Terminal Blood Research Articles

MON-228 FSH/AMH Ratio and Adipocyte Size Are Important Indicators for Ovarian Cyst Formation

High-fat, high-sugar (HFHS) diet induces a number of metabolic dysfunctions. Furthermore, there is evidence that diet-induced obesity correlates with diminished reproductive outcomes, though the mechanism remains unclear. We utilized a female, HFHS rat model to determine the effects of adiposity on ovarian morphology and hormonal balance. Because animals were on an ad libitum diet, we observed a gradient of obesity and adiposity, as well as ovarian dysfunction. We explored the distribution of these variables in order to establish a potential mechanism for reproductive dysfunction in our HFHS model. To determine the effect of a HFHS diet on ovarian structures, adiposity, and follicle-relevant hormones, female Sprague-Dawley rats were separated into two diet groups. The control group (n=14) received standard chow and water ad libitum while the HFHS group (n=16) received 60% of calories from fat, 30% sucrose solution, and water ad libitum. Weights were recorded every other day, and estrous cycles were monitored through daily vaginal cytology. During the course of the study, a fraction of animals ceased cycling. Terminal blood samples were taken on diestrus-2. AMH and FSH levels in the plasma were measured using ELISA. Ovaries and abdominal fat pads were collected and sectioned for histological analysis. At the conclusion of the study, HFHS rats gained significantly more weight than controls and displayed larger abdominal and gonadal fat pads. Rats were divided into three groups based on estrous cycle status: non-cycling (n=7), HFHS cycling (n=8), and control cycling (n=7). Non-cycling and HFHS rats displayed different distributions in adipocyte size relative to controls, indicating a shift towards larger cell size. On average, non-cyclers and HFHS had more cysts then controls, and non-cyclers had fewer corpora lutea compared to HFHS and controls. While AMH and FSH levels did not differ between groups, the ratio of FSH to AMH significantly correlated with follicular structures. The FSH/AMH ratio displayed strong negative correlations with total follicle number (r=-0.72, p<0.0005) and late antral follicles (r=-0.78, p<0.00005), also correlating positively with the number of cysts per section (r=0.74, p<0.001). Independently, adipocyte size was also predictive of cysts per section (r=0.49, p<0.02). Taken together, however, the FSH/AMH ratio and adipocyte size strongly predicted number of cysts per section in multiple regression. Our results suggest that morphological changes resulting from diet-induced obesity occur along a spectrum, modulated by hormonal balance and adiposity. These results establish the FSH/AMH ratio and adipocyte size as important markers for the onset of ovarian dysfunction, particularly cyst formation. In addition, the FSH/AMH ratio predicts follicle counts in the ovary, providing a combined measure for prediction of follicle status.

Lectin activity of Pseudomonas aeruginosa vaccine candidates PSE17-1, PSE41-5 and PSE54

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections most commonly in immunocompromised, cystic fibrosis (CF) and burns patients. The pilin and Pseudomonas lectins 1 (PA-IL) and 2 (PA-IIL) are known glycan-binding proteins of P. aeruginosa that are involved in adherence to host cells, particularly CF host airways. Recently, new P. aeruginosa surface proteins were identified by reverse vaccinology and tested in vivo as potential vaccine antigens. Three of these, namely PSE17-1, PSE41-5 and PSE54, were screened for glycan binding using glycan arrays displaying glycan structures representative of those found on human cells. Surface plasmon resonance was used to confirm the lectin activity of these proteins, and determined affinities with several host glycans to be in the nanomolar range. PSE17-1 binds hyaluronic acid and sialyl Lewis A and X. PSE41-5 binds terminal β-linked galactose structures, Lewis and ABO blood group antigens. PSE54 binds to ABO blood group antigens and some terminal β-linked galactose. All three proteins are novel lectins of P. aeruginosa with potential roles in infection of host cells.

