Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive. RESULTS: We demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC. CONCLUSIONS: We are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

Regulation of human oocyte maturation in vivo during the final maturation of follicles.

STUDY QUESTION: Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo? SUMMARY ANSWER: The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY: Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-ß family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016-2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE: All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12-17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12-17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA: None. LIMITATIONS, REASONS FOR CAUTION: Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus-oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS: Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared.

Proteomic analysis of brain metastatic lung adenocarcinoma reveals intertumoral heterogeneity and specific alterations associated with the timing of brain metastases.

BACKGROUND: Brain metastases are associated with considerable negative effects on patients' outcome in lung adenocarcinoma (LADC). Here, we investigated the proteomic landscape of primary LADCs and their corresponding brain metastases. MATERIALS AND METHODS: Proteomic profiling was conducted on 20 surgically resected primary and brain metastatic LADC samples via label-free shotgun proteomics. After sample processing, peptides were analyzed using an Ultimate 3000 pump coupled to a QExactive HF-X mass spectrometer. Raw data were searched using PD 2.4. Further data analyses were carried out using Perseus, RStudio and GraphPad Prism. Proteomic data were correlated with clinical and histopathological parameters and the timing of brain metastases. Mass spectrometry-based proteomic data are available via ProteomeXchange with identifier PXD027259. RESULTS: Out of the 6821 proteins identified and quantified, 1496 proteins were differentially expressed between primary LADCs and corresponding brain metastases. Pathways associated with the immune system, cell-cell/matrix interactions and migration were predominantly activated in the primary tumors, whereas pathways related to metabolism, translation or vesicle formation were overrepresented in the metastatic tumors. When comparing fast- versus slow-progressing patients, we found 454 and 298 differentially expressed proteins in the primary tumors and brain metastases, respectively. Metabolic reprogramming and ribosomal activity were prominently up-regulated in the fast-progressing patients (versus slow-progressing individuals), whereas expression of cell-cell interaction- and immune system-related pathways was reduced in these patients and in those with multiple brain metastases. CONCLUSIONS: This is the first comprehensive proteomic analysis of paired primary tumors and brain metastases of LADC patients. Our data suggest a malfunction of cellular attachment and an increase in ribosomal activity in LADC tissue, promoting brain metastasis. The current study provides insights into the biology of LADC brain metastases and, moreover, might contribute to the development of personalized follow-up strategies in LADC.

Infecciones del tracto urinario en pacientes con esclerosis múltiple y los diferentes sistemas de vaciado vesical / Urinary tract infections in patients with multiple sclerosis and different methods of bladder evacuation

Introducción y objetivos: Evaluar la incidencia y la evolución de las infecciones del tracto urinario (ITU) en pacientes con esclerosis múltiple (EM) y su relación con el sistema de vaciado vesical. Materiales y métodos: Se incluyeron en el estudio pacientes con disfunciones miccionales neurógenas debido a la EM (n=111). Durante un año de seguimiento, se realizó una evaluación clínica con cultivo de orina cada 4 meses o ante la presencia de síntomas. El grupo de control incluyó a pacientes con ITU sintomática sin enfermedad neurológica o autoinmune. Se evaluó estadísticamente la incidencia de bacteriuria sintomática y asintomática, el efecto del drenaje urinario en la incidencia de ITU y el efecto del tratamiento antibiótico. Resultados: Cincuenta y cuatro pacientes con EM completaron el protocolo. La incidencia media de bacteriuria sintomática y asintomática en el grupo de EM fue del 12,5% y del 29,6%, respectivamente. Se observó una tendencia decreciente en la incidencia de la bacteriuria sintomática y una tendencia creciente en la incidencia de la asintomática. La erradicación de la ITU en los pacientes sintomáticos con EM fue significativamente menor que en los controles (37,75% frente a 92,93%, p<0,05). Los agentes causales fueron significativamente diferentes en ambos grupos (p=0,0005). No se rechazó la hipótesis de que la incidencia de ITU en los pacientes con EM es independiente del sistema de evacuación vesical (p>0,99 en las visitas 0, 1 y 3; p=0,078 en la visita 2). Conclusiones: Existe una diferencia significativa entre los agentes causales de la ITU en ambos grupos. La erradicación de la bacteriuria en los pacientes sintomáticos con EM es difícil en comparación con la población normal. No disponemos de pruebas suficientes para confirmar la relación entre la incidencia de ITU y el sistema de evacuación vesical. (AU)

