Deciphering radiological stable disease to immune checkpoint inhibitors.

BACKGROUND: 'Stable disease (SD)' as per RECIST is a common but ambiguous outcome in patients receiving immune checkpoint inhibitors (ICIs). This study aimed to characterize SD and identify the subset of patients with SD who are benefiting from treatment. Understanding SD would facilitate drug development and improve precision in correlative research. PATIENTS AND METHODS: A systematic review was carried out to characterize SD in ICI trials. SD and objective response were compared to proliferation index using The Cancer Genome Atlas gene expression data. To identify a subgroup of SD with outcomes mirroring responders, we examined a discovery cohort of non-small-cell lung cancer (NSCLC). Serial cutpoints of two variables, % best overall response and progression-free survival (PFS), were tested to define a subgroup of patients with SD with similar survival as responders. Results were then tested in external validation cohorts. RESULTS: Among trials of ICIs (59 studies, 14 280 patients), SD ranged from 16% to 42% in different tumor types and was associated with disease-specific proliferation index (ρ = -0.75, P = 0.03), a proxy of tumor kinetics, rather than relative response to ICIs. In a discovery cohort of NSCLC [1220 patients, 313 (26%) with SD to ICIs], PFS ranged widely in SD (0.2-49 months, median 4.9 months). The subset with PFS >6 months and no tumor growth mirrored partial response (PR) minor (overall survival hazard ratio 1.0) and was proposed as the definition of SD responder. This definition was confirmed in two validation cohorts from trials of NSCLC treated with durvalumab and found to apply in tumor types treated with immunotherapy in which depth and duration of benefit were correlated. CONCLUSIONS: RECIST-defined SD to immunotherapy is common, heterogeneous, and may largely reflect tumor growth rate rather than ICI response. In patients with NSCLC and SD to ICIs, PFS >6 months and no tumor growth may be considered 'SD responders'. This definition may improve the efficiency of and insight derivable from clinical and translational research.

Effects of calcium ammonium nitrate fed to dairy cows on nutrient intake and digestibility, milk quality, microbial protein synthesis, and ruminal fermentation parameters.

We evaluated the effects of supplemental calcium ammonium nitrate (CAN) fed to dairy cows on dry matter (DM) intake, nutrient digestibility, milk quality, microbial protein synthesis, and ruminal fermentation. Six multiparous Holstein cows at 106 ± 14.8 d in milk, with 551 ± 21.8 kg of body weight were used in a replicated 3 × 3 Latin square design. Experimental period lasted 21 d, with 14 d for an adaptation phase and 7 d for sampling and data collection. Cows were randomly assigned to receive the following treatments: URE, 12 g of urea/kg of DM as a control group; CAN15, 15 g of CAN/kg of DM; and CAN30, 30 g of CAN/kg of DM. Supplemental CAN reduced DM intake (URE 19.0 vs. CAN15 18.9 vs. CAN30 16.5 kg/d). No treatment effects were observed for apparent digestibility of DM, organic matter, crude protein, ether extract, and neutral detergent fiber; however, CAN supplementation linearly increased nonfiber carbohydrate digestibility. Milk yield was not affected by treatments (average = 23.1 kg/d), whereas energy-corrected milk (ECM) and 3.5% fat-corrected milk (FCM) decreased as the levels of CAN increased. Nitrate residue in milk increased linearly (URE 0.30 vs. CAN15 0.33 vs. CAN30 0.38 mg/L); however, treatments did not affect nitrite concentration (average: 0.042 mg/L). Milk fat concentration was decreased (URE 3.39 vs. CAN15 3.35 vs. CAN30 2.94%), and the proportion of saturated fatty acids was suppressed by CAN supplementation. No treatment effects were observed on the reducing power and thiobarbituric acid reactive substances of milk, whereas conjugated dienes increased linearly (URE 47.6 vs. CAN15 52.7 vs. CAN30 63.4 mmol/g of fat) with CAN supplementation. Treatments had no effect on microbial protein synthesis; however, molar proportion of ruminal acetate and acetate-to-propionate ratio increased with CAN supplementation. Based on the results observed, supplementing CAN at 30 g/kg of DM should not be recommended as an optimal dose because it lowered DM intake along with ECM and 3.5% FCM, although no major changes were observed on milk quality and ruminal fermentation.

Analysis of outcomes of emergency general and gastrointestinal surgery during the COVID-19 pandemic.

