Effects of renal denervation on renal pelvic contractions and connexin expression in rats.

AIMS: The renal pelvis shows spontaneous rhythmic contractile activity. We assessed to what extent this activity depends on renal innervation and studied the role of connexins in pelvic contractions. METHODS: Rats underwent unilateral renal denervation or renal transplantation. Renal pelvic pressure and diuresis were measured in vivo. Spontaneous and agonist-induced contractions of isolated renal pelves were investigated by wire myography. Rat and human renal pelvic connexin mRNA abundances and connexin localization were studied by real-time PCR and immunofluorescence respectively. RESULTS: Renal denervation or transplantation increased renal pelvic pressure in vivo by about 60 and 150%, respectively, but did not significantly affect pelvic contraction frequency. Under in vitro conditions, isolated pelvic preparations from innervated or denervated kidneys showed spontaneous contractions. Pelves from denervated kidneys showed about 50% higher contraction frequencies than pelves from innervated kidneys, whereas contraction force was similar in pelves from denervated and innervated kidneys. There was no denervation-induced supersensitivity to noradrenaline or endothelin-1. Renal denervation did not increase pelvic connexin37, 40, 43 or 45 mRNA abundances. Gap junction blockade had no effect on spontaneous pelvic contractile activity. CONCLUSIONS: The denervation-induced effect on pelvic pressure may be the consequence of the enhanced diuresis. The mechanisms underlying the denervation-induced effects on pelvic contraction frequency remain unknown. Our data rule out a major role for two important candidates, by showing that renal denervation neither induced supersensitivity to contractile agonists nor increased connexin mRNA abundance in the pelvic wall.

Antigen-specific expansion of human regulatory T cells as a major tolerance mechanism against mucosal fungi.

Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.

Azole-resistant invasive aspergillosis in a patient with acute myeloid leukaemia in Germany.

We report the first culture-proven case of invasive aspergillosis (IA) caused by azole-resistant Aspergillus fumigatus in a patient with acute myeloid leukaemia in Germany. IA presented as breakthrough infection under posaconazole prophylaxis. Analysis of the resistance mechanism revealed the TR/L98H mutation in the cyp51A gene, which indicates an environmental origin of the strain. This case underscores the need for monitoring azole resistance in Aspergillus spp. and for routine susceptibility testing of moulds.

Current issues in the clinical management of invasive candida infections--the AGIHO, DMykG, ÖGMM and PEG web-based survey and expert consensus conference 2009.

The objectives of this study were to identify unsolved issues in the management of invasive candidiasis, identify controversies and achieve consensus. The German Speaking Mycological Society (Deutschsprachige Mykologische Gesellschaft, DMykG e.V.) asked other German infectious diseases (ID) and mycological societies to submit unsolved issues concerning the diagnosis and treatment of fungal infections. Based on these contributions, a digital web-based questionnaire of 12 questions on Candida infections was designed to be completed by experts of the participating societies. Controversial results were identified by a mathematical model and were discussed at a consensus conference during the 43rd Annual Meeting of the DMykG e.V. in Cologne, Germany. Forty-two individuals completed the questionnaire. Analysis showed a strong consensus on treatment indications, choice of antifungals for clinical situations, handling of central venous catheters, duration of treatment and role of susceptibility testing. Opinions diverged on: initial treatment of haemodynamically stable neutropenic and haemodynamically unstable non-neutropenic patients, step down to oral treatment and the differential role of the echinocandins. These questions were presented for discussion at the expert consensus conference. In three of four questions, consensus was achieved. A two-step approach - web-based survey plus classical panel discussion - allows to capture expeditiously the opinions of a large and diverse group of individuals, to identify controversial issues and to resolve them in a personal, interactive setting. Thus, expert consensus was achieved on nine of 12 important questions on how to treat invasive candidiasis.

Clinical effectiveness of posaconazole prophylaxis in patients with acute myelogenous leukaemia (AML): a 6 year experience of the Cologne AML cohort.

