Miniaturized modules for light sheet microscopy with low chromatic aberration.

Two miniaturized fibre-coupled modules for light sheet-based microscopy are described and compared with respect to image quality, chromatic aberration and beam alignment. Whereas in one module the light sheet is created by an achromatic cylindrical lens, reflection by a spherical mirror and concomitant astigmatic distortion are used to create the light sheet in the second module. Test experiments with fluorescent dyes in solution and multicellular tumour spheroids are reported, and some details on construction are given for both systems. Both modules are optimized for imaging individual cell layers of 3D biological samples and can be adapted to fit commercial microscopes.

Chamber bioaerosol study: human emissions of size-resolved fluorescent biological aerosol particles.

Humans are a prominent source of airborne biological particles in occupied indoor spaces, but few studies have quantified human bioaerosol emissions. The chamber investigation reported here employs a fluorescence-based technique to evaluate bioaerosols with high temporal and particle size resolution. In a 75-m(3) chamber, occupant emission rates of coarse (2.5-10 µm) fluorescent biological aerosol particles (FBAPs) under seated, simulated office-work conditions averaged 0.9 ± 0.3 million particles per person-h. Walking was associated with a 5-6× increase in the emission rate. During both walking and sitting, 60-70% or more of emissions originated from the floor. The increase in emissions during walking (vs. while sitting) was mainly attributable to release of particles from the floor; the associated increased vigor of upper body movements also contributed. Clothing, or its frictional interaction with human skin, was demonstrated to be a source of coarse particles, and especially of the highly fluorescent fraction. Emission rates of FBAPs previously reported for lecture classes were well bounded by the experimental results obtained in this chamber study. In both settings, the size distribution of occupant FBAP emissions had a dominant mode in the 3-5 µm diameter range.

Motivation of patients with inflammatory bowel disease to participate in a clinical trial.

Background: Clinical trials are designed to investigate innovative diagnostic and therapeutic strategies for patients. However, factors that influence patients with inflammatory bowel disease (IBD) and willingness to participate in a clinical trial are unknown. Methods: We developed a questionnaire and asked IBD patients about their willingness to hypothetically participate in a clinical trial and their current health-related quality of life by using the IBDQ. Results: Of 201 distributed questionnaires, 166 were returned and included in the analysis. One-hundred-one (61 %) patients declared their willingness to participate in a clinical trial hypothetically offered in their current situation, whereas 65 (39 %) declined. Among all patients, a trustful relationship between patient and doctor was most important for trial participation. The willingness to help others and to support medical progress were other key issues mentioned. In contrast, those patients inclined to refuse trial participation feared impairment of their current health status, potential side effects, medical examinations, and the expenditure of time and effort. Conclusion: In our cohort of IBD patients, approximately two-thirds were willing to participate in a clinical trial. We were able to identify a number of factors that should help physicians to directly address fears and break down barriers in order to increase the number of patients willing to participate in clinical trials.

Sample holder for axial rotation of specimens in 3D microscopy.

In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results.

Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma-Pinus symbiosis: improved methodologies for assessing gene expression in situ.

Ectomycorrhizal (EM) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next-generation sequencing show great promise for studying EM symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of RNA from mycorrhizae. Here we apply an optimized method for combined DNA/RNA extraction using field-collected EM fungal-pine root clusters, together with protocols for taxonomic identification of expressed ribosomal RNA, and inference of EM function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal RNA genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine including Amphinema, Russula and Piloderma spp. One taxon, Piloderma croceum, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressed Piloderma genes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast, Piloderma gene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field-collected samples.

[Spontaneous and secondary bacterial peritonitis in cirrhotic patients with ascites]. / Spontan-bakterielle und sekundäre Peritonitis bei Patienten mit Leberzirrhose und Aszites.

