CD19 CAR T-Cell Therapy in Autoimmune Disease - A Case Series with Follow-up.

BACKGROUND: Treatment for autoimmune diseases such as systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis often involves long-term immune suppression. Resetting aberrant autoimmunity in these diseases through deep depletion of B cells is a potential strategy for achieving sustained drug-free remission. METHODS: We evaluated 15 patients with severe SLE (8 patients), idiopathic inflammatory myositis (3 patients), or systemic sclerosis (4 patients) who received a single infusion of CD19 chimeric antigen receptor (CAR) T cells after preconditioning with fludarabine and cyclophosphamide. Efficacy up to 2 years after CAR T-cell infusion was assessed by means of Definition of Remission in SLE (DORIS) remission criteria, American College of Rheumatology-European League against Rheumatism (ACR-EULAR) major clinical response, and the score on the European Scleroderma Trials and Research Group (EUSTAR) activity index (with higher scores indicating greater disease activity), among others. Safety variables, including cytokine release syndrome and infections, were recorded. RESULTS: The median follow-up was 15 months (range, 4 to 29). The mean (±SD) duration of B-cell aplasia was 112±47 days. All the patients with SLE had DORIS remission, all the patients with idiopathic inflammatory myositis had an ACR-EULAR major clinical response, and all the patients with systemic sclerosis had a decrease in the score on the EUSTAR activity index. Immunosuppressive therapy was completely stopped in all the patients. Grade 1 cytokine release syndrome occurred in 10 patients. One patient each had grade 2 cytokine release syndrome, grade 1 immune effector cell-associated neurotoxicity syndrome, and pneumonia that resulted in hospitalization. CONCLUSIONS: In this case series, CD19 CAR T-cell transfer appeared to be feasible, safe, and efficacious in three different autoimmune diseases, providing rationale for further controlled clinical trials. (Funded by Deutsche Forschungsgemeinschaft and others.).

[Physician Numbers and Employment in Bavaria: Trends in statistics of the Bavarian Medical Association and the Association of Statutory Health Insurance Physicians of Bavaria]. / Entwicklung der Ärzt*innenzahlen und Beschäftigungsverhältnisse in Bayern ­ Trends in den Ärzt*innenstatistiken der Bayerischen Landesärztekammer und der Kassenärztlichen Vereinigung Bayerns.

BACKGROUND: Although the number of places at medical schools and physicians in Germany has increased continuously over the past 25 years, there is a threat of a shortage of physicians. Based on data from the Bavarian Medical Association (BLÄK) and the Association of Statutory Health Insurance Physicians of Bavaria (KVB), an analysis of the number of physicians in Bavaria over a longer period of time was carried out in order to understand current developments and possible starting points for the future organization of medical care. The figures were analyzed with regard to the distribution of physicians by outpatient and inpatient sector as well as with regard to the development of the number of employees, the scope of employment and the gender distribution in the outpatient sector. METHODS: Data were taken from the annually published and systematically compiled numbers of physicians from the BLÄK (2000 to 2022) as well as the outpatient billing data of practicing and employed physicians in Bavaria (2010 to 2022), processed by the KVB. Descriptive analyses were performed. RESULTS: Since 2000, the number of physicians in Bavaria has risen by 83% in the inpatient setting and by 35% in the outpatient setting. As a result, more physicians have been working in hospitals than in outpatient care since 2010. In the outpatient setting the trend is moving away from establishing one's own practice and full-time work towards salaried and part-time employment. Employed physicians have lower average working hours than self-employed physicians. The proportion of women among physicians has steadily increased, with female physicians more likely to be employed and working part-time compared with male physicians. Nevertheless, part-time employment is also prominent among male physicians in some specialties today. CONCLUSION: The trend towards practicing in salaried and part-time positions continues unabated and is represented across all specialties, suggesting that more physicians are needed to maintain the number of working hours over time. In addition to incentives and subsidies, this reality must be taken into account when planning care. At the same time, it is questionable whether increasing medical school places without managing them according to need is the right way to address the shortage of physicians in outpatient care when an ever-increasing proportion of physicians is working in inpatient care.