Betaine insufficiency and reduced betaine dependent remethylation are associated with hyperhomocysteinemia, oxidative stress and mitochondrial damage in early Polycystic Kidney Disease

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD.MethodsWe investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy.ResultsSerum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling.ConclusionIn PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression.Support or Funding InformationR25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Curcumin Does Not Attenuate the Preeclamptic Phenotype in the Dahl Salt‐Sensitive Rat

Preeclampsia (PE) is associated with new onset of hypertension and proteinuria and affects 2–8% of pregnancies worldwide. It is a leading cause of maternal and fetal morbidity and mortality with no current therapy to manage the disease. While its etiology is yet to be elucidated, immune system activation of various inflammatory pathways is reported to be a contributing factor. Toll‐like receptor (TLR) activation by damage‐associated molecular patterns (DAMPS) released following poor placentation and oxidative stress has been linked to the development of PE and TLR4 expression is increased in the placenta of preeclamptic women. We performed a whole transcriptomic analysis on placental tissues from gestation day (GD) 14 rats using Affymetrix Rat 2.0ST GeneChip and noted significant dysregulation in multiple genes during pregnancy in the Dahl salt sensitive (S) rat, a model of spontaneous superimposed preeclampsia, compared to the normal pregnant control Sprague Dawley (SD) rat. These genes included upregulation of TLR4 in the placenta of Dahl S rats (1.9 fold, p&lt;0.05), which was subsequently confirmed via quantitative real‐time PCR (2.9 fold, p&lt;0.05). To further investigate this pathway, the goal of this study was to assess the effect of TLR4 inhibition in pregnant Dahl S rats. Curcumin, the anti‐oxidant and anti‐inflammatory active ingredient in the spice turmeric, has been reported to inhibit TLR4 activation, and reduced blood pressure in rodent models of LPS‐induced preeclampsia. We hypothesized that curcumin would inhibit TLR4 activation and reduce blood pressure and uterine artery resistance index during pregnancy in the Dahl S rat. Starting on gestational day (GD) 10, 17 weeks old rats were divided into two groups: a treated group (N=7) that orally received dough containing 400mg/kg of curcumin daily and a control group (N=6) that received the vehicle dough through GD20. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler ultrasound. On GD20, terminal blood pressure measurements were taken, and pups and placentas harvested and measured. Curcumin had no effect on UARI (0.78 vs. 0.82, p=0.4) or mean arterial pressure (146 ± 5 vs. 133 ± 5 mmHg, p=0.08). Fetal outcome assessed by litter size (10 ± 1 vs. 11 ± 1 pups); pup weight (2.23 ± 0.08 vs. 2.30 ± 0.07 g) and pup length (29.44 ± 0.31 vs. 29.6 ± 0.46 cm) was similar between groups. These results suggest that administration of curcumin does not alleviate the preeclamptic phenotype in the Dahl S rat.Support or Funding InformationR01HL1347111R01HL137673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circulating microRNA and automated motion analysis as novel methods of assessing chemotherapy-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy (CiPN) is a serious adverse effect in the clinic, but nonclinical assessment methods in animal studies are limited to labor intensive behavioral tests or semi-quantitative microscopic evaluation. Hence, microRNA (miRNA) biomarkers and automated in-life behavioral tracking were assessed for their utility as non-invasive methods. To address the lack of diagnostic biomarkers, we explored miR-124, miR-183 and miR-338 in a CiPN model induced by paclitaxel, a well-known neurotoxic agent. In addition, conventional and Vium’s innovative Digital Vivarium technology-based in-life behavioral tests and postmortem microscopic examination of the dorsal root ganglion (DRG) and the sciatic nerve were performed. Terminal blood was collected on days 8 or 16, after 20 mg/kg paclitaxel was administered every other day for total of 4 or 7 doses, respectively, for plasma miRNA quantification by RT-qPCR. DRG and sciatic nerve samples were collected from mice sacrificed on day 16 for miRNA quantification. Among the three miRNAs analyzed, only miR-124 was statistically significantly increased (5 fold and 10 fold on day 8 and day 16, respectively). The increase in circulating miR-124 correlated with cold allodynia and axonal degeneration in both DRG and sciatic nerve. Automated home cage motion analysis revealed for the first time that nighttime motion was significantly decreased (P < 0.05) in paclitaxel-dosed animals. Although both increase in circulating miR-124 and decrease in nighttime motion are compelling, our results provide positive evidence warranting further testing using additional peripheral nerve toxicants and diverse experimental CiPN models.