BACKGROUND: Few surgical studies have provided adjusted comparative postoperative outcome data among contemporary patients with and without COVID-19 infection and patients treated before the pandemic. The aim of this study was to determine the impact of performing emergency surgery in patients with concomitant COVID-19 infection. METHODS: Patients who underwent emergency general and gastrointestinal surgery from March to June 2020, and from March to June 2019 in 25 Spanish hospitals were included in a retrospective study (COVID-CIR). The main outcome was 30-day mortality. Secondary outcomes included postoperative complications and failure to rescue (mortality among patients who developed complications). Propensity score-matched comparisons were performed between patients who were positive and those who were negative for COVID-19; and between COVID-19-negative cohorts before and during the pandemic. RESULTS: Some 5307 patients were included in the study (183 COVID-19-positive and 2132 COVID-19-negative during pandemic; 2992 treated before pandemic). During the pandemic, patients with COVID-19 infection had greater 30-day mortality than those without (12.6 versus 4.6 per cent), but this difference was not statistically significant after propensity score matching (odds ratio (OR) 1.58, 95 per cent c.i. 0.88 to 2.74). Those positive for COVID-19 had more complications (41.5 versus 23.9 per cent; OR 1.61, 1.11 to 2.33) and a higher likelihood of failure to rescue (30.3 versus 19.3 per cent; OR 1.10, 0.57 to 2.12). Patients who were negative for COVID-19 during the pandemic had similar rates of 30-day mortality (4.6 versus 3.2 per cent; OR 1.35, 0.98 to 1.86) and complications (23.9 versus 25.2 per cent; OR 0.89, 0.77 to 1.02), but a greater likelihood of failure to rescue (19.3 versus 12.9 per cent; OR 1.56, 95 per cent 1.10 to 2.19) than prepandemic controls. CONCLUSION: Patients with COVID-19 infection undergoing emergency general and gastrointestinal surgery had worse postoperative outcomes than contemporary patients without COVID-19. COVID-19-negative patients operated on during the COVID-19 pandemic had a likelihood of greater failure-to-rescue than prepandemic controls.

A new species in the Mycosphaerellaceae from Cecidomyiidae leaf galls on Avicennia marina in South Africa.

During studies to investigate the health of mangrove trees in South Africa, high numbers of Avicennia marina were found with leaf galls caused by unidentified adults and larvae of midges (Cecidomyiidae). Fungal fruiting structures were commonly observed on the abaxial areas of the galls. To determine the identity of the fungi associated with the gall midges, phylogenetic analyses using multigene sequence data were used. The nuclear large subunit (LSU), internal transcribed spacer (ITS), and a portion of the actin gene region (ACT), were amplified and analyzed. The results revealed that the fungal fruiting structures represent a new taxon in the Mycosphaerellaceae described here as Zasmidium mangrovei sp. nov. This is the first report of a species in the Mycosphaerellaceae associated with cecidomyiid leaf galls on A. marina.

Short communication: Molecular markers for epithelial cells across gastrointestinal tissues and fecal RNA in preweaning dairy calves.

The objective of this study was to compare the transcription of gene markers for gastrointestinal (GI) epithelial cells, including fatty acid binding protein 2 (FABP2) and cytokeratin 8 (KRT8), and tight junction complex genes (TJP1, CLDN1, CLDN4) in fecal RNA against several GI tract tissue sections in dairy calves. Eight healthy Jersey calves were euthanized at 5 wk of age, and postmortem samples were collected from rumen, duodenum, jejunum, ileum, large intestine, cecum, and feces for total RNA isolation. Tissues and fecal samples were immediately frozen in liquid nitrogen until RNA isolation. A real-time quantitative PCR analysis was performed using a single standard curve composited of equal amounts of all samples, including cDNA from fecal and GI tract tissues. The mRNA expression of the tight junctions TJP1, CLDN1, and CLDN4 was greater in fecal RNA compared with lower GI tract tissues (i.e., duodenum, jejunum, ileum, large intestine, and cecum). Similar to fecal RNA, rumen tissue had greater expression of tight junctions CLDN1 and CLDN4 than lower GI tract tissues. Similarly, rumen tissue had greater expression of TPJ1 than all lower GI tract tissues except duodenum. The expression of TJP1 and CLDN4 was greater in fecal RNA than in rumen tissue; in contrast, CLDN1 mRNA expression was greater in rumen tissue than in the fecal RNA. The expression of FABP2 was greater in duodenum in comparison to all tissue except ileum. The mRNA expression of FABP2 in fecal samples was similar to jejunum and ileum. The expression of KRT8 in fecal samples was similar to duodenum, large intestine, and cecum. The fecal RNA had a greater expression of KRT8 in comparison to jejunum and ileum. The rumen tissue had the lowest mRNA expression of KRT8. The expression levels of FABP2, KRT8, and tight junction genes observed in fecal transcripts suggest that a considerable amount of RNA derived from GI tract epithelial cells can be detected in fecal RNA, which is in agreement with previous data in neonatal dairy calves and other biological models including humans, rodents, and primates. The greater expression of tight junctions in fecal RNA in comparison to sections of the low GI remains to be understood, and due to the importance of tight junctions in GI physiology, further clarification of this effect is warranted. The similarities in mRNA expression of FABP2 and KRT8 between fecal RNA and intestinal sections add up to the accumulating evidence that fecal RNA can be used to investigate molecular alterations in the GI tract of neonatal dairy calves. Further research in this area should include high-throughput transcriptomic analysis via RNA-seq to uncover novel molecular markers for specific sections of the GI tract of neonates.

Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows.

Feeding yeast culture fermentation products has been associated with improved feed intake and milk yield in transition dairy cows. These improvements in performance have been further described in terms of rumen characteristics, metabolic profile, and immune response. The objective of this study was to evaluate the effects of a commercial yeast culture product (YC; Culture Classic HD, Phibro Animal Health) on performance, blood biomarkers, rumen fermentation, and rumen bacterial population in dairy cows from -30 to 50 d in milk (DIM). Forty Holstein dairy cows were enrolled in a randomized complete block design from -30 to 50 DIM and blocked according to expected calving day, parity, previous milk yield, and genetic merit. At -30 DIM, cows were assigned to either a basal diet plus 114 g/d of ground corn (control; n = 20) or a basal diet plus 100 g/d of ground corn and 14 g/d of YC (n = 20), fed as a top-dress. Cows received the same close-up diet from 30 d prepartum until calving [1.39 Mcal/kg of dry matter (DM) and 12.3% crude protein (CP)] and lactation diet from calving to 50 DIM (1.60 Mcal/kg of DM and 15.6% CP). Blood samples and rumen fluid were collected at various time points from -30 to 50 d relative to calving. Cows fed YC compared with control showed a trend for increased energy-corrected milk (+3.2 kg/d). Lower somatic cell counts were observed in YC cows than in control. We detected a treatment × time interaction in nonesterified fatty acids (NEFA) that could be attributed to a trend for greater NEFA in YC cows than control at 7 DIM, followed by lower NEFA in YC cows than control at 14 and 30 DIM. In the rumen, YC contributed to mild changes in rumen fermentation, mainly increasing postpartal valerate while decreasing prepartal isovalerate. This was accompanied by alterations in rumen microbiota, including a greater abundance of cellulolytic (Fibrobacter succinogenes) and lactate-utilizing bacteria (Megasphaera elsdenii). These results describe the potential benefits of supplementing yeast culture during the late pregnancy through early lactation, at least in terms of rumen environment and performance.

Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows.

The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

Enzymatic effects of Pleurotus ostreatus spent substrate on whole-plant corn silage and performance of lactating goats.

Pleurotus ostreatus (oyster mushroom) synthesizes enzymes that degrade lignin, cellulose, and hemicellulose. The objectives of this study were to evaluate the effect of Pleurotus ostreatus spent substrate (POSS) on whole-plant corn silage (WPCS) chemical composition, antioxidant capacity, lignin monomers, and in vitro digestibility, as well as the performance of lactating goats fed corn silage treated with different levels of POSS. In experiment 1, 4 levels of lignocellulolytic enzymes were tested in a complete randomized design: 0, 10, 20, and 30 mg of lignocellulosic enzymes per kilogram of fresh matter, 4 replicates per treatment (vacuum-sealed bags). The bags were opened 60 d after ensiling. In experiment 2, corn silage treated with 3 enzyme levels (0, 10, or 30 mg/kg of fresh matter) was fed to lactating goats as part of the total mixed ration. Nine lactating Saanen goats (62.68 ± 7.62 kg BW; 44 ± 8 d in milk; 2.91 ± 0.81 kg of milk/day, mean ± SD) were assigned to three 3 × 3 Latin squares. Data were analyzed using the GLIMMIX procedure of SAS (version 9.4, SAS Institute Inc.), and means were compared by linear and quadratic orthogonal contrast. In experiment 1, neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, and cellulose quadratically decreased in the WPCS treated with POSS. At the nadir point, POSS decreased NDF by 14.1%, ADF by 19.5%, lignin by 9.07%, and cellulose by 22.1% compared with the untreated silage. Therefore, POSS led to a quadratic increase in in vitro dry matter digestibility of WPCS (+8.88% at the vertex) compared with the untreated silage. In experiment 2, POSS quadratically increased the in vivo total-tract ADF digestibility. Also, the concentration of polyphenols in the milk of goats linearly increased with the addition of POSS, and no differences were observed among treatments for milk yield and composition. In summary, adding 10 mg of lignocellulolytic enzymes from POSS per kilogram of fresh matter of whole-plant corn at ensiling had a more evident reduction in lignin and cellulose concentration, leading to greater in vitro digestibility, as well as greater in vivo ADF digestibility; however, milk yield was not different among treatments.