BACKGROUND: Large randomized controlled trials have shown significant decreases in morbidity and mortality in leukaemia patients with posaconazole prophylaxis. However, the value of prophylaxis has been questioned in centres with a low incidence of invasive fungal diseases (IFDs) and pre-emptive treatment strategies. METHODS: We prospectively evaluated the epidemiology of IFDs in acute myelogenous leukaemia (AML) patients undergoing first remission-induction chemotherapy before and after posaconazole prophylaxis had been introduced as a standard of care. Patients admitted from January 2003 to December 2005 received topical polyenes as antifungal prophylaxis (first group), while those admitted between January 2006 and December 2008 received 200 mg of oral posaconazole three times daily (second group). Other diagnostic and therapeutic standard operating procedures remained unchanged. RESULTS: A total of 82 patients in the polyene prophylaxis group and 77 in the posaconazole prophylaxis group were included in the final analysis. Baseline characteristics were well matched between groups. Patients receiving topical polyene prophylaxis were more likely to experience breakthrough IFDs (19.5% and 3.9%; P = 0.003) or breakthrough aspergillosis (13.4% and 2.6%; P = 0.018) than patients receiving systemic posaconazole prophylaxis. They also had more febrile days (mean 10.7 +/- 9.66 and 7.3 +/- 5.73; P = 0.007), longer need for inpatient treatment (mean 53.0 +/- 24.16 and 46.0 +/- 14.39; P = 0.026) and a shorter fungal-free survival (78.7 and 90.4 days; P = 0.024). No significant differences were observed for persistent fever, pneumonia, lung infiltrates indicative of invasive pulmonary aspergillosis, or attributable and overall mortality. CONCLUSIONS: After introduction of posaconazole prophylaxis for patients with AML, the number of febrile days, the incidence rate of IFDs and aspergillosis and the duration of hospitalization decreased significantly.

Low NaCl intake elevates renal medullary endothelin-1 and endothelin A (ETA) receptor mRNA but not the sensitivity of renal Na+ excretion to ETA receptor blockade in rats.

AIMS: This study was performed to investigate the effects of NaCl intake on renal mRNA expression of pre-pro-endothelin-1 (ET-1), endothelin A (ET(A)) and endothelin B (ET(B)) receptors as well as on renal ET-1 content in rats. We further tested for NaCl intake-dependent differences in the contribution of the ET system to renal sodium handling. METHODS: Male Sprague-Dawley rats with telemetric devices were randomized to 0.15%, 0.60% and 1.80% NaCl diets with or without losartan. Renal sodium balance and arterial pressure were monitored. Renal blood flow and fractional sodium excretion (FENa) were measured in response to acute infusion of ET(A) and ET(B) blockers into the inner stripe of the outer renal medulla. RESULTS: Medullary pre-pro-ET-1, ET(A) and ET(B) receptor mRNA was 50%, 81% and 33% higher in rats on 0.15% vs. 1.80% NaCl. Losartan reduced medullary gene expression in rats on 0.15% NaCl. Medullary ET-1 content was 983 +/- 88 and 479 +/- 42 ng mg(-1) protein in rats on 0.15% and 1.80% NaCl (P < 0.001). Chronic ET(A) receptor blocker treatment reduced arterial pressure by 8-10 mmHg in rats on 0.15% vs. 1.80% NaCl without affecting renal sodium balances. Acute medullary ET(A) or ET(B) receptor blockade did not alter medullary blood flow and FENa in animals on either diet. CONCLUSION: In rats renal medullary ET-1 content and mRNA expression of three ET system components are inversely related to NaCl intake. Higher expression levels on low NaCl intake are AT(1) receptor dependent but are not associated with increased sensitivity of renal sodium handling to ET(A) receptor blockade.

Towards a medically approved technology for alginate-based microcapsules allowing long-term immunoisolated transplantation.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules.