Introduction and objectives: To evaluate the incidence and course of urinary tract infections (UTI) in patients with multiple sclerosis (MS) and their relationship to the method of bladder evacuation. Materials and methods: Patients with neurogenic bladder dysfunction due to MS (n=111) were enrolled in the study. During one-year follow-up, clinical examination with urine culture was performed every 4 months or whenever symptoms occurred. The control group included patients with symptomatic UTI, without neurological or autoimmune disease. Incidence of symptomatic and asymptomatic bacteriuria, the effect of urine drainage on UTI incidence, and the effect of antibiotics were statistically evaluated. Results: Fifty-four MS patients completed the protocol. The mean incidence of symptomatic and asymptomatic bacteriuria in the MS group was 12.5% and 29.6%, respectively. A decreasing trend in the incidence of symptomatic, and an increasing trend in the incidence of asymptomatic bacteriuria was observed. Eradication of UTI in symptomatic MS patients was significantly lower than in controls (37.75% vs. 92.93%, P<.05). Causative agents significantly differed in both groups (P=.0005). The hypothesis that the incidence of UTIs in MS patients is independent of the method of bladder evacuation was not rejected (P>.99 at visit 0, 1 and 3, P=.078 at visit 2). Conclusions: There is a significant difference between the causative agents of UTI in both groups. Eradication of bacteriuria in symptomatic MS patients is difficult when compared to the normal population. We have insufficient evidence to confirm the relationship between the incidence of UTI and the method of bladder evacuation. (AU)

Urinary tract infections in patients with multiple sclerosis and different methods of bladder evacuation.

INTRODUCTION AND OBJECTIVES: To evaluate the incidence and course of urinary tract infections (UTI) in patients with multiple sclerosis (MS) and their relationship to the method of bladder evacuation. MATERIALS AND METHODS: Patients with neurogenic bladder dysfunction due to MS (n=111) were enrolled in the study. During one-year follow-up, clinical examination with urine culture was performed every 4 months or whenever symptoms occurred. The control group included patients with symptomatic UTI, without neurological or autoimmune disease. Incidence of symptomatic and asymptomatic bacteriuria, the effect of urine drainage on UTI incidence, and the effect of antibiotics were statistically evaluated. RESULTS: 54 MS patients completed the protocol. The mean incidence of symptomatic and asymptomatic bacteriuria in the MS group was 12.5% and 29.6%, respectively. A decreasing trend in the incidence of symptomatic, and an increasing trend in the incidence of asymptomatic bacteriuria was observed. Eradication of UTI in symptomatic MS patients was significantly lower than in controls (37.75% vs. 92.93%, P<0.05). Causative agents significantly differed in both groups (P=0.0005). The hypothesis that the incidence of UTIs in MS patients is independent of the method of bladder evacuation was not rejected (P>0.99 at visit 0, 1 and 3, P=0.078 at visit 2). CONCLUSIONS: There is a significant difference between the causative agents of UTI in both groups. Eradication of bacteriuria in symptomatic MS patients is difficult when compared to the normal population. We have insufficient evidence to confirm the relationship between the incidence of UTI and the method of bladder evacuation.

Primary laparoscopic fundoplication in selected patients with gastroesophageal reflux disease.