Pneumococcal serotypes in children, clinical presentation and antimicrobial susceptibility in the PCV13 era.

The aim was to analyse invasive pneumococcal disease (IPD) serotypes in children aged ⩽17 years according to clinical presentation and antimicrobial susceptibility. We conducted a prospective study (January 2012-June 2016). IPD cases were diagnosed by culture and/or real-time polymerase chain reaction (PCR). Demographic, microbiological and clinical data were analysed. Associations were assessed using the odds ratio (OR) and 95% confidence intervals (CI). Of the 253 cases, 34.4% were aged <2 years, 38.7% 2-4 years and 26.9% 5-17 years. Over 64% were 13-valent pneumococcal conjugate vaccine (PCV13) serotypes. 48% of the cases were diagnosed only by real-time PCR. Serotypes 3 and 1 were associated with complicated pneumonia (P < 0.05) and non-PCV13 serotypes with meningitis (OR 7.32, 95% CI 2.33-22.99) and occult bacteraemia (OR 3.6, 95% CI 1.56-8.76). Serotype 19A was more frequent in children aged <2 years and serotypes 3 and 1 in children aged 2-4 years and 5-17 years, respectively. 36.1% of cases were not susceptible to penicillin and 16.4% were also non-susceptible to cefotaxime. Serotypes 14, 24F and 23B were associated with non-susceptibility to penicillin (P < 0.05) and serotypes 11, 14 and 19A to cefotaxime (P < 0.05). Serotype 19A showed resistance to penicillin (P = 0.002). In conclusion, PCV13 serotypes were most frequent in children aged ⩽17 years, mainly serotypes 3, 1 and 19A. Non-PCV13 serotypes were associated with meningitis and occult bacteraemia and PCV13 serotypes with pneumonia. Non-susceptibility to antibiotics of non-PCV13 serotypes should be monitored.

Consumer knowledge and perceptions of milk fat in Denmark, the United Kingdom, and the United States.

This study examined the relationship between consumers' country of origin: Denmark (DK), the United Kingdom (UK), and the United States (US) and their knowledge and perceptions of milk fat. Adult participants answered a web-based survey, and data were collected online from December 2018 to April 2019, in DK, the UK, and the US. A total of 694 participants completed the online survey. Most respondents were between 18 and 39 years old, female, highly educated, and employed. Most UK respondents consumed milk daily (73%), whereas in DK (56%) and the US (46%) consumption was significantly lower. Whole milk and semi-skim milk were consumed less by respondents in DK (20 and 36%, respectively) compared with the UK (50 and 49%, respectively) and the US (47 and 50%, respectively). Danish respondents (53%) consumed more skim milk than respondents from the UK (16%) and the US (19%). Concern about milk fat was higher in DK (60%) than in the UK (34%) and the US (31%). More respondents considered milk fat to be "healthy" or "very healthy" in the UK (31 and 10%, respectively) and the US (37 and 19%, respectively), than in DK (23 and 6%, respectively). Nutritional benefit was the most important reason for perceiving milk fat as healthy in the 3 countries. Awareness of milk saturated fat was higher among respondents from the UK (53%) than from DK (44%) and the US (38%). Results suggest that consumers in DK are different in their perceptions of milk fat, but consumers in the UK and the US share common characteristics.

Thiamine ameliorates inflammation of the ruminal epithelium of Saanen goats suffering from subacute ruminal acidosis.

This study aimed to examine the role of thiamine in the local inflammation of ruminal epithelium caused by high-concentrate diets. Eighteen mid-lactating (148 ± 3 d in milk; milk yield = 0.71 ± 0.0300 kg/d) Saanen goats (body weight = 36.5 ± 1.99 kg; body condition score = 2.73 ± 0.16, where 0 = emaciated and 5 = obese) in parity 1 or 2 were selected. The goats were randomly divided into 3 groups (n = 6/group): (1) control diet (concentrate:forage 30:70), (2) high-concentrate diet (HC; concentrate:forage 70:30), and (3) high-concentrate diet with 200 mg of thiamine/kg of dry matter intake (THC; concentrate:forage 70:30). Goats remained on experimental diets for 8 wk. On the last day of 8 wk, ruminal and blood samples were collected to determine ruminal parameters, endotoxin lipopolysaccharide, and blood inflammatory cytokines. Goats were slaughtered to collect ruminal tissue to determine gene and protein expression of toll-like receptor 4 (TLR4) signaling pathways. Thiamine supplementation increased ruminal pH (6.03 vs. 5.42) compared with the HC group. Propionate (21.08 vs. 31.61 mM), butyrate (12.08 vs. 19.39 mM), lactate (0.52 vs. 0.71 mM), and free lipopolysaccharide (42.16 vs. 55.87 × 103 endotoxin units/mL) concentrations in ruminal fluid were lower in THC goats compared with HC goats. Similar to plasma interleukin 1ß (IL-1ß) concentration (209.31 vs. 257.23 pg/mL), blood CD8+ percentage (27.57 vs. 34.07%) also decreased in response to thiamine. Compared with HC goats, THC goats had lower ruminal epithelium activity of the enzymes myeloperoxidase and matrix metalloproteinase (MMP) 2 and 9. In contrast to HC, THC had downregulated mRNA expression of nuclear factor-κB (NFKB), TLR4, IL1B, MMP2, and MMP9 in ruminal epithelium. Thiamine supplementation led to lower relative protein expression of IL-1ß, NF-κB unit p65, and phosphorylated NF-κB unit p65 in ruminal epithelium. Taken together, these results suggest that thiamine supplementation mitigates HC-induced local inflammation and ruminal epithelial disruption.

