity of 69% and a sensitivity of 53% for diagnosing bone metastases. Best results, however, were seen for CA27.29. A cut-off value of 30 U/ml resulted in a specificity of 62% and a sensitivity of 81%. CONCLUSIONS: CA27.29 was the best parameter for the discrimination of stage IV breast cancer with bone metastases. The primary advantage of DPD lies in the monitoring of bone metastases under specific therapy.

Effect of interferon alpha on hepatitis B virus replication and gene expression in transiently transfected human hepatoma cells.

BACKGROUND/AIMS: Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFNalpha), which results in efficient reduction of the viral load only in 10-20% of treated patients. The mechanisms induced by IFNalpha resulting in reduction of viremia in responding patients are unknown. The aim of this study was to characterize HBV-specific IFNalpha-induced intracellular inhibitory mechanisms and IFNalpha-sensitive HBV targets. METHODS: To determine the antiviral activity, cells transiently transfected with HBV DNA were treated with IFNalpha and thereafter, viral products were quantified at different time points. RESULTS: Time-dependent reduction of RNA, replicative DNA-intermediates, core protein and secreted HBsAg/HBeAg levels was observed in IFNalpha-treated cells. Viral RNA levels were reduced most effectively early post-treatment whereas those of core protein and replicative intermediates decreased later. By expression of subgenomic HBV sequences, an RNA target region mediating IFNalpha-induced RNA degradation was mapped. CONCLUSIONS: These data indicate that HuH7 cells transiently transfected with HBV-DNA represent a system well suited for detailed analysis of IFNa-induced antiviral mechanisms and HBV targets. At least two IFNalpha-induced HBV-specific antiviral activities are active in this system: one reduces the levels of core protein and replicative intermediates, the other leads to posttranscriptional degradation of HBV-RNA. Based on the established in vitro system a detailed characterization of the IFNalpha-sensitive RNA-region and of factors mediating this intracellular antiviral effect is feasible. This may lead to the development of novel strategies for therapy of chronic hepatitis.

The course of the urinary DPD-crosslink secretion in metastatic breast cancer--a possibility for response assessment.

BACKGROUND: Urinary deoxypyridinoline (DPD)-crosslinks have been shown to be a highly specific parameter for type I collagen metabolism. MATERIALS AND METHODS: In a prospective breast cancer study, urine samples were collected in after-care patients and in patients with bone metastases. DPD-crosslinks were measured every three weeks using a fully automated chemiluminescence immunoassay. Bone metastases were confirmed by bone scan and/or x-ray, and were followed-up over six months. To validate the test, a receiver operating characteristics (ROC)-curve was set up to find the DPD cut-off concentration which separates patients with no evidence of disease (NED) from patients with bone metastases. RESULTS: 73 breast cancer patients (41 with NED, 32 with bone metastases) were included into the ROC analysis. At a DPD cut-off value of 8 nmol/mmol creatinine, we found the best sensitivity (84.4%) for the detection of bone metastases with a specificity of 70.7%. Patients with stable bone disease under intravenous pamidronate treatment (90 mg q3w) and specific therapy had a significant (p = 0.007) fall of the DPD-crosslinks in comparison to the progressive subset with 72.7% falling below 8 nmol/mmol. CONCLUSIONS: We conclude that the net bone turnover is not increased at a DPD-crosslinks elimination < 8 nmol/mmol.

Absence of mutations in the YMDD motif/B region of the hepatitis B virus polymerase in famciclovir therapy failure.

BACKGROUND/AIMS: Nucleoside analogues such as lamivudine and famciclovir are potent drugs for treatment of chronic hepatitis B virus infection. Breakthrough infections during lamivudine therapy are associated with mutations in the YMDD motif and putative B region of the HBV polymerase. This study investigated whether failure of famciclovir therapy is also associated with presence or emergence of particular mutations in the HBV polymerase. METHODS: We analyzed longitudinally the sequence of the priming and polymerase domain in seven patients with primary non-response to therapy and two patients with a breakthrough during therapy. Two patients who responded to therapy served as a control. RESULTS: The YMDD motif and the B region were conserved in all isolates. V-->I changes at position 555 just downstream of the YMDD motif were observed before and during therapy in a virus subpopulation of two patients with a primary non-response. In patients with a breakthrough, 378-V-->I and 424-N-->D mutations emerged in the N terminal part of the polymerase domain during follow-up. Lamivudine rescue therapy initiated in four patients, including a patient infected with YMDD(555-V-->I) variants, efficiently reduced viremia. CONCLUSIONS: These data indicate that failure of famciclovir therapy can occur independently of mutations in the YMDD motif or B region of the HBV polymerase and provide a rationale for rescue therapy with lamivudine.