BACKGROUND: Despite proton pump inhibitors being a powerful therapeutic tool, laparoscopic fundoplication (LF) has proven successful in the treatment of gastroesophageal reflux disease (GERD), through mechanical augmentation of a weak antireflux barrier and the advantages of minimally invasive access. A critical patient selection for LF, based on thorough preoperative assessment, is important for the management of GERD-patients. The purpose of this study is to provide an overview on the management of GERD-patients treated by primary LF in a specialized center and to illustrate the possible outcome after several years. METHODS: Patients were selected after going through diagnostic workup consisting of patient's history and physical examination, upper gastrointestinal endoscopy, assessment of gastrointestinal Quality of Life Index, screening for somatoform disorders, functional assessment by esophageal manometry, (impedance)-24-hour-pH-monitoring, and selective radiographic studies. The indication for LF was based on EAES-guidelines. Either a floppy and short Nissen fundoplication was performed or a posterior Toupet-hemifundoplication was chosen. A long-term follow-up assessment was attempted after surgery. RESULTS: In total, n = 1131 patients were evaluated (603 males; 528 females; mean age; 48.3 years; and mean body mass index: 27). The mean duration between onset of symptoms and surgery was 8 years. Nissen: n = 873, Toupet: n = 258; conversion rateerativ: 0.5%; morbidity 4%, mortality: 1 (1131). Mean follow-up (n = 898; 79%): 5.6 years; pre/post-op results: esophagitis: 66%/12.1%; Gastrointestinal Quality of Life Index: median: 92/119; daily proton pump inhibitors-intake after surgery: 8%; and operative revisions 4.3%. CONCLUSIONS: In conclusion, our data show that careful patient selection for laparoscopic fundoplication and well-established technical concepts of mechanical sphincter augmentation can provide satisfying results in the majority of patients with severe GERD.

Characterization of oxidation of glutathione by cytochrome c.

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis-elevated pH, Ca2+ level and Mg2+-increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

How effective is laparoscopic redo-antireflux surgery?

BACKGROUND: The failure-rate after primary antireflux surgery ranges from 3 to 30%. Reasons for failures are multifactorial. The aim of this study is to gain insight into the complex reasons for, and management of, failure after antireflux surgery. METHODS: Patients were selected for redo-surgery after a diagnostic workup consisting of history and physical examination, upper gastrointestinal endoscopy, quality-of-life assessment, screening for somatoform disorders, esophageal manometry, 24-hour-pH-impedance monitoring, and selective radiographic studies such as Barium-sandwich for esophageal passage and delayed gastric emptying. Perioperative and follow-up data were compiled between 2004 and 2017. RESULTS: In total, 578 datasets were analyzed. The patient cohort undergoing a first redo-procedure (n = 401) consisted of 36 patients after in-house primary LF and 365 external referrals (mean age: 62.1 years [25-87]; mean BMI 26 [20-34]). The majority of patients underwent a repeated total or partial laparoscopic fundoplication. Major reasons for failure were migration and insufficient mobilization during the primary operation. With each increasing number of required redo-operations, the complexity of the redo-procedure itself increased, follow-up quality-of-life decreased (GIQLI: 106; 101; and 100), and complication rate increased (intraoperative: 6,4-10%; postoperative: 4,5-19%/first to third redo). After three redo-operations, resections were frequently necessary (morbidity: 42%). CONCLUSIONS: Providing a careful patient selection, primary redo-antireflux procedures have proven to be highly successful. It is often the final chance for a satisfying result may be achieved upon performing a second redo-procedure. A third revision may solve critical problems, such as severe pain and/or inadequate nutritional intake. When resection is required, quality of life cannot be entirely normalized.

Severity of GERD and disease progression.

BACKGROUND: Many factors may play a role in the severity and progression of gastroesophageal reflux disease (GERD) since pathophysiology is multifactorial. Data regarding the progression of GERD are controversial: some reports of increased esophageal acid exposure (EAE) and mucosal damage were considered as evidence for a stable disease course, while others interprete these findings as disease progression. The aim of this study is to analyze a large patient-population with persisting symptoms indicative of GERD under protonpumpinhibitor-therapy and identify components characterizing disease severity and progression. METHODS: Patients with symptoms indicative of GERD were included in the study in a tertiary referral center (Frankfurt, Germany). All selected patients were under long-term protonpumpinhibitor-therapy with persistant symptoms. All patients underwent investigations to collect data on their physical status, EAE, severity of esophagitis, anatomical changes, and esophageal functional defects as well as their relation to the duration of the disease. Incidence over time was plotted as survival curves and tested with Log-rank tests for the four main disease markers. Multivariate modeling with COX-regression model was used to estimate the general impact of the four main disease markers on the time course of the disease. In order to elucidate possible causal relationships over time, a path analysis (structural equation model) was calculated. RESULTS: From the database with 1480 data sets, 972 patients were evaluated (542 males, 430 females). The mean age was 50.5 years (range18-89). The mean body mass index was 27.2(19-48). The mean time between the onset of symptoms and the diagnostic investigations was 8.2 years (1-50). A longer disease history for GERD was significantly associated with a higher risk for LES-incompetence. The mean duration from symptom onset to the time of clinical investigation was 9 years for patients with LES-incompetence (n = 563), compared to a mean of 6 years for those with mechanically intact LES (n = 95). A longer period from symptom onset to diagnostics was significantly associated with higher acid exposure. The pathway analysis was significant for the following model: 'history' (P < 0.001➔LES-incompetence & Hiatal Hernia➔(p < 0,001)➔pH-score (P < 0.001).Conclusion: LES-incompetence, the functional deterioration of the LES, and the anatomical alteration at the esophagogastric junction (Hiatal Hernia) as well as an increased EAE were associated with a long history of suffering from GERD. Path modeling suggests a causal sequence overtime of the main disease-parameters, tentatively allowing for a prediction of the course of the disease.

