NOX5-induced uncoupling of endothelial NO synthase is a causal mechanism and theragnostic target of an age-related hypertension endotype.

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5-but not NOX1, NOX2, or NOX4-with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed-upon aging-severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi.

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA3, GA4, and GA7) but also pure GA4 or GA7. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA3, GA4, and GA7 explaining the varying GA levels of genetically almost identical mutant strains.

A polyurethane-based surgical adhesive for sealing blood vessel anastomoses-A feasibility study in pigs.

Peri- and postoperative anastomotic leakage from blood vessel anastomosis is a common and potentially life-threatening complication. As an adjunctive therapy providing an additional layer of safety, a new biodegradable, polyurethane-based adhesive was developed. It consists of two components: an isocyanate-functionalized prepolymer and an amino-based curing agent. The adhesive was investigated in a porcine animal model to seal sutured blood vessel anastomoses of arteries, veins, aortas and prosthetic aortic graft replacements. The material-determined properties of the adhesive like viscosity, processing and polymerization time as well as bonding strength were well suited for this application. The adhesive stopped perioperative suture-line bleedings and stayed on all anastomoses until sacrifice. Hematological and serological inflammation marker assessments were unobtrusive. The histological evaluation showed a mild to moderate local tissue reaction to the adhesive constituting a physiological, non-adverse tissue-biomaterial interaction. The adhesive did not interfere with vascular wound healing. The adhesive demonstrated to be suitable to improve the outcome of cardiovascular surgeries by securing the classical sutured anastomoses in a fast, easy and safe manner. However, further studies are required to quantitatively evaluate efficacy in terms of anastomotic leakage prevention as well as long-term tissue compatibility and degradation.

Staging of rectal cancer by EUS: depth of infiltration in T3 cancers is important.

BACKGROUND: EUS is an established method for staging of rectal cancer. Nevertheless, there are few data about the significance of infiltration depth measured by EUS. OBJECTIVE: Assessment of accuracy of T and N staging by EUS with attention to infiltration depth as provided by EUS. DESIGN: Part retrospective, part prospective study. SETTING: Community and tertiary referral hospital, covering the period before neoadjuvant therapy for advanced rectal cancer was established. PATIENTS: Eighty-three patients (60% men) with untreated rectal cancer. INTERVENTION: EUS examination. MAIN OUTCOME MEASUREMENTS: We examined the correlation between EUS findings and postoperative histology. T3 cancers as diagnosed by EUS were classified into minimally invasive (1-2 mm) or advanced (>2 mm) tumors depending on the depth of infiltration beyond the muscularis propria. RESULTS: Accuracy of T staging and N status was 76% and 63%, respectively. Overstaging by EUS was more common in minimally invasive T3 by EUS (uT3) (8 of 16 [50%]) compared with advanced uT3 tumors (1 of 24 [4%]) (P=.01). Accuracy of EUS discrimination between T1/2 and T3/4 in rectal cancer for all but minimally invasive uT3 rectal tumors was 88%. LIMITATIONS: Partly retrospective analysis. CONCLUSIONS: EUS examination of rectal carcinoma determines T stage with high accuracy. Additionally, it provides information beyond T and N staging. The 50% probability of overstaging patients with minimally invasive uT3N0 by EUS may argue for managing these cancers as stage I disease, ie, to refer the patient for surgery without neoadjuvant therapy.

Myocarditis in newborn wild-type BALB/c mice infected with the murine gamma herpesvirus MHV-68.

OBJECTIVES: Animal models of human Epstein-Barr virus (EBV) infection include EBV infection of primates and infection of mice with MHV-68, a further gamma herpesvirus (gamma-HV). We aimed at extending the MHV-68 model to study gamma-HV-related cardiac disease. METHODS: Newborn wild-type BALB/c- (n=107), wild-type C57BL/6- (n=17) and immunodeficient B6-(Rag1) mice (n=18) were infected by nasal inoculation and evaluated for histopathological changes as well as tissue viral loads. RESULTS: From day 5 on BALB/c mice showed myocardial viral replication. Whereas focal inflammation occurred simultaneously, necrosis was first observed 9 days post-infection. The maximum rates of necrosis (40%) and of focal inflammation (33%) were found after 10 to 12 and 33 to 35 days, respectively. Some animals developed persistent viral activity and inflammation throughout the observation period of three months. Inflammation was mainly related to T cell infiltrates. Although C57BL/6 mice also showed myocardial inflammation, necrosis was not found suggesting differences in the susceptibility to the virus in distinct mouse strains. In immunodeficient animals higher myocardial viral loads were observed compared to wild-type mice but no cardiac lesions, which suggests that the antiviral immune response contributed to the lesions. CONCLUSIONS: The model system presented here is the first to allow detailed studies on cardiac disease caused by gamma-HV infections and may facilitate the development of more specific treatment options for human cardiac EBV infection.

Conserved arginine and aspartate residues are critical for function of MjNhaP1, a Na+/H+ antiporter of M. jannaschii.

Recently MjNhaP1 was identified as a pH-regulated Na(+)/H(+) antiporter of Methanococcus jannaschii [Hellmer, J. et al. (2002) FEBS Lett. 527, 245-249]. The antiporter is active at pH 6.0 and displays continuously decreasing activity towards alkaline pH. We have performed a site-directed mutagenesis study on all histidines as well as on conserved Asp, Glu and Arg residues of MjNhaP1, and analyzed the mutated proteins for activity. The mutants fall into three classes, i.e. normally active mutants, mutants with intermediate activity and mutants which are completely inactive. None of the histidine residues appears to be essential unlike in the bacterial proteins. The results point at an important role of a number of aspartate and arginine residues.

