Changes in the Relationship between Ionized and Total Calcium in Clinically Healthy Dairy Cows in the Period around Calving

We aimed to establish a model for prediction of iCa from tCa, using multivariable regressions with diverse blood constituents. Blood was taken from 14 cows at days −2, 0, 2, 4, 7, and 14 relative to parturition. Cows were clinically healthy, and no hypocalcaemia prophylaxis and treatment were applied. Total calcium and further parameters were determined from frozen serum. Ionized calcium, blood gases, and electrolytes were determined from heparin-stabilized blood samples. Linear regression between iCa and tCa was estimated. Precision improved only slightly using a multivariable model. Best precision was achieved when estimating the iCa:tCa ratio from other blood constituents. To identify the reason behind the poorly predictive value of tCa for iCa, the relative changes of iCa and tCa around calving were calibrated to the respective values of day −2 (=100%) for each cow. An increase in the iCa:tCa ratio was observed from 0.43 at day −2 to 0.48 at day 0, followed by a gradual decrease towards 0.43 at day 7. We conclude that routine measurement of iCa should be implemented in the diagnosis of hypocalcaemia. An optimized estimate of iCa from tCa with non-esterified fatty acids (NEFA), beta-hydroxybutyric acid, cholesterol, and phosphorous as co-predictors is still poorly satisfying

Systematic genetic analysis of pediatric patients with autoinflammatory diseases

Monogenic autoinflammatory diseases (AID) encompass a growing group of inborn errors of the innate immune system causing unprovoked or exaggerated systemic inflammation. Diagnosis of monogenic AID requires an accurate description of the patients’ phenotype, and the identification of highly penetrant genetic variants in single genes is pivotal. We performed whole exome sequencing (WES) of 125 pediatric patients with suspected monogenic AID in a routine genetic diagnostic setting. Datasets were analyzed in a step-wise approach to identify the most feasible diagnostic strategy. First, we analyzed a virtual gene panel including 13 genes associated with known AID and, if no genetic diagnosis was established, we then analyzed a virtual panel including 542 genes published by the International Union of Immunological Societies associated including all known inborn error of immunity (IEI). Subsequently, WES data was analyzed without pre-filtering for known AID/IEI genes. Analyzing 13 genes yielded a definite diagnosis in 16.0% (n = 20). The diagnostic yield was increased by analyzing 542 genes to 20.8% (n = 26). Importantly, expanding the analysis to WES data did not increase the diagnostic yield in our cohort, neither in single WES analysis, nor in trio-WES analysis. The study highlights that the cost- and time-saving analysis of virtual gene panels is sufficient to rapidly confirm the differential diagnosis in pediatric patients with AID. WES data or trio-WES data analysis as a first-tier diagnostic analysis in patients with suspected monogenic AID is of limited benefit

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Effect of menthol from the group of plant bioactive lipid compounds in hypocalcemia prophylaxis of early lactating dairy cows with special focus on ionized calcium