Use of proteomics to identify biomarkers associated with chronic kidney disease and long-term outcomes in patients with myocardial infarction.

BACKGROUND: Patients with chronic kidney disease (CKD) have poor outcomes following myocardial infarction (MI). We performed an untargeted examination of 175 biomarkers to identify those with the strongest association with CKD and to examine the association of those biomarkers with long-term outcomes. METHODS: A total of 175 different biomarkers from MI patients enrolled in the Swedish Web-System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies (SWEDEHEART) registry were analysed either by a multiple reaction monitoring mass spectrometry assay or by a multiplex assay (proximity extension assay). Random forests statistical models were used to assess the predictor importance of biomarkers, CKD and outcomes. RESULTS: A total of 1098 MI patients with a median estimated glomerular filtration rate of 85 mL min-1 /1.73 m2 were followed for a median of 3.2 years. The random forests analyses, without and with adjustment for differences in demography, comorbidities and severity of disease, identified six biomarkers (adrenomedullin, TNF receptor-1, adipocyte fatty acid-binding protein-4, TNF-related apoptosis-inducing ligand receptor 2, growth differentiation factor-15 and TNF receptor-2) to be strongly associated with CKD. All six biomarkers were also amongst the 15 strongest predictors for death, and four of them were amongst the strongest predictors of subsequent MI and heart failure hospitalization. CONCLUSION: In patients with MI, a proteomic approach could identify six biomarkers that best predicted CKD. These biomarkers were also amongst the most important predictors of long-term outcomes. Thus, these biomarkers indicate underlying mechanisms that may contribute to the poor prognosis seen in patients with MI and CKD.

Importance of bicarbonate transport in pH control during amelogenesis - need for functional studies.

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT-7 ameloblast cells. HAT-7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT-7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation-stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.

Effects of monensin on glucose metabolism in transition dairy cows.

Eight multiparous periparturient Holstein cows fitted with ruminal cannula were used in a split plot design to evaluate the effects of monensin on plasma glucose metabolism. Diets were top-dressed daily with 0 mg/cow of monensin (control) or 300 mg/cow of monensin (MON) both pre- and postpartum. Plasma glucose kinetic parameters on d -13 ± 2.0 and 19 ± 1.6 relative to parturition were determined by using stable isotopes. Na-1-13C3-Propionate (labeled propionate) was infused into the rumen to measure glucose synthesis originating from ruminal propionate, and U-13C-glucose (labeled glucose) was injected into the jugular vein to determine total glucose kinetics. A sampling period of 480 min following labeled glucose injection was implemented. A compartmental analysis was employed to determine steady state glucose kinetic parameters. To develop a steady state glucose model, the Windows version of SAAM software (WinSAAM) was used. A 4-compartment model was adequate to comprehensively describe plasma glucose metabolism. The main model compartments consisted of propionate and plasma glucose. The time frame of the 480-min sampling period post-tracer glucose infusion allowed accurate quantification of glucose metabolism. The model estimated that glucose input from sources other than ruminal propionate decreased with MON, from 2.26 to 1.09 g/min postpartum. Gluconeogenesis, expressed as the propionate contribution to the plasma glucose pool, increased in cows fed MON (22 vs. 31%), whereas glucose oxidation, expressed as the glucose disposal rate, significantly decreased (1.67 vs. 0.92 g/min). In conclusion, MON may improve the energy status of transition cows by (1) improving the efficiency of propionate to produce glucose and (2) decreasing glucose oxidation in body tissues.

