Osimertinib and anti-HER3 combination therapy engages immune dependent tumor toxicity via STING activation in trans.

Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors due to toxicity, relying only on palliative chemotherapy and the third-generation tyrosine kinase inhibitor (TKI) osimertinib. This new drug extends lifespan by 9-months vs. second-generation TKIs, but unfortunately, cancers relapse due to resistance mechanisms and the lack of antitumor immune responses. Here we explored the combination of osimertinib with anti-HER3 monoclonal antibodies and observed that the immune system contributed to eliminate tumor cells in mice and co-culture experiments using bone marrow-derived macrophages and human PBMCs. Osimertinib led to apoptosis of tumors but simultaneously, it triggered inositol-requiring-enzyme (IRE1α)-dependent HER3 upregulation, increased macrophage infiltration, and activated cGAS in cancer cells to produce cGAMP (detected by a lentivirally transduced STING activity biosensor), transactivating STING in macrophages. We sought to target osimertinib-induced HER3 upregulation with monoclonal antibodies, which engaged Fc receptor-dependent tumor elimination by macrophages, and STING agonists enhanced macrophage-mediated tumor elimination further. Thus, by engaging a tumor non-autonomous mechanism involving cGAS-STING and innate immunity, the combination of osimertinib and anti-HER3 antibodies could improve the limited therapeutic and stratification options for advanced stage lung cancer patients with mutant EGFR.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Immunization against gonadotropin-releasing factor (GnRF) in market gilts: Effect on growth and carcass parameters, and impact of immunization timing.

Reproductive cycling in fattening gilts can be associated with undesirable effects, such as estrus-related aggressive behavior, reduced feed intake and, in production systems where gilts are co-housed with entire males, unwanted pregnancy. Immunization against Gonadotrophin Releasing Factor (IM) can temporarily suppress ovarian activity, including related negative consequences on animal welfare and productivity. Feed intake has been shown to be higher after IM, resulting in both increased growth and increased carcass fat. A series of studies was conducted to confirm these effects on production and look at their dynamics over time. Three trials were performed to a similar design, each involving 240 gilts divided into 4 experimental groups at 12 weeks of age. One group remained untreated while the others had the two dose, IM course completed 8, 6 or 4 weeks before harvest, which was on a single day at 24, 25 or 26 weeks of age depending on the study. Feed intake was measured daily and bodyweight weekly, allowing growth parameters to be calculated on a weekly basis and for specific longer periods. Carcass weight, backfat depth and lean meat percentage were recorded at harvest. No effects were observed before the second application of the immunological product (V2) and completion of the IM course. Starting in the second week after V2 all IM groups showed a marked and consistent increase in Average Daily Feed Intake (ADFI), typically peaking at over 120% of the control group 3 to 4 weeks after V2 and then slowly declining, but still remaining elevated at 8 weeks. Weekly Average Daily Gain (ADG) showed a similar pattern but with a faster decline, resulting in the initially favorable impact on feed efficiency becoming less favorable as the V2 to harvest interval (V2H) progressed. Carcass weights were higher in IM gilts and backfat depths were greater, with the effects increasing with increasing V2H. Correspondingly, carcass lean meat percentage tended to decrease, although the higher carcass weights meant that the absolute weight of lean meat remained similar or higher. Carcass yield was generally unaffected by IM, but some between-group differences were statistically significant, and it is possible that different factors predominated at different times after V2, creating a complex relationship with V2H duration. The optimum IM protocol will depend on local conditions and production objectives but, as a generalization and assuming ad libitum feeding, a shorter V2H will favor efficient growth, while a longer duration will maximize carcass changes, such as increased fat coverage. It is suggested that the growth performance changes seen after IM in gilts might be viewed as a process of adjustment to a heavier and fatter target body type.

Herd-level factors associated with piglet weight at weaning, kilograms of piglets weaned per sow per year and sow feed conversion.

