Response Of Glands Research Articles

Sex-mediated elevation of the specialized pro-resolving lipid mediator levels in a Sjögren's syndrome mouse model.

Our previous results showed that the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) promotes resolution of inflammation in salivary glands in non-obese diabetic (NOD)/ShiLtJ, a mouse model for Sjögren's syndrome (SS). Additionally, mice lacking the RvD1 receptor ALX/FPR2 show defective innate and adaptive immune responses in salivary glands. Particularly, ALX/FPR2 KO mice exhibit exacerbated inflammation in their salivary glands in response to systemic LPS treatment. Moreover, female ALX/FPR2 KO mice show increased autoantibody production and loss of salivary gland function with age. Together, these studies suggest that an underlying SPM dysregulation could be contributing to SS progression. Therefore, we investigated whether SPM production is altered in NOD/ShiLtJ using metabololipidomics and enzyme-linked immunosorbent assay (ELISA). Our results demonstrate that SPM levels were broadly elevated in plasma collected from NOD/ShiLtJ female mice after disease onset, whereas these drastic changes did not occur in male mice. Moreover, gene expression of enzymes involved in SPM biosynthesis were altered in submandibular glands (SMG) from NOD/ShiLtJ female mice after disease onset, with 5-LOX and 12/15-LOX being downregulated and upregulated, respectively. Despite this dysregulation, the abundances of the SPM products of these enzymes (ie, RvD1 and RvD2) were unaltered in freshly isolated SMG cells suggesting that other cell populations (eg, lymphocytes) may be responsible for the overabundance of SPMs that we observed. The elevation of SPMs noted here appeared to be sex mediated, meaning that it was observed only in one sex (females). Given that SS primarily affects females (roughly 90% of diagnosed cases), these results may provide some insights into the mechanisms underlying the observed sexual dimorphism.

MicroRNA profiles were altered in neonatal piglet mammary glands following postnatal infant formula feeding

Postnatal dietary modulation of microRNAs (miRNAs) and effects on miRNA–mRNA interactions in tissues remain unknown. This study aimed to investigate whether dietary factors (formula vs. breastfeeding) affect mammary miRNA expression and to determine if these changes are concurrent with developmental alterations of the mammary gland in neonatal piglets. Female Yorkshire/Duroc piglets were fed sow's milk or cow's milk- or soy-based infant formula (from postnatal day 2 to day 21; n=6/group). Differentially expressed miRNAs were determined using mammary miRNA profiling, followed by miRNA and mRNA expressions characterized by quantitative reverse-transcription polymerase chain reaction. Milk and soy formulas reduced expressions of miR-1, -128, -133a, -193b, -206 and -27a; miRNA down-regulation altered mRNA expressions of genes (e.g., Ccnd1, Tgfb3, Igf1r and Tbx3) that were consistent with enhanced cell proliferation and suppressed apoptotic processes in the developing mammary gland. Interestingly, down-regulation of miR-1, -128 and -27a also correlated with increased mRNA genes such as Hmgcs and Hmgcr encoding cholesterol synthesis in the mammary glands in response to lower circulating cholesterol levels. Infant formula feeding affected mammary miRNA profiles in neonatal piglets, concurrent with increased expression of cell proliferation and cholesterol synthesis genes, suggesting early nutritional modulation of miRNAs may contribute to regulation of proliferative status and cholesterol homeostasis of developing mammary glands during infancy.

Abnormal Regeneration of the Gastric Epithelium May Result in Increased H. pylori‐Induced Gastric Disease in the Elderly