Foregut symptoms, somatoform tendencies, and the selection of patients for antireflux surgery.

A large variety of foregut symptoms can occur in patients with gastroesophageal reflux disease (GERD), which can overlap with other disorders such as somatoform disorders and dyspepsia. Due to unclear diagnostic situations, these patients are often not adequately treated. The aim of this study was the evaluation of patients with foregut symptoms, referred for possible antireflux surgery, regarding their relationship with GERD and somatization tendencies based on control data from an unselected population. Symptom evaluation and somatization screening were initiated both in volunteers and in patients with foregut symptoms and GERD. Unselected volunteers from a village population were also evaluated by symptom analysis and for somatisation tendency. In addition, patients with foregut symptoms were diagnosed for GERD, and symptom analysis and psychodiagnostic evaluation were performed. There is no major significant difference in the symptom-spectrum in patients with foregut symptoms, whether they have a proven pathologic acid exposure from GERD or not. The probability for the risk of somatization was 5.6% in the unselected population of nonpatient volunteers (n = 267). In patients with foregut symptoms (n = 750), the probability for the presence of somatoform tendencies was approximately 20%, independent whether these patients had a documented GERD or a normal esophageal acid exposure, implicating further diagnostic work-up for the selection of patients for antireflux surgery. There is a remarkable symptom load and variety in patients with GERD, in patients with foregut symptoms, and in an unselected population of volunteers. There is no difference in the risk for somatization between patients with foregut symptoms and those with documented GERD. Therapeutic decision making especially prior to antireflux surgery requires an awareness of mental and emotional challenges.

Relationship between tumour size and outcome in pancreatic ductal adenocarcinoma.

BACKGROUND: The size of pancreatic ductal adenocarcinoma (PDAC) at diagnosis is an indicator of outcome. Previous studies have focused mostly on patients with resectable disease. The aim of this study was to investigate the relationship between tumour size and risk of metastasis and death in a large PDAC cohort, including all stages. METHODS: Patients diagnosed with PDAC between 1988 and 2013 were identified from the Surveillance, Epidemiology, and End Results (SEER) database. Tumour size was defined as the maximum dimension of the tumour as provided by the registry. Metastatic spread was assessed, and survival was calculated according to size of the primary tumour using the Kaplan-Meier method. Cox proportional regression modelling was used to adjust for known confounders. RESULTS: Some 58 728 patients were included. There were 187 patients (0·3 per cent) with a tumour size of 0·5 cm or less, in whom the rate of distant metastasis was 30·6 per cent. The probability of tumour dissemination was associated with tumour size at the time of diagnosis. The association between survival and tumour size was linear for patients with localized tumours, but stochastic in patients with regional and distant stages. In patients with resected tumours, increasing tumour size was associated with worse tumour-specific survival, whereas size was not associated with survival in patients with unresected tumours. In the adjusted Cox regression analysis, the death rate increased by 4·1 per cent for each additional 1-cm increase in tumour size. CONCLUSION: Pancreatic cancer has a high metastatic capacity even in small tumours. The prognostic impact of tumour size is restricted to patients with localized disease.

Hypoxia regulates global membrane protein endocytosis through caveolin-1 in cancer cells.

Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment.

Early Versus Delayed Autologous Stem Cell Transplantation and Interferon Maintenance in Multiple Myeloma: Single-Center Experience of 18 Years.