To understand the production factors that affect conclusive parameters of sow herd performance can improve the use of the resources and profitability of farm. The objective of this study was to identify associations and quantify the effects of a set of factors related to piglet weight at weaning (PWW), kilograms of piglets weaned per sow per year (kgPWSY) and sow feed conversion (SFC). Data from 150 farms were collected, for a total study population of 135 168 sows, including gilt replacement, breeding (mating), gestation and farrowing/lactation phases. A questionnaire focusing on reproductive performance, management, facilities, feeding, health and biosafety was administered. Multiple linear regression models were used to assess associations among factors with each of the three dependent variables. Increased duration of lactation was positively associated with PWW, kgPWSY and SFC. The increase in the number of live born pigs per litter was positively associated with kgPWSY and with SFC. Farms with higher PWW had farrowing room humidifiers, did not surgically castrate male piglets and used quaternary ammonia compounds for farrowing room disinfection. Farms with higher kgPWSY used lined ceilings in farrowing rooms and winter feeds with higher CP percentages in gestation; they also had more farrowings per sow per year. Sow feed conversion was worse in farms with partly slatted floors during gestation, in farms feeding lactating sows six times a day or ad libitum and farms with a higher sow-handler ratio. This study indicates that farms can increase PWW and kgPWSY and improve the SFC by changing one or more management, biosafety and feeding practices or facilities as well as by focusing on improving several performance parameters, particularly increasing the duration of lactation and the number of live born pigs per litter.

Oxidative stress and biomarker responses in the Atlantic halibut after long term exposure to elevated CO2 and a range of temperatures.

CO2 emissions from human activities are increasing, resulting in greater rates of change in the oceans, exceeding any other event in geological and historical records over the past 300 million years. Oceans are warming and pH levels are decreasing. Marine organisms will need to respond to multiple stressors and the potential consequences of global change-related effects in fish needs to be investigated. Fish are affected by many biotic and abiotic environmental variables, including temperature and CO2 fluctuations, and it is therefore critical to investigate how these variables may affect physiological and biochemical processes. We investigated the effects of elevated CO2 levels (pH of 8.0, which served as a control, or 7.6, which is predicted for the year 2100) combined with exposure to different temperatures (5, 10, 12, 14, 16, and 18 °C) in the Atlantic halibut (Hippoglossus hippoglossus) during a three month experiment. Since regulation of reactive oxygen species (ROS) is crucial for physiological processes the focus was on the antioxidant defense system and we assessed the effects on catalytic activities of antioxidant enzymes (SOD, CAT, GR, GST, GPx). In addition we also analyzed effects on cholinesterase enzymes (AChE and BChE), and CYP1A enzyme activities (EROD). The treatments resulted in oxidative stress, and damage was evident in the form of protein carbonyls which were consistently higher in the elevated CO2-treated fish at all temperatures. Analyses of antioxidant enzymes did not show the same results, suggesting that the exposure to elevated CO2 increased ROS formation but not defences. The antioxidant defense system was insufficient, and the resulting oxidative damage could impact physiological function of the halibut on a cellular level.

Performance, health and physiological responses of newly weaned feedlot cattle supplemented with feed-grade antibiotics or alternative feed ingredients.

With increased regulations regarding the use of feed-grade antimicrobials in livestock systems, alternative strategies to enhance growth and immunity of feedlot cattle are warranted. Hence, this experiment compared performance, health and physiological responses of cattle supplemented with feed-grade antibiotics or alternative feed ingredients during the initial 60 days in the feedlot. Angus×Hereford calves (63 steers+42 heifers) originating from two cow-calf ranches were weaned on day -3, obtained from an auction yard on day -2 and road-transported (800 km; 12 h) to the feedlot. Upon arrival on day -1, shrunk BW was recorded. On day 0, calves were ranked by sex, source and shrunk BW, and allocated to one of 21 pens. Pens were assigned to receive (7 pens/treatment) a free-choice total mixed ration containing: (1) lasalocid (360 mg/calf daily of Bovatec; Zoetis, Florham Park, NJ, USA)+chlortetracycline (350 mg/calf of Aureomycin at cycles of 5-day inclusion and 2-day removal from diet; Zoetis) from days 0 to 32, and monensin only (360 mg/calf daily of Rumensin; Elanco Animal Health, Greenfield, IN, USA) from days 33 to 60 (PC), (2) sodium saccharin-based sweetener (Sucram at 0.04 g/kg of diet dry matter; Pancosma SA; Geneva, Switzerland)+plant extracts containing eugenol, cinnamaldehyde and capsicum (800 mg/calf daily of XTRACT Ruminants 7065; Pancosma SA) from days 0 to 32 and XTRACT only (800 mg/calf daily) from days 33 to 60 (EG) or (3) no supplemental ingredients (CON; days 0 to 60). Calves were assessed for bovine respiratory disease (BRD) signs and dry matter intake was recorded from each pen daily. Calves were vaccinated against BRD pathogens on days 0 and 22. Shrunk BW was recorded on day 61, and blood samples collected on days 0, 6, 11, 22, 33, 43 and 60. Calf ADG was greater (P=0.04) in PC v. EG and tended (P=0.09) to be greater in PC v. CON. Feed efficiency also tended (P=0.09) to be greater in PC v. CON, although main treatment effect for this response was not significant (P=0.23). Mean serum titers against bovine respiratory syncytial virus were greater in EG v. PC (P=0.04) and CON (tendency; P=0.08). Collectively, the inclusion of alternative feed ingredients prevented the decrease in feed efficiency when chlortetracycline and ionophores were not added to the initial feedlot diet, and improved antibody response to vaccination against the bovine respiratory syncytial virus in newly weaned cattle.