[Prevalence of circulating autoantibodies in healthy individuals]. / Prävalenz von zirkulierenden Autoantikörpern bei gesunden Individuen.

BACKGROUND AND AIM: Circulating autoantibodies are diagnostic markers for a variety of autoimmune diseases including rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic lupus erythematosus, and autoimmune hepatitis. Since only view studies exist on individuals without known diseases, we analyzed the prevalence of frequently determined autoantibodies in "healthy" individuals. PATIENTS AND METHODS: 111 individuals (43 female, 68 male; mean age 58 +/- 13 years, median 58, range 22-89), in whom either known or actual clinical evidence for autoimmune or internal disease was found, were included. Antinuclear and anti-smooth muscle antibodies (ANA and ASMA, respectively) were detected by immunofluorescence on rat organ sections and Hep 2 cells. Antibodies to liver-kidney-microsomes-1 (anti-LKM-1) and antimitochondrial antibodies (AMA) were detected and semiquantified by immunofluorescence. Additionally, anti-LKM-1 and AMA were determined by ELISA and Western blot. Antibodies against soluble liver antigens (anti-SLA) were quantified by ELISA. Sera with a titer of 1 : 40 or higher were classified as positive. RESULTS AND CONCLUSIONS: Sera of "healthy" adults displayed high frequencies of ANA and ASMA (28/111, 25%, and 48/111, 43%, respectively). Although no sex differences were found for ASMA, sera of healthy women tested more often positive for ANA (p < 0.01). Since at least one in three or four healthy individuals tested positive for ANA or ASMA, the positive predictive value of these autoantibodies is low, and clinical interpretation should include additional information.

Immunokinases, a novel class of immunotherapeutics for targeted cancer therapy.

Certain characteristics of tumor cells make it possible to develop rational strategies for targeting tumors without harming normal cells. These include the presence of cell surface molecules that characterize the current state of the tumor (e.g. CD30 on Hodgkin lymphoma cells) and the genetic and epigenetic changes that activate oncogenes and inactivate tumor suppressor genes (e.g. the inactivation of tumor suppressor gene DAPK2 in Hodgkin lymphoma cells, which blocks apoptosis). We have developed a novel tumor-targeting fusion protein by combining a selective ligand (CD30L) with a constitutively active version of DAPK2 (DAPK2'-CD30L), thus increasing tumor specificity and reducing systemic toxicity. We showed that this immunokinase fusion protein induces apoptosis specifically in CD30(+)/DAPK2(-) tumor cells in vitro and significantly prolonged overall survival in a disseminated Hodgkin lymphoma xenograft SCID mouse model. Therapeutic strategies based on the cell-specific restoration of a defective, tumor-suppressing kinase demonstrate the feasibility of targeted therapy using recombinant immunokinases.

Murine gammaherpesvirus-68 infection of mice: A new model for human cerebral Epstein-Barr virus infection.

Epstein-Barr virus infection may cause severe neurological complications that are not mirrored by animal models. Here, we show that nasal inoculation of newborn BALB/c wild-type mice with MHV-68, a murine gammaherpesvirus, causes cerebral infection with inflammation in 50% of the animals. The inflammatory patterns are strikingly similar to those known from Epstein-Barr virus, including hydrocephalus, meningitis, cerebellitis, focal or diffuse encephalitis, and temporal lobe encephalitis. This offers a new powerful tool to study the virological and immunological characteristics of cerebral gammaherpesvirus infections.

Interleukin-6 plays a crucial role in the hepatic expression of SOCS3 during acute inflammatory processes in vivo.

BACKGROUND/AIMS: Interleukin-6 is mandatory for liver regeneration after injury and for the hepatic expression of acute phase proteins and cytochrome P450 enzymes during inflammation. Due to its crucial contribution to the maintenance of homeostasis IL-6 signaling is tightly controlled. Suppressor of cytokine signaling (SOCS) 3 is a potent IL-6-induced feedback inhibitor terminating IL-6 signal transduction. However, several signaling pathways converge on SOCS3: SOCS3 can be induced by other mediators in vitro, and it does not exclusively inhibit IL-6 signaling. The individual contribution of each cytokine to the induction of SOCS3 in vivo is unknown. METHODS: Using IL-6-deficient mice we analyzed the role of interleukin-6 for the hepatic SOCS3 expression in response to turpentine and LPS as models of aseptic and bacterial inflammation, respectively. RESULTS: In wild-type animals, turpentine and LPS elicited strong induction of SOCS3. IL-6-deficient mice, by contrast, showed severely impaired SOCS3 expression in response to both stimuli: turpentine failed to induce SOCS3 mRNA; in LPS-induced inflammation, the early inductive response 60min after LPS injection was absent, and the delayed expression of SOCS3 was markedly reduced. The residual delayed SOCS3 expression in IL-6-deficient mice was abolished in IL-6/TNFR-1 knockout mice. CONCLUSIONS: Our data strongly argue for a crucial role of IL-6 in the hepatic expression of SOCS3 during acute inflammatory processes in vivo. Although other cytokines are capable of inducing SOCS3 their contribution seems to be minor.