BACKGROUND: Autologous stem cell transplantation (ASCT) has become the mainstay of 1st-line treatment in younger patients with multiple myeloma (MM), but statistical confirmation of its superiority over other therapies, especially in the era of novel agents, is still lacking. METHODS: We reviewed the results of all 548 myeloma ASCTs performed in our institute over the past 18 years. RESULTS: More than one-half of the patients had access to novel agents before their transplantations. Although the age of the transplanted patients increased significantly over the years, treatment-related mortality (TRM) was remarkably low, especially in 1st-line transplanted patients (100-day TRM, 0.3%). The median overall survival (OS) of the entire cohort was 98.4 months. Patients transplanted within 12 months from the start of their therapy had significantly better responses than those having delayed ASCT (complete response rate, 58.1% vs 46.8%; P = .016) and significant post-ASCT progression-free survival (PFS) benefit (30.2 [26.1-34.3] mo vs 23.3 [16.8-29.8] mo; P = .036), but we found no significant overall survival difference. The results were similar in patients treated with or without novel agents before ASCT. During a period of time, interferon maintenance was our standard approach to post-ASCT maintenance. Our analysis showed not only a significant PFS advantage with interferon, but also a highly significant overall survival benefit (150.4 [105.1-195.8] mo vs 86.1 [72.5-99.7] mo; P = .003). CONCLUSIONS: Our findings demonstrate that delayed ASCT can be feasible in selected patients.

Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model.

Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized, presumably by HCO3 (-)ions transported from ameloblasts into the developing enamel matrix. Ameloblasts express a number of transporters and channels known to be involved in HCO3 (-)transport in other epithelia. However, to date, there is no functional evidence for HCO3 (-)transport in these cells. To address questions related to HCO3 (-)export from ameloblasts, we have developed a polarized 2-dimensional culture system for HAT-7 cells, a rat cell line of ameloblast origin. HAT-7 cells were seeded onto Transwell permeable filters. Transepithelial resistance was measured as a function of time, and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. Intracellular pH regulation and HCO3 (-)transport were assessed by microfluorometry. HAT-7 cells formed epithelial layers with measureable transepithelial resistance on Transwell permeable supports and expressed claudin-1, claudin-4, and claudin-8-key proteins for tight junction formation. Transport proteins previously described in maturation ameloblasts were also present in HAT-7 cells. Microfluorometry showed that the HAT-7 cells were polarized with a high apical membrane CO2 permeability and vigorous basolateral HCO3 (-)uptake, which was sensitive to Na(+)withdrawal, to the carbonic anhydrase inhibitor acetazolamide and to H2DIDS inhibition. Measurements of transepithelial HCO3 (-)transport showed a marked increase in response to Ca(2+)- and cAMP-mobilizing stimuli. Collectively, 2-dimensional HAT-7 cell cultures on permeable supports 1) form tight junctions, 2) express typical tight junction proteins and electrolyte transporters, 3) are functionally polarized, and 4) can accumulate HCO3 (-)ions from the basolateral side and secrete them at the apical membrane. These studies provide evidence for a regulated, vectorial, basolateral-to-apical bicarbonate transport in polarized HAT-7 cells. We therefore propose that the HAT-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts.

[Translational research in pediatric rheumatology. Current research approaches to the innate immune system]. / Translationale Forschung in der pädiatrischen Rheumatologie. Aktuelle Forschungsansätze zum angeborenen Immunsystem.

Translational research aims at closely linking basic research and clinical observations so that important mechanistic insights identified in one field should trigger progress in the other. Particularly in the field of pediatric rheumatology this approach has significantly improved the understanding and therapy of several diseases in recent years. One focus of our research in this respect is on the structure, release mechanisms and function of damage associated molecular patterns (DAMP), particularly S100 proteins. Due to their huge potential as inflammation biomarkers for more specific diagnostics these proteins are of particular clinical interest. Overactivated cells of the innate immune system play a crucial role in the development of rheumatic diseases. Innate mechanisms, such as the generation of neutrophil extracellular traps (NETosis) were linked to the pathogenesis of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, it became increasingly more evident that various excessive sterile inflammatory mechanisms and reactions significantly contribute to an activation of adaptive immune responses and thus to the development of autoimmunity. Studying such potentially DAMP-dependent pathways at the interface between innate and adaptive immunity can provide a better understanding of autoinflammatory conditions in pediatric rheumatology and to identify novel targets for optimization of therapy.

Alternative ileal flap for bladder augmentation if mesentery is short.