Alterations in terminal duct diameter and blood flow in the breasts of subjects with polycystic ovary syndrome

Alterations in terminal duct diameter and blood flow in the breasts of subjects with polycystic ovary syndrome

Catha edulis-induced skeletal muscle toxicity in experimental rats via regulation of rhabdomyolysis biomarkers

Background: Recently, there are clinical reports on the potential of Catha edulis (khat) to induce muscular toxicity. C. edulis (khat) is an evergreen shrub and a well-known controversial plant due to the content of natural stimulant, cathine and cathinone. Objective: The main objective of the study is to evaluate the possible effect of C. edulis leaves extract to induce rhabdomyolysis in vivo. Materials and Methods: Sprague Dawley rats were grouped and treated with khat extract at two different doses (250 and 500 mg/kg), while atorvastatin was used as positive control for 28 days. Body weight was measured throughout the study period. Overnight urine was collected from each rat at the 28th day for myoglobin (Myo) analysis. Terminal blood samples were collected from sacrificed animals for the measurement of serum biomarkers and clinical chemistry. The standard clinical pathology assays aspartate aminotransferase (AST), alanine aminotransferase (ALT), and serum creatinine (CR) were monitored. Skeletal muscle, cardiac muscle, and kidney were collected for histopathological examination. Results: Animals received 250 mg/kg khat extract had shown mild-to-no gait disorders, while at higher dose extract (500 mg/kg) had reduced the body weight of rats with marked increase of gait disorders compared to control. CR, AST, and ALT were elevated in high-dose administration and in rats received ethanol. The tested biomarkers such as heart-type fatty acid-binding protein (HFABP) 3, Troponin I Type 1 slow skeletal (TNNI1), and Myo were significantly increased in khat high dose and statin treatment, but not in low-dose extract and alcohol. The increase in HFABP and TNN1 results were well reflected in histopathological findings of skeletal myofiber degeneration and in the hemorrhages and pyknosis of nucleus observed in the cardiac muscle. Conclusion: These results provide evidence that khat chewing contributes to the development of muscle toxicity and probable rhabdomyolysis. The current subject thus warrants detailed studies which could emphasize on the cardiac complications and muscular toxicity mechanisms.

NCOA4 Mediates Mobilization of Hepatic Iron Stores after Blood Loss

The intracellular protein NCOA4 mediates the autophagic degradation of ferritin in vitro (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014); mice with global Ncoa4 disruption show hyperferremia, microcytic anemia, and ferritin accumulation in multiple organs, including liver (Bellelli et al., Cell Rep 2016). Here, we dissect the requirement for NCOA4 in hepatic iron mobilization after acute blood loss, using Ncoa4-targeting siRNA that was conjugated to triantennary N-acetylgalactosamine (GalNAc-Ncoa4 siRNA) to promote uptake by hepatocytes. On experimental day 0, 8-week-old female C57BL/6N mice underwent a single 500 μl phlebotomy, which was followed immediately by intraperitoneal volume replacement (saline) and concomitant subcutaneous injection of either GalNAc-Ncoa4 siRNA (3 mg/kg) or saline vehicle. The phlebotomized mice then underwent a second, terminal blood collection and organ harvest at either 3 days later (when mice subjected to this phlebotomy protocol are known to reach their hematocrit nadir) or 7 days later (when mice subjected to this phlebotomy protocol are known to exhibit substantial hematocrit recovery). To provide an experimental baseline, a group of 8-week-old mice that were not phlebotomized and did not receive a subcutaneous injection of either saline or GalNAc-Ncoa4-siRNA were analyzed on experimental day 0.Injection of GalNAc-Ncoa4 siRNA immediately after phlebotomy resulted in marked hepatic Ncoa4 knockdown at both 3 and 7 days after phlebotomy. Compared to non-phlebotomized mice (NPM), phlebotomized mice (PM) treated with either saline or GalNAc-Ncoa4 siRNA showed similar reductions in red blood cell count, hemoglobin concentration, and hematocrit at day 3, confirming that similar blood volumes were removed. Compared to NPM, PM treated with saline showed significantly lower levels of ferritin subunits by immunoblotting of liver lysates, consistent with a model in which phlebotomy induces the degradation of ferritin complexes in hepatocytes. By contrast, in PM treated with GalNAc-Ncoa4 siRNA, hepatic ferritin subunit levels did not decrease after blood loss, and at post-phlebotomy day 7, hepatic ferritin subunit levels were significantly higher in PM treated with GalNAc-Ncoa4 siRNA than in PM treated with saline. By post-phlebotomy day 7, mean liver non-heme iron concentration (LIC) was also significantly lower in PM injected with saline compared to NPM, suggesting that non-heme iron had been mobilized from their livers in the setting of increased iron demand for erythropoiesis. By contrast, in PM treated with GalNAc-Ncoa4 siRNA, mean LIC failed to decrease after phlebotomy. Hepatic Tfrc mRNA and protein were upregulated in PM treated GalNAc-Ncoa4 siRNA compared to saline, suggesting that an acute reduction in NCOA4 activity reduces cytosolic iron levels in hepatocytes. Interestingly, when compared to NPM, saline-treated PM showed a trend towards higher hepatic Ncoa4 mRNA when assessed at either day 3 (P=0.09) or day 7 (P=0.06) after blood loss, raising the possibility that hepatic NCOA4 requirements increase in response to phlebotomy. Although phlebotomy failed to lower hepatic iron stores in PM with hepatocellular Ncoa4 knockdown, hematological recovery at day 7 was similar in both treatment groups. At day 7 after phlebotomy, splenic non-heme iron stores were similarly depleted in both treatment groups. Interestingly, compared to PM treated with saline, PM treated with GalNAc-Ncoa4 siRNA showed lower hepatic hepcidin expression at day 7, raising the possibility that greater dietary iron absorption may compensate for the lack of hepatic iron mobilization in this group to allow for normal hematological recovery. Collectively, these findings suggest that NCOA4 activity is required to mobilize iron from the liver when extrahepatic iron demands increase following acute blood loss. However, in our model with hepatocellular Ncoa4 knockdown, dietary iron uptake and/or iron release from extrahepatic storage sites may compensate for the lack of hepatic iron mobilization to support stress erythropoiesis. While ferritin degradation has been shown to occur through both lysosomal and proteasomal pathways in vitro, our findings suggest that if NCOA4 expression is reduced following acute blood loss, other cellular pathways in hepatocytes cannot compensate to mobilize iron from the liver. DisclosuresNo relevant conflicts of interest to declare.