BackgroundIncreased incidence of gastric ulcer disease is often related to frequent nonsteroidal anti‐inflammatory drug (NSAID) use and Helicobacter pylori (H. pylori) infection. Studies have shown H. pylori adheres to the gastric epithelium through binding of bacterial Sialic Acid‐binding Adhesin (SAbA) to glycosylated gastric epithelial mucin receptor sialyl‐Lewis X (S‐LewX). Our group has reported that CD44v9 emerges during repair of the gastric epithelium after injury where it is co‐expressed with markers of Spasmolytic Polypeptide/TFF2‐Expressing Metaplasia (SPEM), a reparative phenotype which has been shown to emerge in response to gastric injury. H. pylori localizes specifically to SPEM glands within the corpus of the stomach. We hypothesize that SAbA adhesin and S‐LewX facilitate H. pylori preferential localization and adhesion to SPEM glands in response to injury.HypothesisThe aged gastric epithelium exhibits sustained SPEM glands expressing S‐LewX in response to injury. H. pylori preferential localization and adhesion to SPEM glands may contribute to the severity of disease in the aged epithelium.MethodsGastric ulcers were induced in young (2–3 months old) and aged (>8 months old) C57/BL6 mice using an acetic acid‐induced injury model. Twenty‐one days after ulcer injury, mice were inoculated with H. pylori or with Brucella broth. Gastric organoids were generated from biopsies collected from young (14–30 years) or elderly (>55 years) human patients and transferred to monolayer cultures for in vivo H. pylori adherence assays. Infected monolayers were treated with sialic acid analogs 3′‐sialyllactose or 6′‐sialyllactose. Flow cytometry and immunofluorescence was used to analyze the co‐localization and adherence of H. pylori to S‐LewX‐expressing SPEM glands.ResultsSixty days post‐injury, MUC5AC re‐localized to the surface of the gastric epithelium with complete regeneration of the gastric epithelium, expression of parietal, chief and endocrine cells and no evidence of SPEM in the young mice. The aged gastric epithelium exhibited decreased and irregular MUC5AC localization and the persistence of SPEM glands that expressed both CD44v9 and S‐LewX. Twenty‐one days post‐injury, H. pylori adherence was limited to the surface epithelium in young mice. However, in the aged mice, metaplasia was present, chief cells were decreased, and H. pylori adherence was localized at the base of the epithelial glands and co‐localized with CD44v9/S‐LewX‐expressing glands. Adherence assays demonstrated that H. pylori adherence was preferential to □‐2,3‐sialyl‐glycans.ConclusionFollowing injury in aged gastric tissues, the mucous layer becomes altered such that SPEM persists and there is loss of normal mucin expression and gastric cell lineages. H. pylori adherence to CD44v9‐positive cells is enhanced by S‐LewX and abnormal regeneration appears to influence the severity of H. pylori‐associated disease.Support or Funding InformationNIH (NIDDK) 5R01DK083402‐08 grant (YZ); Albert C. Yates Fellowship, Univ. of Cincinnati (MD)

Mechanosensitivity of TRPV Channels: Implications for Vasopressin Neuron Activity

Vasopressin is synthesised by magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and is secreted from the posterior pituitary gland in response to increased plasma osmolality. Vasopressin maintains body fluid balance by promoting renal water retention. Mechanosensitive TRPV channels facilitate vasopressin neuron responses to plasma osmolality and express various TRPV subunits, including ΔN‐TRPV1, TRPV2 and TRPV4. Most TRPV channels express more than one type of TRPV subunit but the subunit composition of the TRPV channels expressed by vasopressin neurons is unknown, as is the potential contribution of any changes in composition to vasopressin neuron function. Here, we tested the hypothesis that a change in TRPV channel composition alters the mechanosensitivity of ΔN‐TRPV1‐containing channels. To test this hypothesis TRPV and vasopressin expression in the SON of female Sprague Dawley rats was visualised using immunohistochemistry and will also be visualised with RNAscope. Xenopus oocytes and HEK293 cells were used for heterologous expression of TRPV channels and membrane current recorded via electrophysiology. TRPV2 was expressed on ~33% of SON vasopressin neurons and TRPV4 was expressed on ~40%. However, the TRPV1 antibody caused non‐specific staining and instead RNAscope will be used. Xenopus oocytes expressing ΔN‐TRPV1, TRPV2 and TRPV4 were recorded via two‐electrode‐voltage‐clamp (TEVC). However, TRPV channels in Xenopus oocytes do not appear to be functional because there was no change in membrane current during application of TRPV antagonists and agonists, independent of injected RNA amount (1–20ng) and incubation time (1–4 days). Transfection of TRPV channels into HEK293 cells with co‐expressed fluorescent protein; ΔN‐TRPV1‐GFP, TRPV2‐mCherry and TRPV4‐CFP was performed. Maximum ΔN‐TRPV1‐GFP expression occurred from 10–20 h, however increased cell death was observed after 24 h. In conclusion, vasopressin neurons express TRPV channels but Xenopus oocytes appear to lack specific cytoskeletal proteins for mechanical gating such as beta‐tubulin. HEK‐293 cells express the required cytoskeletal proteins for TRPV trafficking, membrane tethering and gating and we are in process of testing whether ΔN‐TRPV1‐GFP transfection in HEK‐293 cells forms mechanosensitive channels.