Altered rumen fermentation patterns in lactating dairy cows supplemented with phytochemicals improve milk production and efficiency.

Tannins and other phytochemicals are known to improve RUP in the diet by binding protein and then limiting ruminal degradation, which may improve milk yield and milk protein synthesis. The objective of this study was to evaluate the effects of dietary phytochemicals (tannins and Capsicum species) as rumen modifiers on production parameters and milk efficiency in dairy cows. Twenty-four multiparous Holstein cows (96 ± 16 d in milk; mean ± standard deviation) were used in a replicated 3 × 3 Latin square design balanced to measure carryover effects. Cows were blocked according to days in milk, milk production, and body weight and randomly assigned to 1 of 3 groups (n = 8/group). Each group was assigned to a unique treatment sequence across the 3 periods in the Latin square. The experiment consisted of a 14-d covariate period and three 30-d treatment periods. Cows received a basal diet supplemented with soybean meal pellets (SB) as the control diet, phytochemicals (RUM; Rumiviv, CCPA, Janzé, France) pelleted with soybean meal, or expeller soybean meal (ESBM; SoyPlus, West Central Soy, Ralston, IA). Milk production and dry matter intake during the last 4 d of each period were used for statistical analysis. Blood and rumen fluid samples were collected on d 27 of each period. Rumen fluid was analyzed for ammonia N and volatile fatty acids as well as ruminal bacteria via quantitative PCR amplification of 16S ribosomal DNA genes. Greater milk yield (37.9 vs. 36 kg/d), energy-corrected milk (39.7 vs 37.1 kg/d), and protein yield (1.15 vs. 1.08 kg/d) were observed in RUM compared with SB, but these parameters were similar between RUM and ESBM. Concentrations of total volatile fatty acids (118.1 vs. 101.5 mM) were greater in RUM in comparison to SB and ESBM diets. Cows fed RUM had greater ß-hydroxybutyrate (0.49 vs. 0.42 mmol/L) than SB and ESBM. Selenomonas ruminantium, Succinimonas amylolytica, and Streptococcus bovis in rumen fluid were lower in RUM fed cows in comparison to SB and ESBM. Increased total volatile fatty acids and lower ruminal abundance of bacteria associated with low feed efficiency in RUM cows can partially explain the improvements observed in milk yield and milk efficiency. Overall, these data suggest that feeding a combination of tannin mixture and Capsicum can significantly affect rumen fermentation characteristics via partial manipulation of rumen microbiota, and these effects were reflected in improved milk production and efficiency.

Short communication: Comparative gene expression analysis on the enrichment of polymorphonuclear leukocytes and gastrointestinal epithelial cells in fecal RNA from nondiarrheic neonatal dairy calves.

Increased understanding of the biology of the gastrointestinal tract (GIT) in neonatal dairy calves during their adaptation to an extrauterine environment will decrease health problems such as diarrhea while increasing feed efficiency and average daily gain in preweaned dairy calves. Within this context, a noninvasive method, based on fecal RNA, to study the GIT in neonatal dairy calves through the isolation of RNA from fecal samples for quantitative reverse-transcription PCR analysis can provide valuable information on GIT biological adaptations during the preweaning period. We aimed to evaluate the potential enrichment of RNA from immune cells or GIT epithelial cells during fecal RNA isolation. Eight neonatal Holstein calves less than 3 wk old (14.9 ± 5.5 d of age at sampling ± standard deviation) and a fecal score of 2.0 ± 0.7 (mean ± standard deviation) were used. During a single sampling, fecal and blood samples were taken simultaneously from each calf before the morning feeding. Fecal samples were immediately frozen in liquid nitrogen until RNA isolation, whereas polymorphonuclear leukocytes (PMN) were isolated from blood samples before RNA isolation. An quantitative reverse-transcription PCR analysis was performed using a single standard curve composited of equal amounts of all samples including cDNA from fecal and PMN. The genes myeloperoxidase (MPO) and L-selectin (SELL) were selected for their specific known function in PMN, whereas keratin 8 (KRT8) and aquaporin 3 (AQP3) have been associated with epithelial enterocytes. Our results showed a contrasting gene expression profile between PMN and fecal RNA; whereas greater mRNA expression of SELL was observed in PMN, a greater KRT8 expression was observed in fecal RNA. The mRNA expression of AQP3 tended to be greater in PMN than fecal RNA. Additionally, MPO was not amplified in fecal RNA. Our findings suggest that under nondiarrheic conditions RNA isolated from stool samples of neonatal dairy calves will have a considerable number of GIT epithelial cells, which confirms the reliability of this method under these conditions. However, further research needs to be done to determine if the same effects are observed during diarrhea or throughout the preweaning period of dairy calves.