Genetic deletion of ADP-activated P2Y12 receptor ameliorates lithium-induced nephrogenic diabetes insipidus in mice.

Therapeutic use of lithium in bipolar disorder is limited by the development of nephrogenic diabetes insipidus (NDI). We reported that pharmacological blockade of P2Y12 receptor (R) with clopidogrel or prasugrel significantly ameliorated lithium-induced NDI in rodents. Using mice genetically lacking P2Y12 -R we evaluated whether the observed amelioration is mediated through P2Y12 -R METHODS: P2ry12-/- mouse line (C57/BL6) was rederived from cryopreserved embryos of the knockout (KO) mice generated by Deltagen Inc. Syngeneic wild type (WT) mice obtained by heterozygous crossing were inbred. Groups of adult WT and KO mice were fed lithium-added (40mmol LiCl/kg food) or regular diet, and euthanized after 2 or 4weeks. Twenty-four hour urine samples and terminal blood and kidney samples were analyzed. At both time points, lithium-induced polyuria and decrease in aquaporin-2 (AQP2) protein abundance in the kidney medulla were less marked in KO vs WT mice. Immunofluorescence microscopy revealed that lithium-induced alterations in the cellular disposition of AQP2 protein in the medullary collecting ducts of WT mice were blunted in KO mice. Serum lithium, sodium and osmolality were similar in both genotypes after lithium treatment. After 2weeks, lithium induced marked increases in urinary excretion of Na, K, and arginine vasopressin in WT mice but not in KO mice. Taken together, our data show that similar to pharmacological blockade, deletion of P2Y12 -R significantly ameliorates lithium-induced NDI, without reducing serum lithium levels. Hence, targeting P2Y12 -R with currently available drugs in the market offers a novel and safer method for treating NDI.

Antidiabetic activities of chloroform fraction of Anthocleista vogelii Planch root bark in rats with diet- and alloxan-induced obesity-diabetes

Ethnopharmacological relevanceAnthocleista vogelii Planch is a medicinal plant traditionally used in West Africa for the management and treatment of diabetes mellitus. Aim of the studyTo determine the antidiabetic activities of chloroform fraction (CF) of Anthocleista vogelii Planch root bark in rats with diet- and alloxan-induced obesity-diabetes. Materials and methodsInhibitory activities of CF against α-amylase and α-glucosidase activities were determined in vitro. Three weeks old rats were fed with high-fat diet for 9 weeks to induce obesity prior to further induction of diabetes using alloxan (150 mg/kg body weight, i.p.). Blood glucose levels and body weight were measured every 7 days throughout the experiment. Glucose tolerance was assessed in normal and CF-treated rats on day 21. Terminal blood samples were collected from sacrificed animals for the measurement of serum insulin levels. Pancreases were excised from treated and untreated animals for histopathological examination. ResultsLCMS/MS chromatographic profile of CF via positive and negative modes revealed 13 and 23 compounds respectively. Further analysis revealed quebrachitol (QCT), loganin, sweroside, oleoside 11-methyl ester and ferulic acid, which have been previously reported for their antidiabetic activities, as constituents of CF. CF inhibited activities of α-amylase (IC50 = 51.60 ± 0.92 µg/ml) and α-glucosidase (IC50 = 5.86 ± 0.97 µg/ml) in a dose-dependent manner. Treatment of animals with obesity-diabetes with 100 and 200 mg/kg CF significantly improved glucose tolerance (P < 0.001) and enhanced serum insulin levels (P < 0.05) compared to diabetic control rats. ConclusionsAntidiabetic activities of CF might be mediated via inhibition of α-amylase and α-glucosidase activities, elevation of serum insulin concentration, and enhancement of insulin and leptin sensitivity in obesity-diabetes rats. This study further substantiates the traditional use of A. vogelii in the management and treatment of diabetes in Africa and encourages further studies to investigate its mechanism of action.

