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.

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.

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.

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.

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.

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.

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.

[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.

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.

Effect of dietary protein level and rumen-protected amino acid supplementation on amino acid utilization for milk protein in lactating dairy cows.

This study investigated the effect of metabolizable protein (MP) supply and rumen-protected (RP) Lys and Met supplementation on productivity, nutrient digestibility, urinary N losses, apparent total-tract digestibility of dietary AA, and the efficiency of AA utilization for milk protein synthesis in dairy cows. The experiment was conducted with 8 ruminally cannulated Holstein cows in a replicated 4×4 Latin square design trial with 21-d periods. Treatments were (1) MP-adequate diet (AMP; MP balance of -24 g/d); (2) MP-deficient diet (DMP; MP balance of -281 g/d); (3) DMP supplemented with 100 g of RPLys/cow per day (estimated digestible Lys supply=24 g/d; DMPL; MP balance of -305g/d); and (4) DMPL supplemented with 24 g of RPMet/cow per day (estimated digestible Met supply=15 g/d; DMPLM; MP balance of -256g/d). Diet had no effect on total-tract nutrient digestibility, milk production, and milk composition, but the DMP diets decreased urinary N excretion and the ammonia emitting potential of manure. Plasma Met concentration was increased by DMPLM compared with AMP. Supplementation with RPLys had no effect on plasma Lys. Concentration of most AA in milk protein was increased or tended to be increased by DMPLM compared with DMPL. Except for the AA supplemented as RPAA (i.e., Met and Lys), apparent total-tract digestibility of all dietary AA was generally greater for the DMP diets and ranged from 33% (Arg, AMP diet) to 67% (Thr, DMPL diet). Apparent recovery of dietary AA in milk protein followed the same trends, being greater for the DMP diets than AMP and generally lower for Lys and Met with the RPAA-supplemented diets versus AMP and DMP. The RPAA were apparently not used for milk protein synthesis in the conditions of this experiment. The AA recoveries in milk protein varied from around 17% (Ala) to 70% (Pro). Milk protein recoveries of essential AA (EAA) were around 54% for the DMP diet and 49% for AMP. The estimated efficiency of utilization of digestible EAA for milk protein synthesis was generally greater for the DMP diets compared with AMP. In this trial, blood plasma Lys and Met were labeled by abomasal pulse-dose of 15N-Lys and 13C-Met (respectively). Analysis of the 15N-Lys and 13C-Met decay curves in plasma indicated trends for a faster extraction of Lys and Met from plasma for the MP-deficient diets, compared with AMP. Overall, this study confirmed conclusions from previous analyses that the efficiency of utilization of dietary EAA will increase with decreasing MP-AA supply.

Inflammatory activation after experimental cardiac tamponade.

BACKGROUND/PURPOSE: Cardiac tamponade is a medical emergency situation associated with a high rate of life-threatening complications, even after immediate interventions. Our aim was to characterize the acute inflammatory consequences of this event in a clinically relevant large animal model. METHODS: Cardiac tamponade was induced for 60 min in anesthetized, ventilated and thoracotomized minipigs by intrapericardial fluid administration, the mean arterial pressure (MAP) being maintained in the interval of 40-45 mm Hg (n = 8). A further group (n = 7) served as sham-operated control. The global macrohemodynamics, including the right- and left-heart end-diastolic volumes (RHEDV and LHEDV), the pulmonary vascular resistance index (PVRI) and the superior mesenteric artery (SMA) flow, were monitored for 240 min, and the intestinal microcirculatory changes (pCO2 gap) were evaluated by indirect tonometry. Blood samples were taken for the determination of cardiac troponin T and vasoactive inflammatory mediators, including histamine, nitrite/nitrate, big-endothelin, superoxide and high-mobility group box protein-1 levels in association with intestinal leukocyte and complement activation. RESULTS: The cardiac tamponade induced significant decreases in MAP, cardiac output, LHEDV and SMA flow, while the PVRI and the pCO2 gap increased significantly. After the removal of fluid from the pericardial sac, the MAP and the LHEDV were decreased, while the PVRI and the pCO2 gap remained elevated when compared with those in the sham-operated group. In the posttamponade period, the abrupt release of inflammatory mediators was accompanied by a significant splanchnic leukocyte accumulation and complement activation. CONCLUSIONS: The macrocirculatory and splanchnic microcirculatory disturbances were accompanied by a significant proinflammatory reaction; endothelin and the complement system may be significant components of the inflammatory cascade that is activated in this porcine model of pericardial tamponade.