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Surviving high-intensity field pulses: strategies for improving robustness and performance of electrotransfection and electrofusion.

Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.

Assessing microbial degradation of o-xylene at field-scale from the reduction in mass flow rate combined with compound-specific isotope analyses.

In recent years, natural attenuation (NA) has evolved into a possible remediation alternative, especially in the case of BTEX spills. In order to be approved by the regulators, biodegradation needs to be demonstrated which requires efficient site investigation and monitoring tools. Three methods--the Integral Groundwater Investigation method, the compound-specific isotope analysis (CSIA) and a newly developed combination of both--were used in this work to quantify at field scale the biodegradation of o-xylene at a former gasworks site which is heavily contaminated with BTEX and PAHs. First, the Integral Groundwater Investigation method [Schwarz, R., Ptak, T., Holder, T., Teutsch, G., 1998. Groundwater risk assessment at contaminated sites: a new investigation approach. In: Herbert, M. and Kovar, K. (Editors), GQ'98 Groundwater Quality: Remediation and Protection. IAHS Publication 250, pp. 68-71; COH 4 (2000) 170] was applied, which allows the determination of mass flow rates of o-xylene by integral pumping tests. Concentration time series obtained during pumping at two wells were used to calculate inversely contaminant mass flow rates at the two control planes that are defined by the diameter of the maximum isochrone. A reactive transport model was used within a Monte Carlo approach to identify biodegradation as the dominant process for reduction in the contaminant mass flow rate between the two consecutive control planes. Secondly, compound-specific carbon isotope analyses of o-xylene were performed on the basis of point-scale samples from the same two wells. The Rayleigh equation was used to quantify the degree of biodegradation that occurred between the wells. Thirdly, a combination of the Integral Groundwater Investigation method and the compound-specific isotope analysis was developed and applied. It comprises isotope measurements during the integral pumping tests and the evaluation of delta13C time series by an inversion algorithm to obtain spatially integrated mean isotope values at the control planes. It was shown that the Rayleigh equation is applicable to spatially integrated mean isotope values in order to obtain the mean biodegradation between the consecutive control planes. All three approaches yielded consistently a 98-99% degradation of o-xylene.

Direct measurement of the Josephson supercurrent in an ultrasmall Josephson junction.

We have measured the supercurrent flowing through a nonhysteretic, ultrasmall, voltage-biased Josephson junction. In contrast with experiments performed so far on hysteretic Josephson junctions, we find a supercurrent peak whose maximum I(s max) increases as the temperature T decreases. The asymptotic T = 0 value of I(s max) agrees with the junction Ambegaokar-Baratoff critical current, as predicted by theory.

Basic investigations on zeolite application for electrochemical analysis.

Basic investigations on the electrochemical behavior of alumosilicate zeolites were conduced in gas atmosphere and aqueous solution using impedance spectroscopy under in situ conditions where "zeolitically" bound water molecules are present in the channel and cage system. Natural stilbite (STI) and heulandite (HEU), zeolites of structure types with the same 4 - 4 = 1 building units, were used for these investigations. At a given temperature, well defined water partial pressures were applied in the gas atmosphere (in situ conditions) and the effect of polar organic molecules on conductivity was investigated. Furthermore, the effect of complete water saturation of the channel system on the Arrhenius type activation energy of conductivity was investigated in aqueous solution in comparison. The ion-exchange behavior of zeolite materials could be monitored under these conditions. These results show that, dependent on the different parameters affecting the zeolite conductivity behavior, they are applicable in the field of electrochemical analysis if these basic results on single crystals are transferable to polycrystalline materials.

The role of staging bronchoscopy in the preoperative assessment of a solitary pulmonary nodule.