Die Hypokalzämie der Milchkuh ist ein großes Problem im peripartalen Zeitraum. Entscheidend für die Diagnostik ist eine sichere Bestimmung der Kalziumkonzentration. In der Praxis ist nach wie vor die Bestimmung des Gesamtkalziums (tCa) weit verbreitet, obwohl einige Studien zu dem Ergebnis kamen, dass eine verlässliche Aussage nur durch die Bestimmung des ionisierten Kalziums (iCa) erbracht werden kann. Einige Untersuchungen wiesen bereits darauf hin, dass es zu einer Änderung des iCa-Anteils am tCa um den Geburtszeitraum kommt. Diese Feststellung sollte in unserer Studie für Kühe ohne gezielte Hypokalzämieprophylaxe geprüft werden. Unser Ziel war es zudem, unter Berücksichtigung verschiedener Blutkennwerte ein Modell zur Vorhersage von iCa aus tCa unter Verwendung multivariabler Regressionen zu erstellen. Dazu wurden die Blutwerte für Blutgase sowie verschiedene Elektrolyte, Enzyme und Metaboliten von 14 Kühen der Rasse Holstein Friesian verwendet. Alle Kühe waren klinisch gesund und erhielten keine Hypokalzämieprophylaxe oder -therapie. Die Blutentnahme erfolgte an den Tagen -2, 0, 2, 4, 7 in Bezug auf die Abkalbung. Bestimmt wurden tCa, iCa, Albumin, Gesamtprotein, Blutgase, einige Enzyme und Elektrolyte. Zunächst wurde eine lineare Regression zwischen iCa und tCa modelliert, deren Vorhersagkraft unbefriedigend war. Eine leichte Verbesserung ergab sich durch die Verwendung eines multivariablen Modells mit weiteren Blutkennwerten. Die Präzision konnte anschließend bei einem multivariablen Modell zur Schätzung des iCa:tCa-Verhältnisses mit Verwendung weiterer Faktoren (nicht veresterte Fettsäuren, Beta-Hydroxybuttersäure, Cholesterin, Phosphor) verbessert werden. Insgesamt zeigte sich die Vorhersagekraft jedoch auch mit dem verbesserten Modell als unbefriedigend. Ursächlich waren relative Veränderungen von iCa und tCa um den Kalbungszeitraum. Dies wurde anhand einer Kalibrierung der relativen Veränderungen von iCa und tCa auf die jeweiligen Werte von Tag -2 (=100%) nachgewiesen. Es konnte eine Zunahme des iCa:tCa-Verhältnisses von 0,43 am Tag -2 auf 0,48 am Tag 0 beobachtet werden, sowie ein langsamer Rückgang auf 0,43 bis zum Tag 7. Damit lässt sich bestätigen, dass es um die Kalbung zu einer Verschiebung des Verhältnisses zwischen iCa und tCa kommt. Die generelle Annahme, dass das iCa 50% des tCa beträgt, erwies sich somit als höchst unpräzise. Auch die Vorhersage durch ein Modell mit Einbeziehung weiterer Blutkennwerte ist unbefriedigend. Um eine genaue Aussage über den Kalziumhaushalt zu treffen, erscheint es sinnvoll direkt das iCa zu bestimmen. Hierbei ist eine standardisierte Heparinisierung des frischen Vollblutes vorzunehmen, da es durch Erhöhungen der Heparinkonzentration zu erniedrigten Werten für iCa kommt. In einem weiteren Versuch widmeten wir uns der Prophylaxe der Hypokalzämie mittels eines PBLC-haltigen Futterzusatzstoffes. Einige Studien weisen darauf hin, dass das PBLC Menthol die Eigenschaft besitzt, die Kalziumaufnahme im Pansen zu erhöhen. In der statistischen Auswertung unserer Studie verwendeten wir insgesamt 29 Tiere der Rasse Braunvieh (BS) und 41 Tiere der Rasse Holstein Friesian (HF). Die Tiere wurden nach Rasse in je eine Kontroll- und Fütterungsgruppe geteilt. Letztere erhielt von 8 Tagen vor dem erwarteten Abkalben bis 80 Tage nach dem Abkalben 1,7 g/d mentholreiches PBLC. Untersucht wurden die Auswirkungen auf Blutmineralien, Milchleistung und Milchzusammensetzung sowie der Effekt auf den Body Condition Score (BCS) in der peripartalen Periode. Eine signifikante Rasse x Behandlungs-Interaktion zeigte sich für iCa, die auf einer Erhöhung des iCa um 0,03 mmol‧l-1 ausschließlich bei den HF-Tieren beruhte. Bei der Prävalenz für subklinische Hypokalzämie war ebenfalls nur bei den HF-Tieren ein positiver Effekt durch die Fütterung erkennbar, der aufgrund der geringen Gruppengröße jedoch nicht statistisch absicherbar war. Die Beschränkung des Effektes auf den Kalziumhaushalt von HF-Tieren könnte in der geringeren Anfälligkeit des BS gegenüber Hypokalzämie begründet sein. Einen direkten Einfluss auf den BCS konnte die Behandlung nicht zeigen. Auffällig war jedoch, dass sich die PBLC-supplementierten HF- und BSGruppen nicht voneinander unterschieden, während die Kontrollgruppe der HF eine deutlichere Abnahme des BCS nach der Kalbung zeigte als die Kontrollgruppe des BS. Dies deutet bei PBLC-supplementierten HF-Tieren auf eine besseren Kompensation der negativen Auswirkungen einer höheren Milchleistung in Bezug auf die Körperkondition hin. Einen positiven Einfluss zeigte die Fütterung des PBLC auf die Milchleistung beider Rassen. Die Milchzusammensetzung blieb davon unberührt. Ursache ist hier wahrscheinlich eine erhöhte Futteraufnahme durch die Zugabe von