Effect of Origanum vulgare L. leaves on rumen fermentation, production, and milk fatty acid composition in lactating dairy cows.

This experiment investigated the effects of dietary supplementation of Origanum vulgare L. leaf material (OR) on rumen fermentation, production, and milk fatty acid composition in dairy cows. The experimental design was a replicated 4 × 4 Latin square with 8 rumen-cannulated Holstein cows and 20-d experimental periods. Treatments were control (no OR supplementation), 250 g/cow per day OR (LOR), 500 g/d OR (MOR), and 750 g/d OR (HOR). Oregano supplementation had no effect on rumen pH, volatile fatty acid concentrations, and estimated microbial protein synthesis, but decreased ammonia concentration and linearly decreased methane production per unit of dry matter intake (DMI) compared with the unsupplemented control: 18.2, 16.5, 11.7, and 13.6g of methane/kg of DMI, respectively. Proportions of rumen bacterial, methanogen, and fungal populations were not affected by treatment. Treatment had no effect on total-tract apparent digestibility of dietary nutrients, except neutral detergent fiber digestibility was slightly decreased by all OR treatments compared with the control. Urinary N losses and manure odor were not affected by OR, except the proportion of urinary urea N in the total excreted urine N tended to be decreased compared with the control. Oregano linearly decreased DMI (28.3, 28.3, 27.5, and 26.7 kg/d for control, LOR, MOR, and HOR, respectively). Milk yield was not affected by treatment: 43.4, 45.2, 44.1, and 43.4 kg/d, respectively. Feed efficiency was linearly increased with OR supplementation and was greater than the control (1.46, 1.59, 1.60, and 1.63 kg/kg, respectively). Milk composition was unaffected by OR, except milk urea-N concentration was decreased. Milk fatty acid composition was not affected by treatment. In this short-term study, OR fed at 250 to 750 g/d decreased rumen methane production in dairy cows within 8h after feeding, but the effect over a 24-h feeding cycle has not been determined. Supplementation of the diet with OR linearly decreased DMI and increased feed efficiency. Oregano had no effects on milk fatty acid composition.

Effect of essential oils on ruminal fermentation and lactation performance of dairy cows.

Three experiments (Exp.) were conducted to study the effects of dietary addition of an essential oil product (EO) based on eugenol and cinnamaldehyde (0, control, or 525 mg/d of Xtract 6965; Pancosma SA, Geneva, Switzerland) on ruminal fermentation, total-tract digestibility, manure gas emissions, N losses, and dairy cow performance. In Exp. 1 and 3, the EO supplement was added to the vitamin-mineral premix. In Exp. 2, EO was top-dressed. Experiments 1 and 2 were crossover designs with 20 multiparous Holstein cows each (including 4 and 8 ruminally cannulated cows, respectively) and consisted of two 28-d periods. Intake of dry matter did not differ between treatments. Most ruminal fermentation parameters were unaffected by EO. Concentrations of ammonia (Exp. 1), isobutyrate (Exp. 1 and 2), and isovalerate (Exp. 1) were increased by EO compared with the control. Apparent total-tract digestibility of nutrients was similar between treatments, except total-tract digestibility of neutral-detergent fiber, which was increased or tended to be increased by EO in Exp. 1 and 2. Manure emissions of ammonia and methane were unaffected by EO. Blood plasma and milk urea-N concentrations and urinary N losses were increased by EO compared with the control in Exp. 1, but not in Exp. 2. Average milk yield, 3.5% fat-corrected milk yield, and milk fat, protein, and lactose concentrations were unaffected by treatment. Urinary excretion of purine derivatives, a marker for microbial protein production in the rumen, was greater in cows receiving the EO diet in Exp. 1, but not in Exp. 2. In Exp. 3, 120 Holstein cows were grouped in pens of 20 cows/pen in a 12-wk experiment to study production effects of EO. Dry matter intake, milk yield (a trend for a slight decrease with EO), milk components, milk urea N, and feed efficiency were similar between treatments. Results from these studies indicate that supplementing dairy cows with 525 mg/d of Xtract 6965 had moderate effects on ruminal fermentation, but consistently increased ruminal isobutyrate concentration and tended to increase total-tract digestibility of neutral-detergent fiber. Under the conditions of these experiments, Xtract 6965 fed at 525 mg/d did not affect milk production or composition.