Development of a Predictive Model for Deceased Donor Organ Yield.

Optimizing organ yield (number of organs transplanted per donor) is a modifiable way to increase the number of organs available for transplant. Historically, models to predict donor organ yield have been developed based ordinary least squares regression and ordinal logistic regression; however, alternative modeling methodology may be superior to conventional approaches.1,2 In this preliminary analysis, rather than treating organ yield as a continuous outcome, we modeled the number of organs transplanted per donor as counts. We aimed to compare different linear and nonlinear statistical models for count responses to predict deceased donor organ yield. We used data from the OPTN database from 2000 to 2016 to parameterize our exploratory models. The initial set of predictors for deceased donor organ yield was derived from published studies.1-3 We included adult deceased donors between 18 and 84 years of age that had at least 1 organ procured for transplantation. 75 350 records met inclusion criteria. We used 80% of the data for derivation in a cross-validation analysis and the remainder of the data as a validation set. The cross-validation analysis was replicated 50 times, and the random holdouts consisted of 20% of the derivation cohort. The following models were evaluated: ordinary least squares regression,1 ordinal logistic regression,2 Poisson regression, negative binomial regression, general additive models, classification and regression trees, random forests, bootstrap aggregated classification and regression trees, boosted classification and regression trees, Bayesian additive regression trees (BART), multivariate adaptive regression splines, artificial neural networks, and mean-only models. Among the models, BART resulted in the lowest error on predicting the number of organs transplanted per deceased donor. Two-sample t tests showed that the BART had significantly lower mean absolute error (MAE) when predicting deceased donor organ yield (all P < 0.001). On average, this model presented a MAE of 0.867 throughout the cross-validation analysis, and a MAE of 0.856 when tested in the validation set. The BART showed that deceased donor organ yield had a negative nonlinear relationship with age, body mass index, terminal blood urea nitrogen, terminal laboratory creatinine, aspartate aminotransferase, terminal laboratory total bilirubin; a positive nonlinear relationship with organ recovery time, partial pressure of oxygen levels, and last serum sodium; and more complex nonlinear relationships with alanine aminotransferase and the ratio of partial pressure arterial oxygen and fraction of inspired oxygen. Bayesian additive regression trees would improve prediction from at least 63 organs per 1000 donors (compared with an ordinary least squares regression1) to at most 120 organs per 1000 donors (compared with an ordinal logistic regression2). Through the use of BART, we were able to obtain higher predictive accuracy for organ yield. This model allows for nonlinear relationships among the predictors and the number of organs transplanted per deceased donor, which likely explains the superior performance compared with conventional models. In conclusion, our preliminary analysis shows that the BART methodology is superior in predicting deceased donor organ yield and can potentially serve as an aid to assess organ procurement organization performance, reduce geographic disparities, and in forecasting future organ availability. A forthcoming article will include the finalized analysis.

Avidity of α-fucose on human milk oligosaccharides and blood group–unrelated oligo/polyfucoses is essential for potent norovirus-binding targets

There is agreement with respect to norovirus infection routes in humans regarding binding of the pathogen to gastrointestinal epithelia via recognition of blood group-active mucin-typeO-glycans as the initiating and essential event. Among food additives playing a potential role in applications to protect newborns, human milk oligosaccharides (HMOs) as competitors are of major importance. By focusing on fractions of high-molecular mass HMOs with high fucose contents, we attempted to identify the structural elements required for norovirus GII.4 (Sydney 2012, JX459908) capsid binding in neoglycolipid-based arrays. We provide evidence that HMO fractions with the strongest binding capacities contained hepta- to decasaccharides expressing branches with terminal blood group H1 or Lewis-b antigen. H2 antigen, as recognized by UEA-I lectin, is apparently not expressed in high-mass HMOs. Beyond affinity, sterical and valency effects contribute more to virus-like particle binding, as revealed for oligovalent fucose conjugates of α-cyclodextrin and oligofucoses from fucoidan. Accordingly, high-mass HMOs with oligovalent fucose can exhibit stronger binding capacities compared with monovalent fucose HMOs. The above features were revealed for the most clinically relevant and prevalent GII.4 strain and are distinct from other strains, like GII.10 (Vietnam 026, AF504671), which showed a preference for blood group Lewis-a positive glycans.