Peritonitis is one of the most frequent infectious complications in patients with liver cirrhosis and ascites. In more than 95 % it occurs as spontaneous bacterial peritonitis (SBP) as a result of bacterial translocation from intestinal bacteria and bacterial products into mesenteric lymph nodes and subsequent systemic circulation. Identified risk factors that justify antibiotic prophylaxis for SBP include a prior episode of SBP, gastrointestinal haemorrhage and low ascitic fluid protein in combination with renal or advanced liver failure. SBP requires conservative therapy, which should be empirically performed using third-generation cephalosporins and adjunctive albumin therapy under consideration of individual and nosocomial risk factors for antimicrobial resistance. In contrast to SBP, secondary bacterial peritonitis is a rare ( < 5 % of all cases of peritonitis) and more unfavourable disease. It occurs as a result of hollow organ perforation or intra-abdominal inflammatory or ischaemic processes. Analysis of ascitic fluid may help substantiating the suspicion of secondary peritonitis and should entail a meticulous diagnostic work-up including abdominal computed tomography. Because of the high mortality rate (60 - 80 %) of secondary peritonitis antibiotic regimens with anti-anaerobic activity, prompt surgical treatment and interdisciplinary postoperative care are necessary to improve patient outcomes.

Soluble urokinase plasminogen activator receptor is compartmentally regulated in decompensated cirrhosis and indicates immune activation and short-term mortality.

OBJECTIVE: Patients with decompensated cirrhosis are susceptible to bacterial infections, which are associated with organ failure and a high mortality rate. Reliable biomarkers are needed to identify patients who require intensified treatment. Our objective was to study the regulation and prognostic relevance of elevated concentrations of soluble urokinase plasminogen activator receptor (suPAR) in patients with advanced cirrhosis. DESIGN, SETTING AND PARTICIPANTS: We examined the associations between serum and ascitic fluid (AF) suPAR and liver function, bacterial infection, and short-term mortality in 162 consecutive patients with decompensated cirrhosis undergoing diagnostic paracentesis in a tertiary health care centre in Germany. MAIN OUTCOME MEASURE: Twenty-eight-day mortality. RESULTS: Circulating suPAR levels were increased in patients with decompensated cirrhosis and correlated with the severity of liver dysfunction and systemic inflammation but were not indicative of bacterial infection. Circulating suPAR levels >14.4 ng mL(-1) predicted 28-day mortality, even after adjustment for liver function and confounders [HR = 3.05 (1.35-6.90); P = 0.0076] equal to the MELD score (AUC = 0.71; 95% CI = 0.61-0.81; P < 0.001). Cut-off levels derived from cohorts without liver disease were not applicable due to the low specificity. AF suPAR levels were elevated during spontaneous bacterial peritonitis (SBP), but not during episodes in which bacteria or bacterial DNA was translocated into the ascites. AF suPAR levels correlated poorly with systemic suPAR but were associated with a more severe course of SBP and a worse outcome. In vitro experiments revealed that monocytes, and to a lesser extent neutrophils, secrete suPAR after Toll-like-receptor ligation, which led to rapid urokinase plasminogen activator receptor cleavage followed by increased synthesis. CONCLUSION: Blood and ascitic suPAR levels provide distinct, but relevant prognostic information on the severity of complications in patients with end-stage liver disease.

[Empirical antibiotic therapy in intra-abdomial infections: cases and evidence-based therapeutic recommendations]. / Kalkulierte Antibiotikatherapie bei intraabdominellen Infektionen - Fallbeispiele und evidenzbasierte Therapieempfehlungen.

Intra-abdominal infections (IAI) are a common problem in visceral medicine. In Germany more than 150 000 patients are treated each year for IAI with courses ranging from uncomplicated disease to severe life-threatening manifestations. IAI represent the second most common cause of septic shock and the second most common cause of infection-related mortality in intensive care. Due to increasing antimicrobial resistance, changes in pathogen spectra and increasing patient co-morbidities, recommendations for empirical antibiotic therapy have to be continuously updated: Whereas inadequate empirical treatment is associated with poor prognosis, unselected broad-spectrum therapy may increase antimicrobial resistances. Illustrated by clinical cases of typical intra-abdominal infections, this article reviews recommendations for antibiotic therapy based on national and international guidelines under consideration of local resistance rates and patient-specific factors to provide a basis for improved therapy of this common problem.