In order to assess the role of a staging fiberoptic bronchoscopy in the preoperative assessment of an indeterminate solitary pulmonary nodule (SPN), we reviewed our experience in 33 SPNs identified among 1,269 bronchoscopies performed at the Albert Einstein Medical Center between 1985 and 1989. All lesions were less than 4 cm in greatest diameter and were not associated with symptoms of weight loss, chest pain, hemoptysis, localized wheezing, or hoarseness. A tissue diagnosis was established in 25 patients, 23 of whom had a malignant SPN. This study failed to detect a single case in which a fiberoptic bronchoscopic examination of the airway discovered a lesion that would preclude surgery and potentially curative resection. We recommend the abandonment of a staging bronchoscopy in the evaluation of a patient with an indeterminant SPN in whom history, physical examination, laboratory, and imaging studies fail to document contraindications to surgery. No additional useful information is derived and a substantial cost savings to the patient can be realized if the procedure is eliminated.

Effect of hyperbaric oxygen exposure on oxygen tension within the medullary canal in the rabbit tibial osteomyelitis model.

The effect on intramedullary oxygen tension of 100% oxygen exposure at 1, 2, 2.4, and 3 atm pressure was studied in 12 New Zealand white rabbits with chronic right tibial osteomyelitis. The model, modified from that described by others, incorporates a multipuncture silastic closure plug placed transcortically in the proximal tibial metaphysis through which platinum needle, polarographic electrode oxygen tension determinations can be made without repeat surgical exposure. In 40% of the control, left, noninfected tibial metaphyses the baseline oxygen tension with the animals breathing room air at sea level was suboptimal for leukocyte bacterial killing. This oxygen tension was depressed further in the infected right tibia. Medullary canal oxygen tension increased in response to hyperbaric oxygen exposure in both the infected and noninfected tibiae. Whereas the amount of the oxygen tension increase varied with the presence of infection and depth of dive, neither the time for oxygen tension to plateau nor the time required for return to baseline tension after completion of hyperbaric oxygen exposure varied with the presence of infection or depth of dive. After completion of hyperbaric oxygen exposure, the oxygen tension within the medullary canal returned to baseline within 15 min.

Neutron microscopy. The low-damage imaging of specialized organic materials.

It is shown that, insofar as radiation damage is concerned, transmission neutron microscopy using neutrons in the energy range approximately 0.0001-1.0 eV is extremely attractive for the imaging of specialized organic materials. By "specialized organic materials" is meant organic specimens composed entirely of specific isotopes that have been selected on the basis of their favorable properties with regard to radiation damage. In connection with such specimens, it is demonstrated that at a resolution of, for example, 100 A, neutrons will have an advantage over soft X-rays in terms of radiation damage, provided that the inherent (neutron) bright field image contrast turns out to be greater than 10(-5). Suggestions relating to (a) the comprehensive calculation of the radiation damage sustained by specialized organic specimens under slow neutron irradiation, (b) the construction of a theory of image formation in the neutron microscope, (c) the development of neutron lenses/focusing devices, and (d) the development of a brighter neutron source (essential for neutron microscopy) are outlined in some detail. The paper concludes with two appendices, which provide important background material.

Neurolinguistic programming: a systematic approach to change.

Neurolinguistic programming (NLP) integrates advances in cybernetics, psychophysiology, linguistics, and information services. It has been used in business, education, law, medicine and psychotherapy to alter people's responses to stimuli, so they are better able to regulate their environment and themselves. There are five steps to an effective NLP interaction. They include 1. establishing rapport; the therapist must match his verbal and non-verbal behaviors to the patient's, 2. gathering information about the patient's present problem and goals by noting his verbal patterns and non-verbal responses, 3. considering the impact that achieving the patient's goals will have on him, his work, family and friends, and retaining any positive aspects of his current situation, 4. helping the patient achieve his goals by using specific techniques to alter his responses to various stimuli, and 5. ensuring the altered responses achieved in therapy are integrated into the patient's daily life. NLP has been used to help patients with medical problems ranging from purely psychological to complex organic ones.