PURPOSE: To date the clam ileocystoplasty is the preferred method of bladder augmentation in children when the urodynamic problem is non-compliance and/or detrusor overactivity. The key to this technique is the incision of the bladder wall deep into the pelvis down to the trigone in order to avoid a diverticulum like neobladder and to provide adequate margin for augmentation. The detubularised ileum flap therefore has to reach to the bottom of the divided bladder on a reliable vascular pedicle without significant tension. A short ileal mesentery caused by previous surgery, peritonitis, peritoneal dialysis or ventriculo-peritoneal shunt may complicate surgery and compromise outcome. We hypothesized we can rely on the communication of the intramural vessels within the intestine and can detubularise the ileum adjacent to the mesentery rather than along the antimesenteric line and this could be combined with ligation of some vasa recta (VR) in order to create alternative ileum flaps, which reach further into the pelvis. Our aim was to assess the viability of the alternative flaps detubularised along the paramesenteric line and measure how many VR could be sacrificed beyond the tertiary arcades. MATERIALS AND METHODS: After ethical approval adjacent ileal segments were detubulirased along the antimesenteric line (Group 1) and along the paramesenteric line (Group 2) in 5 minipigs in general anaesthesia. Ligation of 0,1,2,3 and 4 VR has been performed starting from the free end of the segments. The length of the ileal flaps was recorded. The microcirculation of flap edges was detected by in vivo microscopy using orthogonal polarising spectral imaging (Cytoscan A/R Cytometrics, PA, USA). Clam ileocystoplasty was performed with the ileum detubularised along the paramesenteric line without ligation of VR. Specimens of the augmented bladder were obtained after 4 weeks and stained with Hematoxilin + Eosin. RESULTS: No alteration in capillary red blood cell velocity (RBCV) and perfusion rate (PR) was observed after paramesenteric detubularisation. The flaps in Group 2 reached 20.25 ± 0.5 mm longer vs. CONTROL: This is 98% of the mean bowel width (20.5 ± 0.57 mm) measured in the animals. Ligation of each VR further increased the length of both flaps (mean: 10.59 ± 3.18 mm) however ligation of more than 2 VR gradually decreased the microcirculation in both groups. All animals augmented with alternative flap survived, there was no urine leak or suture break down. Histology confirmed viable bowel flaps. CONCLUSION: Paramesenteric detubularisation of the ileum is fully tolerated and results in longer reaching ileal flap vs. CONTROL: Only limited ligation of VR is tolerated. DISCUSSION: This study showed the first time that clam ileocystoplasty is feasible with ileal flap detubularised along the paramesenteric line. The use of the animal model and the relative short postoperative observation are the main limitations of this study.

The potential and perspective of peroral endoscopic esophageal myotomy for achalasia.

The therapeutic spectrum of this disorder consists of medical therapy, endoscopic balloon dilatation, endoscopic Botox injection, open or laparoscopic cardia myotomy, and most recently transesophageal endoscopic myotomy (POEM peroral endoscopic myotomy). The most important requirement is a well-experienced team in interventional flexible endoscopy. The endoscopist as well as the assisting staff should have experience in advanced therapeutic endoscopic techniques and hemostasis to handle all necessary endoscopic instruments such as injection needles, needle knife, triangle knife, coagulation graspers, and endoscopic clip handling and closures. In addition, advanced surgical and especially laparoscopic skills and experience as well as surgical knowledge about esophageal disease must be available in case of conversion and/or consultation. Prior to this procedure, the patient undergoes a detailed diagnostic work-up to confirm the diagnosis of achalasia. The procedures are performed in general anesthesia. The patient is brought in a supine position, and the abdomen is free for inspection and palpation during the procedure. The myotomy can be performed in different locations around the esophageal circumference. In Europe, several centers with large experience in esophageal disease, laparoscopy, and especially advanced interventional endoscopy have started to introduce this POEM-technique in their clinical practice. Initial success and low complication rates are quite promising and show a great future perspective for this technique. In the USA, POEM is a procedure with a substantial increase in numbers performed in the past years with a low complication rate. The largest series are performed in Asia with a great clinical success. The perspective of POEM may be the lesser access trauma. Its potential can be also realized in Redo cases, where experienced centers have initial experience with POEM after POEM and POEM after LHMD.