Do proton pump inhibitors increase the risk of dementia? A systematic review, meta-analysis and bias analysis.

AIM: Previous studies on the association between proton pump inhibitor (PPI) intake and the increased risk of dementia has shown discrepancies in their conclusions. We aimed to provide updated evidence based on extensive bias assessments and quantitative sensitivity analyses. METHODS: We searched the databases PubMed, EMBASE, SCOPUS, CENTRAL and clinicaltrials.gov for prospective studies that examined an association between PPI use and dementia, up to February 2022. Each study was assessed using the Cochrane risk of bias assessment tools for non-randomized studies of interventions (ROBINS-I) or randomized trials (RoB2). Pooled risk ratios (RRs) and 95% prediction intervals were computed using random-effects models. Sensitivity analyses were adjusted for small-study bias. RESULTS: We included nine observational studies with 204 108 dementia cases in the primary analysis on the association between PPI use vs. non-use and dementia, and the RR was 1.16 (95% CI = 1.00; 1.35). After adjusting for small-study bias by Copas selection model and Rücker's shrinkage procedure, the RR was 1.16 (1.02; 1.32) and 1.15 (1.13; 1.17), respectively. A subgroup analysis of PPI use vs. non-use regarding Alzheimer's disease risk yielded an RR of 1.15 (0.89; 1.50). The secondary analysis on the risk of dementia by use of PPI vs. histamine-2 receptor antagonist showed an RR of 1.03 (0.66; 1.62). CONCLUSION: This meta-analysis provided no clear evidence for an association between PPI intake and the risk of dementia. Due to discrepancies in sensitivity analyses, however, some risk of dementia by PPI use cannot be ruled out. Since an unequivocal conclusion is still pending, further research is warranted.

Salt-responsive gut commensal modulates TH17 axis and disease.

A Western lifestyle with high salt consumption can lead to hypertension and cardiovascular disease. High salt may additionally drive autoimmunity by inducing T helper 17 (TH17) cells, which can also contribute to hypertension. Induction of TH17 cells depends on gut microbiota; however, the effect of salt on the gut microbiome is unknown. Here we show that high salt intake affects the gut microbiome in mice, particularly by depleting Lactobacillus murinus. Consequently, treatment of mice with L. murinus prevented salt-induced aggravation of actively induced experimental autoimmune encephalomyelitis and salt-sensitive hypertension by modulating TH17 cells. In line with these findings, a moderate high-salt challenge in a pilot study in humans reduced intestinal survival of Lactobacillus spp., increased TH17 cells and increased blood pressure. Our results connect high salt intake to the gut-immune axis and highlight the gut microbiome as a potential therapeutic target to counteract salt-sensitive conditions.

Emulating a target trial of proton pump inhibitors and dementia risk using claims data.

BACKGROUND AND PURPOSE: Understanding the adverse effects of proton pump inhibitors (PPIs) is important due to their widespread use, but the available evidence for an increased dementia risk amongst patients taking PPIs is inconclusive. The present study aimed to estimate the causal effect of PPIs on the risk of dementia by target trial emulation and time-varying exposure modeling. METHODS: Using claims data of 2,698,176 insured people of a large German statutory health insurer, a target trial was conceptualized in which individuals aged 40 years and older were classified as PPI initiators or non-initiators between 2008 and 2018, and were followed until diagnosis of dementia, death, loss to follow-up or end of study. Incidence of dementia (International Classification of Diseases 10 codes F00, F01, F03, F05.1, G30, G31.0, G31.1, G31.9 and F02.8+G31.82) was defined applying a 1-year lag window. Weighted Cox models were used to estimate the effect of PPI initiation versus non-initiation on dementia risk and weighted pooled logistic regression was used to estimate the effect of time-varying use versus non-use. RESULTS: In all, 29,746 PPI initiators (4.4%) and 26,830 non-initiators (1.3%) were diagnosed with dementia. Comparing PPI initiation with no initiation, the hazard ratio for dementia was 1.54 (95% confidence interval 1.51-1.58). The hazard ratio for time-dependent PPI use versus non-use was 1.56 (95% confidence interval 1.50-1.63). Differentiated subtypes, including unspecified dementia, Alzheimer's disease and vascular dementia, showed increased risk by PPI initiation and time-varying PPI use. CONCLUSIONS: This study suggests that PPI initiation and time-varying PPI use may increase overall dementia risk.