Light exposure and cell viability in fluorescence microscopy.

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm⁻² and about 300 J cm⁻² for native cells (corresponding to about 250-3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi- or total internal reflection illumination) and decreased to values between 50 J cm⁻² and less than 1 J cm⁻² upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high-resolution wide field or laser scanning microscopy of single cells, typically 10-20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super-resolution microscopy based on single molecule detection.

Calcineurin inhibitors or anti-TNF-α agents in severe ulcerative colitis: available options and limitations.

A majority of patients with ulcerative colitis (UC) respond to medical therapy and remain in remission. However, approximately 20 % of UC patients experience severe relapses that require hospitalization. For these cases, intravenous corticosteroids are the established first-line treatment and 70 % of patients respond. Patients who do not respond to corticosteroids within 48 to 72 hours should receive calcineurin inhibitors, anti-TNF-α therapy or total proctocolectomy, which must be selected based on clinical, endoscopic and laboratory parameters and patient preference. The selection of second-line medical therapy after steroid failure, surgery and the benefits of medical salvage therapy following second-line therapy failure represent current challenges in the treatment of acute steroid-refractory colitis. Patients and physicians must realize that immunosuppressive therapies, especially in combination, bear the risk of serious and fatal infections. Therefore, decisions regarding short- and long-term treatment strategies must consider the efficacy and potential toxicity of pharmacological interventions and the quality of life after restorative proctocolectomy with ileal pouch-anal anastomosis.

The augmented neutrophil respiratory burst in response to Escherichia coli is reduced in liver cirrhosis during infection.

Several functional abnormalities in phagocytes from patients with liver cirrhosis contribute to an increased risk of infection. An increased resting respiratory burst has been observed in neutrophils from cirrhotic patients. We investigated whether an infection in cirrhosis affects the respiratory burst capacity of neutrophils and monocytes in response to Escherichia coli. This study included 45 hospitalized patients with liver cirrhosis and clinical signs of infection, 39 patients with liver cirrhosis in the absence of infection and 29 healthy subjects. Respiratory burst, lipopolysaccharide-binding protein (LBP), and immunoglobulin (Ig)G-autoantibodies against oxidized low-density lipoproteins (ab-oxLDL) were measured. The fraction of neutrophils spontaneously producing reactive oxygen species (ROS) was elevated in liver cirrhosis (P < 0·01). The neutrophil resting burst increased with Child-Pugh stage (P = 0·02) and correlated with augmented ROS release in response to opsonized E. coli (P < 0·05). Although LBP was increased in patients with cirrhosis (P < 0·01), higher LBP levels correlated with a lower resting burst in neutrophils (r(s) = -0·395; P < 0·01). In the presence of infection, the resting burst was unaltered. However, neutrophil ROS release in response to E. coli was reduced markedly (P = 0·01), and it decreased as serum C-reactive protein (CRP) concentration rose (r(s) = -0·437; P < 0·01), indicating the development of a sepsis-like immune paralysis. A positive correlation between ab-oxLDL and ROS release was observed (P < 0·01). In conclusion, the respiratory burst increases with severity of liver cirrhosis but is restrained by increasing LBP levels. Augmented ROS release in response to E. coli is accompanied by elevated markers of oxidative damage and becomes exhausted in the presence of infection.

Subacute liver failure induced by adalimumab.