Technical note: A novel approach to estimate dry matter intake of lactating dairy cows through multiple on-cow accelerometers.

The objective of this study was to evaluate the feasibility of using multiple 3-dimensional accelerometers to estimated individual dry matter intake (DMI) of lactating dairy cows. Twenty-four Holstein cows in late lactation were assigned into 2 groups, a calibration group (n = 12) and a validation group (n = 12). All cows were fitted with 3 sensors that recorded 3-dimensional acceleration (i.e., x, y, and z) at 10-s intervals, 1 on the lateral side of the left hind leg and 2 attached directly to a halter over the nose and jaw area on the left side. Then, 3 accelerations were generated from each accelerometer (e.g., Leg-X, Leg-Y, and Leg-Z). Six new variables were created based on the change in acceleration in the nose and jaw accelerometers between 2 consecutive time points (e.g., LagJaw-X). For both groups (i.e., calibration and validation), cows were continuously video recorded while data on acceleration and intake of total mixed ration were collected for 10 consecutive days. Cows were fed once daily using an individual gate system, and individual refusals were recorded next day before morning feeding. Cows were fed a common lactating cow diet (17.9% crude protein; 1.70 Mcal/kg of dry matter). In the calibration group, individual eating bouts were obtained based on video recordings and merged with the corresponding accelerometer data. Then, a stepwise regression analysis was conducted using the REG procedure of SAS (SAS Institute, Cary, NC) to determine the ranges in acceleration that accounted for the highest variation in DMI (highest R2) in each acceleration variable. All 32,767 potential acceleration combinations were tested in the validation group using the acceleration ranges predetermined in the calibration group. The CORR procedure of SAS was used to test the Pearson correlation coefficient (r) between the type of DMI [i.e., based on accelerations (DMIaccel) or actual DMI (DMIactual)]. The MIXED procedure of SAS was used to perform a repeated-measures analysis with type (DMIaccel vs. DMIactual), day, and their interaction (T × D) in the model. From this analysis, 8 candidate acceleration models were selected based on high r and similarity (P > 0.15) in terms of T and T × D between DMIaccel and DMIactual. A simulated effect on DMIactual was artificially created in the validation group by dividing this group (n = 12) into high and low intake cows (n = 6/group; DMI of 24.1 vs. 18.7 kg/d), and the candidate models were tested to determine whether they were sensitive enough to detect this effect. From these candidate models, AEN (Leg-X + Jaw-Z + LagJaw-Z) showed a weak correlation (r = 0.36) between DMIaccel and DMIactual, but DMIaccel and DMIactual were highly similar (21.2 vs. 21.4 kg/d of DMI). In addition, this was the only model that could detect the simulated effect on DMIactual (22.1 vs. 20.3 kg/d of DMI) in the validation group. The fact that the simulated effect on DMIactual was detected based only on accelerations is highly significant, and models such as AEN could be substantially improved if they were derived from a greater sample size and included different physiological stages in dairy cows.

Invasive slug Meghimatium pictum (Stoliczka, 1873) infected by Angiostrongylus costaricensis Morera & Céspedes, 1971, and the possible risk of human infection associated with grape consumption.

Many molluscs may be infected with angiostrongylid larvae. Following the histopathological diagnosis of abdominal angiostrongyliasis in a grape farmer from southern Brazil, molluscs in the area were investigated. During a nocturnal search, 245 specimens of slugs were collected and identified as the invasive Chinese slug Meghimatium pictum. Angiostrongylus costaricensis worms were recovered from mice that were experimentally infected with larvae obtained from 11 (4.5%) of the molluscs. This study presents the first report of M. pictum being identified as an intermediate host for A. costaricensis. Most of the slugs were collected from grape plants, which suggests that transmission may be associated with grape consumption.