Who gets prescriptions for proton pump inhibitors and why? A drug-utilization study with claims data in Bavaria, Germany, 2010-2018.

PURPOSE: The German annual drug prescription-report has indicated overuse of proton pump inhibitors (PPIs) for many years; however, little was known about the characteristics of people using PPIs. This study aimed to provide comprehensive utilization data and describe frequencies of potential on- and off-label PPI-indications in Bavaria, Germany. METHODS: Claims data of statutorily insured people from 2010 to 2018 were used. Defined daily doses (DDDs) of PPIs by type of drug, prevalence of PPI-use and DDDs prescribed per 1000 insured people/day were analyzed. For 2018, proportions of users and DDDs per 1000 insured people were calculated by age and sex. To elucidate changes in prescribing practices due to a suspected drug-drug interaction, we examined co-prescribing of clopidogrel and PPIs between 2010 and 2018. For PPI new users, sums of DDDs and frequencies of potential indications were examined. RESULTS: PPI prescribing increased linearly from 2010 to 2016 and gradually decreased from 2016 to 2018. In 2018, 14.7% of women and 12.2% of men received at least one prescription, and 64.8 DDDs (WHO-def.) per 1000 insured people/day were prescribed. Overall, omeprazole use decreased over the observation period and was steadily replaced by pantoprazole, especially when co-prescibed with clopidogrel. An on-label PPI-indication was not reported at first intake in 52.0% of new users. CONCLUSIONS: The utilization of prescribed PPIs has decreased since 2016. However, a large proportion of new PPI-users had no documentation of a potential indication, and the sums of DDDs prescribed often seemed not to comply with guidelines.

[The Development of General Practices in Ambulatory Care in the Last Decades - An Analysis of the Physicians' Registry of Bavaria]. / Entwicklung der hausärztlichen Versorgung innerhalb der letzten Jahrzehnte ­ eine Analyse des Basisdatensatzes der Kassenärztlichen Vereinigung Bayerns.

AIM OF THE STUDY: There is an increasing shortage of general practitioners (GPs) in Germany. An in-depth understanding of the variation regarding the characteristics of the GPs in the last decades might help to optimize primary health care. The aim of the analysis of the characteristic features of GPs as preserved by the registry of the Bavarian Association of Statutory Health Insurance of Physicians (KVB) was to delineate the time interval between passing the medical state examination and establishment of one's own private practice, the development of group practices and employments, and the number of additional qualifications. METHODS: The physicians' registry of the KVB was used to analyse the time intervals, additional qualifications, and development of group practices and employments. Data were analysed descriptively. RESULTS: The time interval from passing the medical state examination and establishing one's own private practice increased remarkably since the 70s from 5 years to 12 years on average. There was an increasing trend towards group practices. The number of newly established practices remained constant around 200 practices per year. Beyond that, there was an increasing trend towards employment in practices. The number of additional qualifications decreased over time, which was in particular true of complementary medicine and sports medicine. CONCLUSIONS: The period of vocational traineeship should be organised efficiently to decrease the length of training. Young GPs should be motivated and receive incentives to establish their own private practice. However, it is also necessary to facilitate employment in the practices.

Influence of probiotics on the periodontium, the oral microbiota and the immune response during orthodontic treatment in adolescent and adult patients (ProMB Trial): study protocol for a prospective, double-blind, controlled, randomized clinical trial.