Short communication: Feed utilization and its associations with fertility and productive life in 11 commercial Pennsylvania tie-stall herds.

The objectives of this study were to quantify the relationships of various definitions of feed utilization with both fertility and productive life. Intake and body measurement data were collected monthly on 970 cows in 11 tie-stall herds for 6 consecutive months. Measures of feed utilization for this study were dry matter intake (DMI), dry matter intake efficiency (DME, defined as 305-d fat-corrected milk/305-d DMI), DME with intake adjusted for maintenance requirements (DMEM), crude protein efficiency (defined as 305-d protein yield/305-d crude protein intake), and 2 definitions of residual feed intake (RFI). The first, RFI(reg), was calculated by regressing daily DMI on daily milk, fat, and protein yields, body weight (BW), daily body condition score (BCS) gain or loss, the interaction between BW and BCS gain or loss, and days in milk. The second, RFI(NRC), was estimated by subtracting 305-d DMI predicted according to their fat-corrected milk and BW from actual 305-d DMI. Data were analyzed with 8-trait animal models and included one measure of feed utilization and milk, fat, and protein yields, BW, BCS, days open (DO), and productive life (PL). The genetic correlation between DME and DO was 0.53 (± 0.19) and that between DME and PL was 0.66 (± 0.10). These results show that cows who had higher feed efficiency had greater DO (undesirable) and greater PL (desirable). Results were similar for the genetic correlation between DO and crude protein efficiency (0.42). Productive life had genetic correlations of -0.22 with BW and -0.48 with BCS, suggesting that larger, fatter cows in this study had shorter PL. Correlations between estimated breeding values for feed utilization and official sire genetic evaluations for fertility were in agreement with the results from the multiple-trait models. Selection programs intended to enhance feed efficiency should factor relationships with functional traits to avoid unfavorable effects on cow fertility.

Immune and production responses of dairy cows to postruminal supplementation with phytonutrients.

This study investigated the effect of phytonutrients (PN) supplied postruminally on nutrient utilization, gut microbial ecology, immune response, and productivity of lactating dairy cows. Eight ruminally cannulated Holstein cows were used in a replicated 4×4 Latin square. Experimental periods lasted 23 d, including 14-d washout and 9-d treatment periods. Treatments were control (no PN) and daily doses of 2g/cow of either curcuma oleoresin (curcumin), garlic extract (garlic), or capsicum oleoresin (capsicum). Phytonutrients were pulse-dosed into the abomasum of the cows, through the rumen cannula, 2 h after feeding during the last 9 d of each experimental period. Dry matter intake was not affected by PN, although it tended to be lower for the garlic treatment compared with the control. Milk yield was decreased (2.2 kg/d) by capsicum treatment compared with the control. Feed efficiency, milk composition, milk fat and protein yields, milk N efficiency, and 4.0% fat-corrected milk yield were not affected by treatment. Rumen fermentation variables, apparent total-tract digestibility of nutrients, N excretion with feces and urine, and diversity of fecal bacteria were also not affected by treatment. Phytonutrients had no effect on blood chemistry, but the relative proportion of lymphocytes was increased by the capsicum treatment compared with the control. All PN increased the proportion of total CD4(+) cells and total CD4(+) cells that co-expressed the activation status signal and CD25 in blood. The percentage of peripheral blood mononuclear cells (PBMC) that proliferated in response to concanavalin A and viability of PBMC were not affected by treatment. Cytokine production by PBMC was not different between control and PN. Expression of mRNA in liver for key enzymes in gluconeogenesis, fatty acid oxidation, and response to reactive oxygen species were not affected by treatment. No difference was observed due to treatment in the oxygen radical absorbance capacity of blood plasma but, compared with the control, garlic treatment increased 8-isoprostane levels. Overall, the PN used in this study had subtle or no effects on blood cells and blood chemistry, nutrient digestibility, and fecal bacterial diversity, but appeared to have an immune-stimulatory effect by activating and inducing the expansion of CD4 cells in dairy cows. Capsicum treatment decreased milk yield, but this and other effects observed in this study should be interpreted with caution because of the short duration of treatment.

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.

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.