Increased activity of lysozyme and complement system in Atlantic halibut exposed to elevated CO2 at six different temperatures.

Ocean acidification and rising seawater temperature are environmental stressors resulting from the continuous increase of the atmospheric CO2 concentration due to anthropogenic activities. As a consequence, marine fish are expected to undergo conditions outside of their tolerance range, leading to physiological challenges with possible detrimental implications. Our research group has previously shown that exposure to elevated CO2 modulated the immune system of the Atlantic halibut. To further investigate this finding, we analysed non-specific immune components in blood plasma of Atlantic halibut (Hippoglossus hippoglossus) juveniles acclimated to six different temperatures (5, 10, 12, 14, 16 and 18 °C), and to water pH of 8.0 (control) or 7.6 (predicted for year 2100) for three months. Plasma ions (K+, Na+, Ca++, Cl-) and lactate concentrations were also measured. The analysis of plasma ions did not show any trends related to temperature or CO2 exposure, and the majority of the experimental fish were able to maintain ionic balance. The results show that both innate immune components (lysozyme and alternative complement system) had increased activities in response to elevated CO2, representing a CO2-related impact on the halibut's immune system. The increased activity of lysozyme and complement system is possibly part of the acclimatization process, and might be protective.

MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate.

OBJECTIVE: The aim of this study was to investigate the contribution of 6 polymorphic variants of the MSX1 gene in non-syndromic cleft lip and/or palate (NSCL/P). METHODS: Three hundred and fifty-eight individuals (158 NSCL/P cases and 200 controls) were genotyped by TaqMan allelic discrimination using predesigned SNP assays. Statistical analyses were conducted using the software spss 15.0 and the r statistical suite. Haplotype block structure and haplotype frequencies were determined using the Haploview. A P-value of 0.05 and confidence interval of 95% were used for all of statistical tests. RESULTS: The patients with non-syndromic cleft lip and/or palate were characterized by similar distribution of MSX1 genotypes and allele in comparison to subjects without oral clefts (P > 0.05). Two haplotype blocks were constructed with polymorphisms of MSX1 gene and haplotypes formed showed a similar frequency in patients with and without oral clefts. CONCLUSIONS: The present study provides no evidence that MSX1 polymorphisms (rs3775261, rs1042484, rs12532, rs6446693, rs4464513 and rs1907998) play a major role in NSCL/P.

Influence of excipients and technological process on anti-inflammatory activity of quercetin and Achyrocline satureioides (Lam.) D.C. extracts by oral route.

The flavonoids quercetin, 3-O-methylquercetin and luteolin play an important role in the anti-inflammatory activity of Achyrocline satureioides ethanol extracts when administered intraperitoneally. The present work describes the oral anti-inflammatory effect of quercetin and A. satureioides extracts and the role played by the solvent concentration, adjuvant and drying processes of freeze-drying (FD) or spray-drying (SD) on the effect. The best anti-edema effect was observed with 250 mg/kg body wt of the freeze-dried powder (FDP), prepared with 40% (v/v) ethanol (FDP40). In contrast, 250 mg/kg body wt of FDP80, prepared with ethanol 80% (ES80), did not significantly inhibit the carrageenan-induced rat paw edema. However, when ES80 was freeze-dried in the presence of polysorbate 80 (FDP80-P80) or spray-dried in the presence of colloidal silicon dioxide (CSD) and P80 (SDP80), both dried extracts became more active. Quercetin suspension in saline did not inhibit paw edema, but the mixture of quercetin with polysorbate 80 was effective in edema inhibition by the oral route. Aqueous extract (ESAQ), freeze-dried (FDPAQ, FDPAQ-P80) or spray-dried (SDPAQ) did not exhibit the edema-inhibition effect. Taken together, the results point to the following order of efficacy (at 4 h, for example): FDP40 > indomethacin > SDP40 > SDP80 = FDP80-80 > Quercetin-P80. Additionally, the FDP40, SDP40 (prepared from 40% v/v ethanol added of CSD) and SDP80 reduced the total leukocyte and polymorphonuclear cell migration in the pleural cavity.