BACKGROUND: Orthodontic treatment with fixed appliances is often necessary to correct malocclusions in adolescence or adulthood. However, oral hygiene is complicated by appliances, and prior studies indicate that they may trigger oral inflammation and dysbiosis of the oral microbiota, especially during the first 3 months after insertion, and, thus, may present a risk for inflammatory oral diseases. In recent periodontal therapeutic studies, probiotics have been applied to improve clinical parameters and reduce local inflammation. However, limited knowledge exists concerning the effects of probiotics in orthodontics. Therefore, the aim of our study is to evaluate the impact of probiotics during orthodontic treatment. METHODS: This study is a monocentric, randomized, double blind, controlled clinical study to investigate the effectiveness of daily adjuvant use of Limosilactobacillus reuteri (Prodentis®-lozenges, DSM 17938, ATCC PTA 5289) versus control lozenges during the first three months of orthodontic treatment with fixed appliances. Following power analysis, a total of 34 adolescent patients (age 12-17) and 34 adult patients (18 years and older) undergoing orthodontic treatment at the University Hospital Erlangen will be assigned into 2 parallel groups using a randomization plan for each age group. The primary outcome measure is the change of the gingival index after 4 weeks. Secondary outcomes include the probing pocket depth, the modified plaque index, the composition of the oral microbiota, the local cytokine expression and-only for adults-serum cytokine levels and the frequencies of cells of the innate and adaptive immune system in peripheral blood. DISCUSSION: Preventive strategies in everyday orthodontic practice include oral hygiene instructions and regular dental cleaning. Innovative methods, like adjuvant use of oral probiotics, are missing. The aim of this study is to analyse, whether probiotics can improve clinical parameters, reduce inflammation and prevent dysbiosis of the oral microbiota during orthodontic treatment. If successful, this study will provide the basis for a new strategy of prophylaxis of oral dysbiosis-related diseases during treatment with fixed appliances. TRIAL REGISTRATION: This trial is registered at ClinicalTrials.gov in two parts under the number NCT04598633 (Adolescents, registration date 10/22/2020), and NCT04606186 (Adults, registration date 10/28/2020).

Structural and functional characterization of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica.

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.

Recombinant protein production and purification of SiiD, SiiE and SiiF - Components of the SPI4-encoded type I secretion system from Salmonella Typhimurium.

In humans, Salmonella enterica infections are responsible for a plethora of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secretes SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Secondary structure content and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure contents were in good agreement with estimates derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 Å resolution.

Defining Metaniches in the Oral Cavity According to Their Microbial Composition and Cytokine Profile.

The human oral microbiota consists of over 700 widespread taxa colonizing the oral cavity in several anatomically diverse oral niches. Lately, sequencing of the 16S rRNA genes has become an acknowledged, culture-independent method to characterize the oral microbiota. However, only a small amount of data are available concerning microbial differences between oral niches in periodontal health and disease. In the context of periodontitis, the cytokine expression in the gingival crevicular fluid has been studied in detail, whereas little is known about the cytokine profile in hard and soft tissue biofilms. In order to characterize oral niches in periodontal health, the oral microbiota and cytokine pattern were analyzed at seven different sites (plaque (P), gingival crevicular fluid (GCF), saliva (S), tongue (T), hard palate (HP), cheek (C) and sublingual area (U)) of 20 young adults using next-generation sequencing and multiplex immunoassays. Site-specific microbial compositions were detected, which clustered into three distinct metaniches ("P-GCF", "S-T-HP" and "C-U") and were associated with niche-/metaniche-specific cytokine profiles. Our findings allow the definition of distinct metaniches according to their microbial composition, partly reflected by their cytokine profile, and provide new insights into microenvironmental similarities between anatomical diverse oral niches.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica.

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion.

Differences in treatment and monitoring of chronic myeloid leukemia with regard to age, but not sex: Results from a population-based study.