Clinical and nerve conduction features in Guillain-Barré syndrome associated with Zika virus infection in Cúcuta, Colombia.

BACKGROUND AND PURPOSE: Zika virus (ZIKV) infection has been associated with an increased incidence of Guillain-Barré syndrome (GBS) but the relative frequency of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) and axonal GBS subtypes is controversial. METHODS: Twenty GBS patients diagnosed according to the Brighton criteria during the ZIKV outbreak in Cúcuta, Colombia, were evaluated clinically and electrophysiologically. The electrodiagnosis of GBS subtypes was made according to a recently described criteria set that demonstrated a high diagnostic accuracy on the basis of a single test. The electrophysiological features of 34 Italian AIDP patients were used as control. RESULTS: All patients had symptoms compatible with ZIKV infection before the onset of GBS and ZIKV infection was laboratory confirmed through a plaque reduction neutralization test (PRNT90 ) in 100% of patients. The median time from onset of ZIKV infection symptoms to GBS was 5 days (interquartile range 1-6 days). Cranial nerve palsy was present in 85% of patients (facial palsy in 75%, bulbar nerve involvement in 60%), autonomic dysfunction in 85%, and 50% of patients required invasive mechanical ventilation. AIDP was diagnosed in 70% of patients. 40% of nerves of AIDP patients showed a prevalent distal demyelinating involvement but this pattern was not different from the Italian AIDP patients without ZIKV infection. CONCLUSIONS: Guillain-Barré syndrome associated with ZIKV infection in Cúcuta is characterized by a high frequency of cranial nerve involvement, autonomic dysfunction and requirement of mechanical ventilation indicating an aggressive and severe course. AIDP is the most frequent electrophysiological subtype. Demyelination is prevalent distally but this pattern is not specific for ZIKV infection.

Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.

Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at -30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from -30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.

Prepartal standing behavior as a parameter for early detection of postpartal subclinical ketosis associated with inflammation and liver function biomarkers in peripartal dairy cows.

A degree of negative energy balance is commonly experienced by cows during early lactation. This physiological state, if pronounced or prolonged, leads to partial oxidation of nonesterified fatty acids as an energy source and, consequently, increasing blood ß-hydroxybutyrate (BHB) concentrations and potentially development of ketosis in postpartal dairy cows. Twenty-four multiparous Holstein cows received a common prepartal and postpartal diet. Cows were fitted with an accelerometer mounted laterally on the distal left hind leg using vet wrap from -30 to 15 d relative to parturition. A retrospective analysis was performed using the postpartal BHB data at 8 time points from 0 to 15 d in milk measured with the Precision Xtra (Abbott Diabetes Care, Alameda, CA). Cows with an average blood BHB <1.4 mmol/L were designated nonketotic (NONKET; n = 12), and those with ≥1.4 mmol/L were designated ketotic (KET; n = 12). A total of 8 samples per cow were used for this analysis. Subsequent analyses of behavioral patterns and blood biomarkers were performed using this group effect. On average, blood BHB reached subclinical levels (1.4 ± 0.3 mmol/L; mean ± standard error of the mean) at 3 d postpartum for all cows in this study. Behavioral patterns were obtained from accelerometer data, and correlation analysis was performed between these behaviors such as standing and lying time from -30 to 3 d relative to parturition and blood BHB concentration at 3 d postpartum. The strongest correlation was obtained between standing time at 3 d before calving and blood BHB at 3 d postpartum. Dry matter intake was greater (ca. 3 kg/d) in NONKET cows than in KET cows. An interaction of group × time for milk yield resulted in an overall increase of 5.7 kg/d in NONKET cows in comparison with KET. The blood concentrations of biomarkers for liver function (γ-glutamyltransferase and glutamic-oxaloacetic transaminase), inflammation (IL-6), and metabolism (nonesterified fatty acids) were increased at various time points in KET cows in comparison with NONKET during the transition period. Overall, lower bilirubin in NONKET cows than in KET further confirmed an impaired liver function in the latter group of cows. Our findings revealed the potential for establishing correlations between prepartal behavioral patterns derived from accelerometer data and postpartal subclinical ketosis, and further confirming the latter by physiological alterations in biomarkers related to inflammation and liver function. Our data also indicate that cows with a predisposition to postpartal subclinical or clinical ketosis will remain standing for fewer hours during the days leading to parturition, which decreased DMI, and this condition was further reflected in lower milk yield.

Antibody-mediated thyroid dysfunction during T-cell checkpoint blockade in patients with non-small-cell lung cancer.