Antioxidant, a pro-oxidant and cytotoxic effects of Achyrocline satureioides extracts.

In this study we compared the antioxidant properties of five different extracts of different composition obtained from Achyrocline satureioides' inflorescences (Compositae), a widely used Brazilian folk medicinal herb. All of the extracts presented significant antioxidant potential identified by TRAP assay, which increased in the presence of human plasma. Characterization of the content of flavonoids in each extract showed that the FDP80 (ethanol 80%) and FFr (enriched flavonoid fraction) extracts contained a higher content of flavonoids. Cytotoxicity of the extracts as determined in Sertoli cell culture showed that FDP80 and FFr were highly toxic at most concentrations tested. The extracts induced a significant increase in lipid peroxidation levels in Sertoli cells. These results suggest that medicinal herb extracts that contain higher flavonoid concentrations and shows higher antioxidant protection in vitro might not always produce the greatest benefit.

Signal to noise and range enhancement of the Brillouin intensity based temperature sensor.

We report on the theory and use of pre-amplification to enhance the measurement range of a spontaneous Brillouin intensity based distributed fiber-optic sensor. One factor that limits temperature resolution is receiver sensitivity, which degrades for long range sensors. Using optical preamplification before photo detection in a 23km sensor improved the signal-to-noise by approximately 17dB using a 20MHz detector. The major source of noise was amplified spontaneous emission beat noise.

LC determination of flavonoids: separation of quercetin, luteolin and 3-O-methylquercetin in Achyrocline satureioides preparations.

The pharmacological activities of the flavonoids show the interest in quantifying these constituents in phytopharmaceutical preparations, as well as in the validation of the analytical methodologies. LC methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. This work was designed, therefore, to develop an LC system to separate quercetin, luteolin and 3-O-methylquercetin and to quantify them in extractive solutions from Achyrocline satureioides. The main validation parameters of the method are also determined. The method showed linearity for quercetin and luteolin in the range 1-10 microg/ml. The aqueous and ethanol 80% extractive solutions showed linear response in the range 2.5-20 microl/ml and ethanol 40% extractive solution in the range 2.5-10 microl/ml. Precision and accuracy were determined for ethanol 80% extractive solution, in the concentration of 10 microl/ml. The LC method showed an excellent performance in separating the flavonoids quercetin, luteolin and 3-O-methylquercetin in A. satureioides extracts, since the presence of interference has been previously evaluated.

The adjuvants aerosil 200 and Gelita-Sol-P influence on the technological characteristics of spray-dried powders from Passiflora edulis var. flavicarpa.

Passiflora edulis (passionflower) is a plant widely used in the Brazilian popular medicine and phytopharmaceutical industry for its sedative activity. This work refers to the development of spray-dried powders (SDPs) from the 40% ethanolic extractive solution of P. edulis aerial parts. The SDPs were prepared with a Büchi 190 Mini-spray dryer using as drying adjuvants Aerosil 200 alone (SDP1), an Aerosil 200: Gelita-Sol-P (1:1) mixture (SDP2) and an Aerosil 200:Gelita-Sol-P (1:3) mixture (SDP3). All the powders were obtained using 40 parts adjuvant and 60 parts extract dried residue. The comparison criteria applied were particle size distribution, hygroscopicity at 95%, 60%, and 35% relative humidity (RH), as well as the flavonoid process recovery. The particle size distributions were analyzed by way of (a) normal distribution parameters, (b) the RRSB grid and (c) considering diameter values in terms of an equivalent sphere. All the powders presented nonnormal distribution, and the RRSB analysis appeared to be, therefore, the analysis method of choice. The total flavonoid recovery was around 80%, and it was practically not affected by the SDP1, SDP2, and SDP3 compositions. At the 60% and 90% RH atmospheres, the SDP3 and SDP2 moisture uptakes were higher than that of the SDP1. All the formulations were, on the contrary, stable at 35% RH, showing a slight moisture loss tendency. The results showed in sum that the SDP prepared using Aerosil 200 as the drying adjuvant alone presented the best technological characteristics of all.

Brillouin-based fiber-optic distributed temperature sensor with optical preamplification.