Most cases of liver toxicity associated with TNF-antagonists have been linked to infliximab and to a lesser extent to etanercept. So far only mild elevations of liver enzymes during therapy with adalimumab have been reported. In general, patients who developed ALT and AST elevations were asymptomatic and the abnormalities decreased or resolved with either continuation or discontinuation of adalimumab, or modification of concomitant medications. In this case report, we are presenting the first case of a patient without previous history of liver disease or concomitant risk factors for liver disease who developed subacute liver failure during therapy with adalimumab for psoriatic arthritis.

Abnormal glucose tolerance: a predictor of 30-day mortality in patients with decompensated liver cirrhosis.

BACKGROUND: In liver cirrhosis an abnormal glucose tolerance correlates with a poor long-term prognosis. The aim of this study was to evaluate whether a disturbed glucose metabolism predicts the short-term prognosis in hospitalized patients with decompensated liver cirrhosis. METHODS: Seventy-eight patients with liver cirrhosis were prospectively enrolled. Disturbed glucose metabolism was determined by a 75-g oral glucose tolerance test (OGTT) according to WHO criteria. Differences in survival were assessed by χ (2) test for 30-day mortality and by multivariate Cox proportional hazards analysis for long-term survival. RESULTS: Impaired glucose tolerance (IGT) was diagnosed in 29 patients (37 %) and diabetes mellitus (DM) in 26 patients (33 %). A pathological OGTT result was a significant negative predictor of a poor 30-day survival compared to NGT (OR 8.6; p=0.03). DM but not IGT was an independent negative predictor of long-term survival (HR=1.89; p=0.04). CONCLUSION: Disturbed glucose metabolism in hospitalized patients with decompensated liver cirrhosis is correlated with increased 30-day mortality. OGTT appears to be useful for identifying cirrhotic patients with poor short term prognosis.

[54-year-old male with hepatic cirrhosis and therapy-associated torsade de pointes tachycardia]. / 54-jähriger Patient mit Leberzirrhose und therapiebedingten Torsade-de-pointes-Tachykardien.

We present a case of a patient with newly diagnosed cirrhosis and kidney failure who underwent cardiopulmonary resuscitation twice after treatment with terlipressin. After the second application a QT interval prolongation and torsade de pointes were documented. Since other causes were excluded, we interpret both events as terlipressin-induced ventricular arrhythmia. Therefore, it seems important to consider this rare but potentially lethal side effect when administering this drug to patients.

Clinical characteristics and outcome of patients with enterococcal liver abscess.

Epidemiology of bacteria isolated from pyogenic liver abscesses change, and an increase in enterococci has been reported in European hospitals. The aim of this study was to investigate the clinical characteristics and outcome of enterococcal PLA. We performed a retrospective analysis of patients with microbiologically confirmed PLA at three German university centers. Indicators of enterococcal PLA were determined using binary logistic regression, and survival analysis was performed using Kaplan-Meier statistics and Cox regression analysis. Enterococci were isolated in 51/133 (38%) patients with PLA. Patients with enterococcal PLA had smaller abscess diameter (4.8 vs. 6.7 cm, p = 0.03) than patients with non-enterococcal PLA, but had more frequent polymicrobial culture results. In univariate logistic regression analysis, alcohol abuse (OR 3.94, 95% CI 1.24-12.49, p = 0.02), hepatobiliary malignancies (OR 3.90, 95% CI 1.86-8.18, p < 0.001) and cirrhosis (OR 6.36, 95% CI 1.27-31.96, p = 0.02) were associated with enterococcal PLA. Patients with enterococcal PLA had a higher mortality than patients with non-enterococcal PLA (hazard ratio 2.92; 95% confidence interval 1.09-7.80; p = 0.03), which remained elevated even after excluding patients with hepatobiliary malignancies, cirrhosis, and transplant recipients in a sensitivity analysis. The increased mortality was associated with non-fecal enterococci but not in patients with Enterococcus faecalis. In this retrospective, multicenter study, enterococcal PLA was common and indicated an increased risk of mortality, underscoring the need for close clinical monitoring and appropriate treatment protocols in these patients.