Background: Programmed cell death protein-1 (PD-1) blockade therapies have demonstrated durable responses and prolonged survival in a variety of malignancies. Treatment is generally well tolerated although immune-related adverse events (irAEs) can occur. Autoimmune thyroid dysfunction is among the most common irAE, but an assessment of the clinical, mechanistic, and immunologic features has not been previously described. Patient and methods: Patients with advanced non-small-cell lung cancer (NSCLC) treated with pembrolizumab at Memorial Sloan Kettering Cancer Center (n = 51) as part of KEYNOTE-001 (NCT01295827) were included. Thyroid function test and anti-thyroid antibodies were assessed prospectively at each study visit, beginning before the first treatment. Frequency of development of thyroid dysfunction, association with anti-thyroid antibodies, clinical course, and relationship with progression-free survival and overall survival to treatment with pembrolizumab was evaluated. Results: Of 51 patients treated, 3 were hypothyroid and 48 were not at baseline. Ten of 48 [21%, 95% confidence interval (CI) 10% to 35%] patients developed thyroid dysfunction requiring thyroid replacement. Anti-thyroid antibodies were present in 8 of 10 patients who developed thyroid dysfunction, compared with 3 of 38 who did not (80% versus 8%, P < 0.0001). Thyroid dysfunction occurred early (median, 42 days) in the pembrolizumab course, and a majority (6 of 10 patients) experienced brief, transient hyperthyroidism preceding the onset of hypothyroidism; no persistent hyperthyroidism occurred. Both hyperthyroidism and hypothyroidism were largely asymptomatic. Overall survival with pembrolizumab was significantly longer in subjects who developed thyroid dysfunction (hazard ratio, 0.29; 95% CI 0.09-0.94; P = 0.04). Conclusions: Thyroid dysfunction during pembrolizumab treatment of NSCLC is common and is characterized by early-onset, frequently preceded by transient hyperthyroidism, closely associated with anti-thyroid antibodies, and may be associated with improved outcomes. The presence of antibody-mediated toxicity in T-cell-directed therapy suggests an under-recognized impact of PD-1 biology in modulating humoral immunity.

Grain challenge affects systemic and hepatic molecular biomarkers of inflammation, stress, and metabolic responses to a greater extent in Holstein than Jersey cows.

Long-term feeding of high-grain diets to dairy cows often results in systemic inflammation characterized by alterations in acute-phase proteins and other biomarkers, both in plasma and immune-responsive tissues like the liver. The molecular and systemic changes that characterize an acute grain feeding challenge remain unclear. The current study involved 6 Holstein and 6 Jersey cows in a replicated 2 × 2 Latin square. Periods (10 d) were divided into 4 stages (S): S1, d 1 to 3, served as baseline with total mixed ration (TMR) ad libitum; S2, d 4, served as restricted feeding, with cows offered 50% of the average daily intake observed in S1; S3, d 5, a grain challenge was performed, in which cows were fed a TMR ad libitum without (CON) or with an additional pellet wheat-barley (1:1; HIG) at 20% of dry matter intake top-dressed onto the TMR; S4, d 6 to 10, served as recovery during which cows were allowed ad libitum access to the TMR. Among the 28 biomarkers analyzed in blood 12 h after grain challenge on d 5, the concentrations of fatty acids and bilirubin increased in HIG Holstein but not Jersey cows. In Holsteins, feeding HIG also increased total protein and albumin while decreasing ceruloplasmin, myeloperoxidase, and alkaline phosphatase concentrations. At the molecular level, hepatic genes associated with inflammation (IL1B, IL6, TNF, TLR4, MYD88, and NFKB1) were upregulated in Holstein cows fed HIG versus CON. Despite such response, expression of the acute-phase proteins SAA and HP in Holsteins fed HIG compared with CON was markedly downregulated. In Holsteins fed HIG versus CON, the marked downregulation of SCD, ELOVL6, and MTTP along with upregulated CPT1A, ACOX1, and APOA5 indicated alterations in fatty acid and lipoprotein metabolism during grain challenge. Genes related to ketogenesis (HMGCS2 and ACAT1) were upregulated in Jerseys, and gluconeogenic genes (PDK4 and PCK1) were upregulated in Holstein cows fed HIG, suggesting alterations in ketone body and glucose production. Expression of phosphorylated p70S6K1, RPS6, and 4EBP1 proteins, as well as total mechanistic target of rapamycin (mTOR) protein, decreased in Holsteins fed HIG, whereas phosphorylated mTOR and 4EBP1 proteins increased in Jerseys fed HIG. From a metabolic and inflammatory biomarker standpoint, data indicate that Jersey cows better tolerated the acute grain challenge. Alterations in mTOR signaling proteins in both Jerseys and Holsteins fed HIG suggest a potential role for exogenous AA in the hepatic adaptations to grain challenge. It remains to be determined if these acute responses to a grain challenge can elicit long-term liver dysfunction, which could negatively affect welfare of the cow.