Optical preamplification has been used in a fiber-optic distributed temperature sensor based on spontaneous Brillouin scattering and the use of direct detection, resulting in improved signal-to-noise ratios. The fiber-based optical preamplifier system comprises a three-port circulator, an erbium-doped fiber amplifier with a small-signal gain of 27 dB, and a fiber Bragg grating with 47-GHz bandwidth. An improvement of 17 dB in the optical signal-to-noise ratio for the Brillouin signal is demonstrated in a 23-km sensor. The limit to the signal-to-noise ratio is attributed to spontaneous-spontaneous beat noise generated at the photodetector by amplified spontaneous emission from the optical amplifier.

Genetics of immunological tolerance: I. Bidirectional selective breeding of mice for oral tolerance.

The polygenic nature of oral tolerance regulation was elucidated by the method of bidirectional selective breeding of mouse strains for tolerance susceptibility (TS) and resistance (TR) starting from a genetically heterogeneous population achieved by the equilibrated intercrossing of eight inbred mouse strains (A/J, DBA/2J, P/J, SWR/J, SJL/J, CBA/J, BALB/cJ and C57BL/6J). Seven days after intragastric administration of 5 mg OVA or BSA, mice were intraperitoneally immunized with 100 microg of the corresponding antigen. The individual antibody titres were measured by haemagglutination. The phenotypes at the highest and lowest extremes were selected for assortative mating, avoiding consanguinity. The second litter of each mating couple was intraperitoneally immunized only to evaluate the immunocompetence of the corresponding generation and to ascertain the non-selection of non-responder mice. A normal distribution of agglutinin titres ranging from 4 to 14 log2 was observed in the F0 population. In the F12 generation, TR and TS strains showed highly significant differences for agglutinin titres (TR=15.06+/-1.80 and TS=8.35+/-2.44), and IgG responses by ELISA. Up to the F12 generation, the mean realized heritability was 0.14+/-0.02. The response to the selection was 0.43 log2 and the selection differential 3.10 log2/generation. A provisional estimation indicated that oral tolerance may be influenced by eight or nine independent loci.

[Prevalence and indirect costs of headache in a Brazilian Company]. / Prevalência e custos indiretos das cefaléias em uma empresa Brasileira.

Employees from a Brazilian oil company research centre (n = 993) were interviewed on the occurrence of headache during a 30 days period. Headache prevalence was 49.8%, with a mean frequency of 4.3 +/- 7.0 attacks per month, lasting 12.2 +/- 21.4 hours each. According to the International Headache Society diagnostic criteria, migraine (5.5%), episodic tension-type headache (26.4%), chronic tension-type headache (1.7%) and headaches not fulfilling the criteria for such disorders (16.2%) were observed. Women suffered comparatively more headache and specifically migraine than men. The pain interfered with work productivity in 10% of the subjects, corresponding to 538.75 hours off. According to an indirect costs estimation for each headache, the company may loose up to US$125.98 per employee annually. Since among headaches migraine has the highest indirect cost, migraine prevention and treatment is particularly important at the working environment. Migraine frequency may be prevented to a large extent, resulting on positive effects in both the quality of life and productivity. The cost-benefit ratio clearly favours therapeutic and preventive programs against chronic headaches.

Allelic associations of two polymorphic microsatellites in intron 40 of the human von Willebrand factor gene.

At intron 40 of the von Willebrand factor (vWF) gene, two GATA-repeat polymorphic sites exist that are physically separated by 212 bp. At the first site (vWF1 locus), seven segregating repeat alleles were observed in a Brazilian Caucasian population, and at the second (vWF2 locus) there were eight alleles, detected through PCR amplifications of this DNA region. Haplotype analysis of individuals revealed 36 different haplotypes in a sample of 338 chromosomes examined. Allele frequencies between generations and gender at each locus were not significantly different, and the genotype frequencies were consistent with their Hardy-Weinberg expectations. Linkage disequilibrium between loci is highly significant with positive allele size association; that is, large alleles at the loci tend to occur together, and so do the small alleles. Variability at each locus appeared to have arisen in a stepwise fashion, suggesting replication slippage as a possible mechanism of production of new alleles. However, we observed an increased number of haplotypes, in contrast with the predictions of a stepwise production of variation in the entire region, suggesting some form of "cooperative" changes between loci that could be due to either gene conversion, or a common control mechanism of production of new variation at these repeat polymorphism sites. The high degree of polymorphism (gene diversity values of 72% and 78% at vWF1 and vWF2, respectively, and of 93% at the haplotype level) makes these markers informative for paternity testing, genetic counseling, and individual-identification purposes.