[Benign prostatic hyperplasia : New and treatment-relevant aspects from the DGU guidelines]. / Benignes Prostatasyndrom : Neues und Praxisrelevantes aus den DGU-Leitlinien.

The diagnosis and treatment of lower urinary tract symptoms (LUTS) due to benign prostatic enlargement plays an important role in daily urological practice. Therefore, a targeted and resource-saving approach is essential. A rational base-line work-up of our patients provides the necessary information for obtaining the diagnosis and only needs to be expanded in individual cases. In addition to drug therapy, the modification of lifestyle and the possibility of watchful waiting must not be underestimated. Simple measures such as a timed fluid intake, double micturition in the case of residual urine development, but also bladder reconditioning can significantly improve the quality of life of our patients. Regarding surgical treatment, laser procedures have found their way into many departments and have established themselves in daily routine as a reference procedure in addition to transurethral resection of the prostate (TUR-P) and simple open prostatectomy. New, minimally invasive procedures-such as prostatic artery embolization (PAE), the Rezum™- (NxThera Inc., Maple-Grove, MN, USA) or the Aquabeam® (Procept, Redwood City, CA, USA) procedure, but also nonablative procedures such as iTind© (TIND, Medi-Tate, Or Akiva, Israel) or Urolift® (Neotract Inc., Pleasanton, CA, USA)-offer new treatment options to those affected, with the potential to maintain patient's sexual function. As a result, individual risk assessment and advice on the advantages and disadvantages of all available treatment options-even more than today-will be an important part of LUTS treatment. An individual approach, similar to that used in the treatment of oncological disease, will become standard also in the treatment of benign prostatic syndrome.

"Nearly a stairway to heaven"--severe dichromate intoxication in a young man.

A 22-year-old man presented with dichromate intoxication in a suicidal attempt. He exhibited signs of liver and renal toxicity and very high serum chromium levels. Since it has been reported in the literature that hemodialysis and hemoperfusion are not sufficient to remove chromium, we tried plasmapheresis considering the fact that chromium salts bind to protein. Five plasmapheresis treatment sessions significantly lowered his serum and urinary chromium concentrations. The patient survived without organ damage despite ingestion of a lethal dichromate dose and high serum chromium concentration. Thus, plasmapheresis should be considered as a potential therapeutic option to reduce chromium concentrations.

Serpentine and non-serpentine ecotypes of Collinsia sparsiflora associate with distinct arbuscular mycorrhizal fungal assemblages.

Although plant adaptation to serpentine soils has been studied for several decades, the mechanisms of plant adaptation to edaphic extremes are still poorly understood. Arbuscular mycorrhizal fungi (AMF) are common root symbionts that can increase the plant hosts' establishment and growth in stressful environments. However, little is known about the role plant-AMF interactions play in plant adaptation to serpentine. As a first step towards understanding this role, we examined the AMF assemblages associated with field populations of serpentine and non-serpentine ecotypes of California native plant Collinsia sparsiflora. We sampled roots of C. sparsiflora from three serpentine and three non-serpentine sites in close proximity (110 m to 1.94 km between sites) and analysed the small subunit ribosomal DNA gene amplified from root DNA extracts using AMF-specific primers. A total of 1952 clones from 24 root samples (four from each site) were sequenced. We used sequence similarity and phylogenetic analysis to determine operational taxonomic units (OTU) resulting in 19 OTUs representing taxa from six AMF genera, including one serpentine-specific OTU. We used Bray-Curtis similarity, multidimensional scaling and analysis of similarity to compare root sample AMF assemblages. These analyses clearly showed that plant ecotypes associated with distinct AMF assemblages; an Acaulospora OTU-dominated serpentine, and a Glomus OTU-dominated non-serpentine assemblages. Species diversity and evenness were significantly higher in serpentine assemblages. Finally, relate analysis showed a relationship between ecotype AMF assemblages and soil nutrients. This study reveals a strong relationship between AMF associates and plant adaptation to edaphic extremes.