PBLC, was jedoch einer weiteren Abklärung bedarf. Zusammenfassend kann geschlussfolgert werden, dass der Einsatz des mentholhaltigen PBLC die Kalziumkonzentration im Blut von HF-Kühen im Zeitraum von 4 Wochen nach der Geburt positiv beeinflussen kann. Zudem wird die Milchleistung sowohl bei den HF-Tieren als auch bei den BS-Tieren erhöht.Hypocalcemia of dairy cows is a very important problem in the peripartum period. A reliable determination of the calcium concentration is essential for diagnosis. In practice, the determination of total calcium (tCa) is still frequently used although some studies indicated that a valid assessment can be made only by the determination of ionized calcium (iCa). Some studies already indicated that there is a change in the proportion of iCa in tCa around calving. The latter proposition was to be tested in our study for cows without specific hypocalcemia prophylaxis. Our further aim also to create a model for predicting iCa from tCa using multivariable regressions, considering concentrations of albumin and total protein, blood gases, as well as different electrolytes, enzymes and metabolites. For this study, 14 cows of Holstein Friesian were sampled, all of which were clinically healthy and not receiving hypocalcemia prophylaxis or therapy. Blood sampling was performed on days -2, 0, 2, 4, 7 relative to calving. Initially, linear regression was modelled but its predictive power was unsatisfactory. A slight improvement was obtained by using a multivariable model with additional blood values. In a further attempt, precision was improved in a multivariable model estimating the iCa:tCa ratio with the use of additional factors (nonesterified fatty acids, betahydroxybutyric acid, cholesterol, phosphorus). Overall, however, the predictive power the multivariable models was also unsatisfactory. The reason could be found in relative changes of iCa and tCa around the calving period. This was demonstrated using a calibration of the relative changes of iCa and tCa to the respective values of day -2 (=100%). An increase in the iCa:tCa ratio from 0.43 at day -2 to 0.48 at day 0 was observed, followed by a slow decrease to 0.43 until day 7, suggesting that there is a shift in the ratio between iCa and tCa around calving. Thus, the literature assumption that iCa is 50% of tCa could not be confirmed. Prediction by a model with inclusion of other blood values was also unsatisfactory. In order to make a precise prediction of the calcium status, it seems useful to determine the iCa directly. Decisive for the determination of the iCa is a standardized heparinization of blood because increased heparin concentrations lead to lower measurement values of iCa. In another experiment, we focused on the prophylaxis of hypocalcemia using a feed additive containing PBLC. Some studies indicated that the PBLC menthol has the effect of increasing calcium absorption in the rumen. In the statistical analysis of our study, we used a total of 29 animals of the Brown Swiss (BS) and 41 animals of the Holstein Friesian (HF) breeds. The animals of each breed were divided into a control and a feeding group. The latter group received 1.7 g/d of menthol-rich PBLC from 8 days before expected calving to 80 days after calving. The effects on blood minerals, milk yield, and milk composition, as well as the effect on body condition score (BCS) in the peripartum period were analyzed. A significant breed x treatment interaction was seen for iCa, evidenced by a 0.03 mmol‧l-1 increase in iCa in HF animals only. A positive effect of PBLC feeding was also indicated regarding the prevalence for subclinical hypocalcemia, in the HF animals; however, this could not be validated statistically because of the low sample size. The restriction of PBLC effects to the calcium balance HF cows could be suspected to be due to the lower predisposition of the BS breed to hypocalcemia. The treatment did not have a direct effect on BCS. However, it was obvious that the PBLC-fed HF and BS feeding groups did not differ from each other whereas the HF control group lost more body condition after calving compared to the BS control group. This may be taken as an indication that PBLC-fed HF animals could better compensate the negative effects of higher milk yield on body condition. A positive effect was shown by feeding the PBLC on the milk yield of both breeds. The milk composition was unaffected. This is possibly caused by increased feed intake due to the addition of PBLC, but this requires further investigation. In conclusion, the use of the menthol-containing PBLC can positively influence the blood calcium concentration of HF cows in the period around calving. In addition, it increases milk yield in both HF and BS animals