Praliciguat, a Clinical-Stage sGC Stimulator, Improved Glucose Tolerance and Insulin Sensitivity and Lowered Triglycerides in a Mouse Diet-Induced Obesity Model

Background: Praliciguat (IW-1973) is a soluble guanylate cyclase (sGC) stimulator in clinical development. In animal models, praliciguat has broad tissue distribution and enhances nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) signaling, which has been shown to elicit hemodynamic, anti-inflammatory, and anti-fibrotic effects. This study assessed the metabolic effects of praliciguat in a mouse DIO model. Methods: Male C57BL/6J mice (12 weeks old at the start of treatment) were either maintained on regular chow (lean mice) or switched to a 60% high fat diet (HFD) starting at age 6 weeks (obese mice). At the start of treatment, obese mice received either praliciguat (6 mg/kg) or vehicle in the HFD. The lean mice remained on regular chow. On day 28, an oral glucose tolerance test (OGTT) was performed after a 3-h fast. On day 29, organs and terminal blood were collected for analysis after a 3-h fast. Results: Praliciguat treatment lowered OGTT glucose area under the curve (-22%, P&amp;lt;0.05), fasting plasma insulin (-53%, P&amp;lt;0.01) and HOMA-IR, an index of insulin resistance (-53%, P&amp;lt;0.01) vs. vehicle treatment. Praliciguat-treated mice also had lower liver (-36%, P&amp;lt;0.05) and fasting circulating triglycerides (-30%, P&amp;lt;0.01) vs. vehicle-treated mice. Furthermore, several fasting biomarkers did not change in treated vs. vehicle including beta-hydroxybutyrate, free fatty acids, plasma glucose, cholesterol, leptin, and adiponectin. Conclusions: Praliciguat improved insulin sensitivity, glucose tolerance, and yielded lower circulating and liver triglycerides in a mouse diet-induced obesity model. Disclosure C.D. Schwartzkopf: None. J. Hadcock: None. J.E. Jones: None. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G.T. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. J. Masferrer: Employee; Self; Ironwood Pharmaceuticals, Inc..

Glycosphingolipids of porcine, bovine, and equine pericardia as potential immune targets in bioprosthetic heart valve grafts.

Pericardial tissue from various animal species is utilized for the production of the bioprosthetic heart valves (BHV) used clinically. Experimental data show that the eventual breakdown of BHV is partly due to immunological interactions with carbohydrate tissue antigens. To understand these processes, we have examined the glycolipid-based carbohydrate antigens in naïve porcine, bovine, and equine pericardia. Total non-acid and acid glycosphingolipid fractions were isolated from porcine, bovine, and equine pericardia, and individual glycolipid compounds were characterized by thin-layer chromatography, mass spectrometry, and binding of monoclonal antibodies, lectins and bacteria in chromatogram binding assays. The non-acid glycolipid fractions from all species contained glycosphingolipids based on the globo- and neolacto-series, including pentaglycosylceramides with terminal Galα3 determinants. Terminal blood group A and H (O) structures based on type 2 core chains were present in porcine pericardium, while the Forssman pentaosylceramide was found in equine pericardium. All acid glycolipid fractions contained sulfatide and several gangliosides with both N-acetyl- and N-glycolyl-neuraminic acid as terminal saccharide chain determinants. Several carbohydrate antigens which are potential targets for the human immune system have been identified in the animal pericardial tissues used for the production of BHV. Which of these antigens are left in the tissues after industrial BHV production processes, as well as their potential role in eventual BHV degradation, remains to be elucidated.