Microbial Spectrum of Intra-Abdominal Abscesses in Perforating Crohn's Disease: Results from a Prospective German Registry.

BACKGROUND: Intra-abdominal abscesses [IAAs] are common life-threatening complications in patients with Crohn's disease [CD]. In addition to interventional drainage and surgical therapy, empirical antibiotic therapy represents a cornerstone of treatment, but contemporary data on microbial spectra and antimicrobial resistance are scarce. METHODS: We recruited 105 patients with CD and IAAs from nine German centres for a prospective registry in order to characterize the microbiological spectrum, resistance profiles, antibiotic therapy and outcome. RESULTS: In 92 of 105 patients, microbial investigations of abscess material revealed pathogenic microorganisms. A total of 174 pathogens were isolated, with a median of 2 pathogens per culture [range: 1-6]. Most frequently isolated pathogens were E. coli [45 patients], Streptococcus spp. [28 patients], Enterococci [27 patients], Candida [13 patients] and anaerobes [12 patients]. Resistance to third-generation cephalosporins, penicillins with beta-lactamase inhibitors and quinolones were observed in 51, 36 and 35 patients, respectively. Seven patients had multiple-drug-resistant bacteria. Thirty patients received inadequate empirical treatment, and this was more frequent in patients receiving steroids or immunosuppression [37%] than in patients without immunosuppression [10%: p = 0.001] and was associated with a longer hospital stay [21 days vs 13 days, p = 0.003]. CONCLUSION: Based on antimicrobial resistance profiles, we herein report a high rate of inadequate empirical first-line therapy for IAAs in CD, especially in patients receiving immunosuppression, and this is associated with prolonged hospitalization.

A prospective multicentre study of the epidemiology and outcomes of bloodstream infection in cirrhotic patients.

OBJECTIVES: To describe the current epidemiology of bloodstream infection (BSI) in patients with cirrhosis; and to analyse predictors of 30-day mortality and risk factors for antibiotic resistance. METHODS: Cirrhotic patients developing a BSI episode were prospectively included at 19 centres in five countries from September 2014 to December 2015. The discrimination of mortality risk scores for 30-day mortality were compared by area under the receiver operator risk and Cox regression models. Risk factors for multidrug-resistant organisms (MDRO) were assessed with a logistic regression model. RESULTS: We enrolled 312 patients. Gram-negative bacteria, Gram-positive bacteria and Candida spp. were the cause of BSI episodes in 53%, 47% and 7% of cases, respectively. The 30-day mortality rate was 25% and was best predicted by the Sequential Organ Failure Assessment (SOFA) and Chronic Liver Failure-SOFA (CLIF-SOFA) score. In a Cox regression model, delayed (>24 hours) antibiotic treatment (hazard ratio (HR) 7.58; 95% confidence interval (CI) 3.29-18.67; p < 0.001), inadequate empirical therapy (HR 3.14; 95% CI 1.93-5.12; p < 0.001) and CLIF-SOFA score (HR 1.35; 95% CI 1.28-1.43; p < 0.001) were independently associated with 30-day mortality. Independent risk factors for MDRO (31% of BSIs) were previous antimicrobial exposure (odds ratio (OR) 2.91; 95% CI 1.73-4.88; p < 0.001) and previous invasive procedures (OR 2.51; 95% CI 1.48-4.24; p 0.001), whereas spontaneous bacterial peritonitis as BSI source was associated with a lower odds of MDRO (OR 0.30; 95% CI 0.12-0.73; p 0.008). CONCLUSIONS: MDRO account for nearly one-third of BSI in cirrhotic patients, often resulting in delayed or inadequate empirical antimicrobial therapy and increased mortality rates. Our data suggest that improved prevention and treatment strategies for MDRO are urgently needed in the liver cirrhosis patients.