There are established guidelines for treatment and monitoring of chronic myeloid leukemia (CML) but little is known about routine care. Data on ICD-10 codes as well as prescribed medications were available for 10.5 million patients in the statutory health insurance system in Bavaria for the years 2010 to 2016. Also, data on the molecular and cytogenetic monitoring were integrated. A total of 1714 adult patients with CML were observed. Only 50.8% received more than 67.5 daily doses per quarter year (target: 91.5) while 18.2% did not receive any tyrosine kinase inhibitor (TKI). The median number of daily doses was at least 80 doses per quarter year for all age groups in men, but decreased to 62 doses in elderly women. With this exception, no differences between men and women were observed. The percentage of patients without any TKI increased with age. The median number of molecular examinations was 3.54 independent of age and sex. Even in a highly developed country, still a considerable number of patients with CML seem to not receive adequate treatment, whereas molecular monitoring can be considered satisfactory.

Colistin- and carbapenem-resistant Klebsiella oxytoca harboring blaVIM-2 and an insertion in the mgrB gene isolated from blood culture.

A carbapenemase-producing colistin-resistant Klebsiella oxytoca isolate was recovered from a blood culture of a female patient without previous report of risk factors to obtain multidrug-resistant Gram-negative bacilli. A combination of biochemical and molecular methods was used to identify the resistance mechanism of this isolate. Carbapenemase production was mediated by Verona integron-encoded metallo-ß-lactamase (VIM)-2. Colistin resistance was not due to plasmid- borne mcr-1 gene, but we found an integration of IS5-like sequence in the mgrB gene of K. oxytoca. This gene is known to be an important regulator of the PhoPQ two-component system, and the disruption of this gene is most likely the cause of lipid A modification resulting in colistin resistance of our isolate. To the best of our knowledge this constitutes the first report of a carbapenemase-producing K. oxytoca with colistin resistance, a case that demonstrates the limited treatment options for infections with multidrug-resistant organisms.

Comparative whole genome analysis of three consecutive Salmonella diarizonae isolates.

Infections of very young children or immunocompromised people with Salmonella of higher subspecies are a well-known phenomenon often associated with contact to cold-blooded animals. We describe the molecular characterization of three S. enterica subsp. diarizonae strains, isolated consecutively over a period of several months from a hospital patient suffering from diarrhea and sepsis with fatal outcome. With the initial isolate the first complete genome sequence of a member of subsp. diarizonae is provided and based on this reference we revealed the genomic differences between the three isolates by use of next-generation sequencing and confirmed by phenotypical tests. Genome comparisons revealed mutations within gpt, hfq and purK in the first isolate as a sign of clonal variation rather than host-directed evolution. Furthermore, our work demonstrates that S. enterica subsp. diarizonae possess, besides a conserved set of known Salmonella Pathogenicity Islands, a variable portfolio of additional genomic islands of unknown function.

Cross talk between the response regulators PhoB and TctD allows for the integration of diverse environmental signals in Pseudomonas aeruginosa.

Two-component systems (TCS) serve as stimulus-response coupling mechanisms to allow organisms to adapt to a variety of environmental conditions. The opportunistic pathogen Pseudomonas aeruginosa encodes for more than 100 TCS components. To avoid unwanted cross-talk, signaling cascades are very specific, with one sensor talking to its cognate response regulator (RR). However, cross-regulation may provide means to integrate different environmental stimuli into a harmonized output response. By applying a split luciferase complementation assay, we identified a functional interaction of two RRs of the OmpR/PhoB subfamily, namely PhoB and TctD in P. aeruginosa. Transcriptional profiling, ChIP-seq analysis and a global motif scan uncovered the regulons of the two RRs as well as a quadripartite binding motif in six promoter regions. Phosphate limitation resulted in PhoB-dependent expression of the downstream genes, whereas the presence of TctD counteracted this activation. Thus, the integration of two important environmental signals e.g. phosphate availability and the carbon source are achieved by a titration of the relative amounts of two phosphorylated RRs that inversely regulate a common subset of genes. In conclusion, our results on the PhoB and TctD mediated two-component signal transduction pathways exemplify how P. aeruginosa may exploit cross-regulation to adapt bacterial behavior to complex environments.

Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.

Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal Escherichia coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.

Inflammation fuels colicin Ib-dependent competition of Salmonella serovar Typhimurium and E. coli in enterobacterial blooms.

The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae ("inflammation-inflicted blooms"). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.

Low-oxygen tensions found in Salmonella-infected gut tissue boost Salmonella replication in macrophages by impairing antimicrobial activity and augmenting Salmonella virulence.

In Salmonella infection, the Salmonella pathogenicity island-2 (SPI-2)-encoded type three secretion system (T3SS2) is of key importance for systemic disease and survival in host cells. For instance, in the streptomycin-pretreated mouse model SPI-2-dependent Salmonella replication in lamina propria CD11c(-)CXCR1(-) monocytic phagocytes/macrophages (MΦ) is required for the development of colitis. In addition, containment of intracellular Salmonella in the gut critically depends on the antimicrobial effects of the phagocyte NADPH oxidase (PHOX), and possibly type 2 nitric oxide synthase (NOS2). For both antimicrobial enzyme complexes, oxygen is an essential substrate. However, the amount of available oxygen upon enteroinvasive Salmonella infection in the gut tissue and its impact on Salmonella-MΦ interactions was unknown. Therefore, we measured the gut tissue oxygen levels in a model of Salmonella enterocolitis using luminescence two-dimensional in vivo oxygen imaging. We found that gut tissue oxygen levels dropped from ∼78 Torr (∼11% O2) to values of ∼16 Torr (∼2% O2) during infection. Because in vivo virulence of Salmonella depends on the Salmonella survival in MΦ, Salmonella-MΦ interaction was analysed under such low oxygen values. These experiments revealed an increased intracellular replication and survival of wild-type and t3ss2 non-expressing Salmonella. These findings were paralleled by blunted nitric oxide and reactive oxygen species (ROS) production and reduced Salmonella ROS perception. In addition, hypoxia enhanced SPI-2 transcription and translocation of SPI-2-encoded virulence protein. Neither pharmacological blockade of PHOX and NOS2 nor impairment of T3SS2 virulence function alone mimicked the effect of hypoxia on Salmonella replication under normoxic conditions. However, if t3ss2 non-expressing Salmonella were used, hypoxia did not further enhance Salmonella recovery in a PHOX and NOS2-deficient situation. Hence, these data suggest that hypoxia-induced impairment of antimicrobial activity and Salmonella virulence cooperate to allow for enhanced Salmonella replication in MΦ.

Tiotropium Respimat(®) vs. HandiHaler(®): real-life usage and TIOSPIR trial generalizability.

AIM: Two inhaler devices (Respimat® and HandiHaler®) are available for tiotropium, a long acting anticholinergic agent. We aimed to analyze drug utilization, off-label usage and generalizability of the TIOSPIR trial results for both devices. METHODS: Patients aged ≥18 years exhibiting at least one documented prescription of tiotropium in the database of the Association of Statutory Health Insurance Physicians, Bavaria, Germany, were included (years 2004-2008). Annual period prevalence rates (PPRs) were calculated stratified by age, gender and inhaler devices. Off-label usage (patients lacking a chronic obstructive pulmonary disease (COPD) diagnosis) and the proportion of patients meeting the inclusion and exclusion criteria of the TIOSPIR trial were analyzed. RESULTS: Between 2004 and 2008, PPRs increased and varied between 49.2 and 74.5 per 10 000 persons for HandiHaler® and between 1.5 and 9.3 per 10 000 persons for Respimat®. Small differences regarding patient characteristics existed between the two inhaler devices. Only about 30% (HandiHaler® 32.1%, Respimat® 30.0%) of the database patients receiving tiotropium could be theoretically included in the TIOSPIR trial. CONCLUSIONS: Comparing the two tiotropium devices, no clinically relevant differences regarding patient and prescribing characteristics were revealed. Results of the TIOSPIR trial were generalizable only to a minority of our study patients, underlining the need for real-life data.