FEATURES OF THE RESPONSE OF ARTERIAL BLOOD FLOW TO THE ORTHOSTATIC LOAD IN YOUNG WOMEN WITH IDIOPATHIC ARTERIAL HYPOTENSION

The aim of the study was to evaluate the response of arterial blood flow to the orthostatic load in young women with idiopathic arterial hypotension (IAH).Material and methods. A comparative analysis of systemic hemodynamics and peripheral arterial blood flow parameters was performed for orthostasis between two groups of women aged 1825: test with IAH (n = 73) and control (n=37). The SBP level in the test group was 90-98 mm Hg, in the control group - 120-129 mm Hg. BP, heart rate and angio-scanning were performed twice: at rest, lying after 15 minutes rest and during the first minute of orthostasis. Blood flow was studied in the right vertebral, radial and posterior tibial arteries. The following parameters were evaluated: diameter during diastole, peak systolic and terminal diastolic blood flow velocity.Results. In young women with IAH, as those with normal arterial pressure, orthostasis was accompanied by an increase in SBP, DBP and an increase in heart rate. With hypotension, an increase in the diameter of the vertebral artery and a decrease in the diameter of the posterior tibial artery were detected. In both groups, orthostasis was accompanied by a decrease in the rate of blood flow, both during systole and diastole.The conclusion. Orthostasis with IAH is accompanied by an increase in SBP, DBP and heart rate. The response of peripheral arteries to orthostasis is characterized by a change in the diameter of the vessels, which indicates a pronounced adaptive response to the physiological stress load.

Comparison of blood sampling methods for plasma corticosterone measurements in mice associated with minimal stress-related artefacts

Accurate measurement of circulating glucocorticoid concentrations in rodents is often hampered by the stress-related activation of the hypothalamic-pituitaryadrenal axis during animal handling. The present study aims to identify methods of blood collection associated with minimal stress and thus artificial increases in plasma glucocorticoid levels. Using two strains of mice, we evaluated common laboratory methods of non-terminal (tail blood sampling with or without restraint; retro-orbital puncture) and terminal blood collection (cardiac puncture) and their immediate and prolonged effect on plasma corticosterone levels.Compared to retro-orbital and cardiac puncture, mice from both the unrestrained and restrained tail snip collection groups displayed the lowest plasma corticosterone levels in both mouse strains. Plasma corticosterone levels in samples obtained from retro-orbital and cardiac puncture collection were up to twenty times higher than those measured in mice undergoing blood collection via tail snip. Repeat tail snip collections (every 30 min for 120 min, or once after 120 min) revealed sustained hypercortisolaemia, compared to the initial collection.We conclude that blood sampling via tail snip without restraint remains the gold-standard method of collection that is associated with minimal stress-related artefacts and hence feasible for single time point corticosterone analyses.

Morbidity and mortality resulting from acute inhalation exposures to hydrogen fluoride and carbonyl fluoride in rats

Objective: Experiments were undertaken to compare morbidity and mortality from brief inhalation exposures to high levels of hydrogen fluoride (HF) and carbonyl fluoride (COF2).Methods: Rats from both sexes were exposed for durations of 5 and 10 min to nominal concentrations of 10,000 to 57,000 ppm HF or 500 to 10,000 ppm COF2. Respiration was monitored before, during, and after exposure. Animals were observed up to 6 days post-exposure. Terminal blood samples were collected for routine clinical chemistry and hematology. Post-mortem lung fluoride concentrations and lung weights were measured, and gross pathology noted.Results: Both gases produced respiratory depression independent of concentration or exposure duration with minute ventilation decreasing to approximately 50% of baseline. Estimated mixed-gender HF and COF2 10-min LC50’s were 48,661 ppm and 1083 ppm, respectively. HF mortalities were generally delayed 3 to 4 days post-exposure, while COF2 mortalities occurred during or briefly after exposure. Lung fluoride levels increased with COF2 dose, though elevated lung weights occurred only at the mid-level exposures. Lung weights were unaffected in the HF-exposed animals, and their lung fluoride concentrations were variable. Clinical chemistry and hematology had few consistent trends with the exception of hemoconcentration primarily in HF-exposed males. These short-term exposure experiments conclude that COF2 is nearly 45 times more lethal than HF in rats.Conclusions: These experiments suggest that hydrolysis to HF cannot solely explain COF2 toxicity. Although HF and COF2 may have common injury mechanisms, they are expressed to markedly different degrees and temporal occurrence.

Role of terminal warm blood cardioplegia in complex congenital heart surgery.