Species richness and biomass explain spatial turnover in ecosystem functioning across tropical and temperate ecosystems

Predicting ecosystem functioning at large spatial scales rests on our ability to scale up from local plots to landscapes, but this is highly contingent on our understanding of how functioning varies through space. Such an understanding has been hampered by a strong experimental focus of biodiversity–ecosystem functioning research restricted to small spatial scales. To address this limitation, we investigate the drivers of spatial variation in multitrophic energy flux—a measure of ecosystem functioning in complex communities—at the landscape scale. We use a structural equation modelling framework based on distance matrices to test how spatial and environmental distances drive variation in community energy flux via four mechanisms: species composition, species richness, niche complementarity and biomass. We found that in both a tropical and a temperate study region, geographical and environmental distance indirectly influence species richness and biomass, with clear evidence that these are the dominant mechanisms explaining variability in community energy flux over spatial and environmental gradients. Our results reveal that species composition and trait variability may become redundant in predicting ecosystem functioning at the landscape scale. Instead, we demonstrate that species richness and total biomass may best predict rates of ecosystem functioning at larger spatial scales.</jats:p

Species richness and biomass explain spatial turnover in ecosystem functioning across tropical and temperate ecosystems

Predicting ecosystem functioning at large spatial scales rests on our ability to scale up from local plots to landscapes, but this is highly contingent on our understanding of how functioning varies through space. Such an understanding has been hampered by a strong experimental focus of biodiversity–ecosystem functioning research restricted to small spatial scales. To address this limitation, we investigate the drivers of spatial variation in multitrophic energy flux—a measure of ecosystem functioning in complex communities—at the landscape scale. We use a structural equation modelling framework based on distance matrices to test how spatial and environmental distances drive variation in community energy flux via four mechanisms: species composition, species richness, niche complementarity and biomass. We found that in both a tropical and a temperate study region, geographical and environmental distance indirectly influence species richness and biomass, with clear evidence that these are the dominant mechanisms explaining variability in community energy flux over spatial and environmental gradients. Our results reveal that species composition and trait variability may become redundant in predicting ecosystem functioning at the landscape scale. Instead, we demonstrate that species richness and total biomass may best predict rates of ecosystem functioning at larger spatial scales

Model: TIP47 recruits LD membranes to the membranous web <i>via</i> interaction with NS5A.

<p><b>In uninfected cells</b>: TIP47 is involved in the retrograde trafficking pathway by interacting with Rab9 at late endosomes, and functions as an “exchangeable” LD-associated protein that is mostly present in the cytosol but moves to LDs upon rapid fat storage. <b>In HCV-infected cells</b>: TIP47 interacts with NS5A-containing replication complexes at the ER and NS5A at LDs, thus making LD membranes accessible for HCV RNA replication for either integration of LDs into the membranous web, or as source of energy for the creation of the membranous web. HCV infection also increases endosome-to-Golgi trafficking <i>via</i> the up-regulation of Rab9 expression.</p

TIP47 associates with LDs during HCV infection.

<p>A) Western blot analysis of cell extracts or isolated lipid droplet fractions from either naïve Huh7.5 or replicon Cells (genotype 1b). B) Quantification of number of LDs (RedO stain) and LD area per cell in either naïve Huh7.5 or replicon cells (genotype 1b) (mean ± standard deviation; n>100, *P<0.05). Quantification was done using Volocity software (Perkin Elmer). C) Indirect immunofluorescence of TIP47 (red) and NS5A (white) in either non-transfected Huh7.5 cells, or replicon Cells (genotype 1b), Lipid droplets were stained with Bodipy493/503 (green). The scale bar = 10 µm. The close-up images are shown with 2.5 fold higher magnification. D) Indirect immunofluorescence of TIP47 (red) and NS5A (white) in either non-transfected Huh7 Lunet cells, or transfected with full-length Luc-JFH1 (genotype 2a). Lipid droplets were stained with Bodipy493/503 (green). The scale bar = 10 µm. The close-up images are shown with 2.5 fold higher magnification. E) Quantification of the percentage of NS5A that colocalizes with TIP47 in either Replicon-expressing cells or Huh7 Lunet cells transfected with Luc-JFH1 (as seen in B) (mean ± standard deviation; n≥30). Quantification was done with the automatic colocalization program in Volocity software (Perkin Elmer). F) Quantification of the percentage of NS5A that colocalizes with LDs in either replicon-expressing cells (as seen in C) or Huh7 Lunet cells transfected with Luc-JFH1 (as seen in D) (mean ± standard deviation; n≥30, ***P<0.001). Quantification was done with the automatic colocalization program in Volocity (Perkin Elmer).</p