Introduction Myocardial protection is vital to ensure successful open heart surgery. Cardioplegic solution is one method to achieve good myocardial protection. Inevitably, ischemia-reperfusion injury occurs with aortic crossclamping. Histidine-tryptophan-ketoglutarate solution is a frequently used cardioplegia for complex congenital heart surgery. We postulated that addition of terminal warm blood cardioplegia before removal of the aortic crossclamp might improve myocardial protection. Method A randomized controlled trial was conducted on 109 cyanotic patients aged, 1 to 5 years who underwent complex biventricular repair. They were divided into a control group of 55 patients who had histidine-tryptophan-ketoglutarate only and a treatment group of 54 who had histidine-tryptophan-ketoglutarate with terminal warm blood cardioplegia. Endpoints were clinical parameters, troponin I levels, and caspase-3 as an apoptosis marker. Results The incidence of low cardiac output syndrome was 34%, with no significant difference between groups (35.2% vs. 33.3%, p = 0.84). The incidence of arrhythmias in our treatment group was lower compared to the control group (36% vs. 12%, p = 0.005). Troponin I and caspase-3 results did not show any significant differences between groups. For cases with Aristotle score ≥ 10, weak expression of caspase-3 in the treatment group post-cardiopulmonary bypass was lower compared to the control group. Conclusion For complex congenital cardiac surgery, the addition of terminal warm blood cardioplegia does not significantly improve postoperative clinical or metabolic markers.

Effects of hydroxytyrosol dose on the redox status of exercised rats: the role of hydroxytyrosol in exercise performance

BackgroundHydroxytyrosol (HT) is a polyphenol found in olive oil that is known for its antioxidant effects. Here, we aimed to describe the effects of a low and high HT dose on the physical running capacity and redox state in both sedentary and exercised rats.MethodsMale Wistar rats were allocated into 6 groups: sedentary (SED; n = 10); SED consuming 20 mg/kg/d HT (SED20; n = 7); SED consuming 300 mg/kg/d HT (SED300; n = 7); exercised (EXE; n = 10); EXE consuming 20 mg/kg/d HT (EXE20; n = 10) and EXE consuming 300 mg/kg/d HT (EXE300; n = 10). All the interventions lasted 10 weeks; the maximal running velocity was assessed throughout the study, whereas daily physical work was monitored during each training session. At the end of the study, the rats were sacrificed by bleeding. Hemoglobin (HGB) and hematocrit (HCT) were measured in the terminal blood sample. Moreover, plasma hydroperoxide (HPx) concentrations were quantified as markers of lipid peroxidation.ResultsIn sedentary rats, HT induced an antioxidant effect in a dose-dependent manner without implications on running performance. However, if combined with exercise, the 300 mg/kg/d HT dosage exhibited a pro-oxidant effect in the EXE300 group compared with the EXE and EXE20 groups. The EXE20 rats showed a reduction in daily physical work and a lower maximal velocity than the EXE and EXE300 rats. The higher physical capacity exhibited by the EXE300 group was achieved despite the EXE300 rats expressing lower HGB levels and a lower HCT than the EXE20 rats.ConclusionsOur results suggest that a high HT dose induces a systemic pro-oxidant effect and may prevent the loss of performance that was observed with the low HT dose.

Interpreting Neuroendocrine Hormones, Corticosterone, and Blood Glucose to Assess the Wellbeing of Anesthetized Rats during Euthanasia.

Current recommendations for assessing animal wellbeing during euthanasia suggest that measuring neuroendocrine hormones-such as ACTH, noradrenaline, and adrenaline-is preferable to measuring corticosterone and blood glucose because of the sensitivity of neuroendocrine hormones to the acute stress associated with rapid methods of euthanasia. However, these neuroendocrine hormones can be stimulated in ways that confound interpretation of welfare assessment in euthanasia studies. Although this property does not negate the usefulness of neuroendocrine hormones as tools of assessment, it is important to differentiate the stress associated with the induction of anesthesia before the loss of consciousness (an animal wellbeing concern) with the physiologic responses that occur after the loss of consciousness (not an animal wellbeing concern). In this study, rats were anesthetized by using a ketamine-xylazine combination. Once the rats achieved a surgical plane of anesthesia, they were exposed to O₂, CO₂, or isoflurane, followed by terminal blood collection to assess concentrations of ACTH, noradrenaline, corticosterone, and blood glucose. Compared with animals exposed to O₂ or isoflurane, rats exposed to CO₂ had significant increases in their serum concentrations of ACTH and noradrenaline, but blood glucose and corticosterone did not differ between groups. These findings indicate that noradrenaline and ACTH should be used with caution to assess animal wellbeing when the method of euthanasia might confound that assessment.