Endogenous β‐endorphin Plays a Pivotal Role in Angiotensin II‐mediated Pressor Response

BackgroundEndorphins are endogenous opioid neuropeptides that are mainly produced from the pituitary gland in response to pain and stress. A number of mediators were reported to stimulate β‐endorphin production including interleukin 1 beta (IL‐1β) and corticotropin‐releasing factor (CRF). Angiotensin II (Ang II) can stimulate β‐endorphin production from anterior pituitary gland, but the exact molecular mechanisms involved in this effect, as well as the role of the released β‐endorphin in Ang II‐mediated pressor response, remain elusive. Our study aimed to fill this knowledge gap.MethodsMale rats (6 per group) were either injected with IL‐1β receptor blocker (IL‐1R antagonist, 100 μg /kg, s.c.), the CRF receptor blocker, astressin (100 μg /kg, Intranasal) or a combination of both prior to Ang II injection (200 μg/kg, ip). Other groups of rats were given naloxone (1.6 mg/kg, s.c.) or telmisartan (10 mg/kg, oral) prior to Ang II injection. Serum and hippocampal β‐endorphin levels, brain IL‐1β and CRF levels were detected. Moreover, markers of oxidative stress including malondialdehyde (MDA) and superoxide dismutase (SOD), the inflammatory marker, C‐reactive protein (CRP) and sympathoexcitation (c‐Fos, L‐glutamate, and phosphorylated MAPK) markers were measured in the hippocampus of different groups.ResultsAng II increased serum and hippocampal β‐endorphin levels by 37.95% and 28.03%, respectively. This response was attenuated when astressin or/and IL‐1R antagonist were given before Ang II injection. Moreover, Ang II induced a pressor response, increased hippocampal IL‐1β (1.4‐fold), CRF (7‐fold), MDA (1.5‐fold) and CRP (3.3‐fold) while deceased SOD (by 31.49%) levels. Also, Ang II injection increased c‐Fos (9.9‐fold), glutamate (3.1‐fold) and phosphorylated‐MAPK (5.6‐fold) levels. The previous effects were abolished when telmisartan was given prior to Ang II injection, while naloxone aggravated Ang II‐mediated pressor response and most of the biochemical changes.ConclusionThese findings suggest that Ang II can induce β‐endorphin release by increasing the levels of both IL‐1β and CRF and that the released endorphin mitigates Ang II‐mediated sympathoexcitatory response. This study reveals new insights on the intermediary role of β‐endorphin in Ang II‐mediated sympathoexcitation and highlights it as a possible target for treating hypertension.

Effect of whole-milk allowance on liveweight gain and growth of parenchyma and fat pads in the mammary glands of dairy heifers at weaning

The rates of development of 2 tissues in mammary glands, parenchyma (PAR) and the mammary fat pad (MFP), in response to nutrition in early life might have a major bearing on lifetime milk production. Historical studies reported that feeding greater amounts of dietary nutrients from postweaning to puberty increased growth rates of heifers and stimulated the growth of MFP at the expense of PAR, which might suggest compromised mammary development and future milk production. The current study sought to determine if a higher volume of whole milk (8 vs. 4 L/d) offered to calves would increase rates of growth and development of PAR in mammary glands at weaning (1 to 12 wk). To measure these tissues, we developed 2 simple methods to assess the size of PAR and MFP at the time of screening using ultrasound. We report that calves offered 8 L/d of whole milk had greater rates of growth until weaning (0.86 ± 0.06 vs. 0.81 ± 0.09 kg/d), compared with calves offered 4 L/d. Ultrasonography showed that despite the faster rates of growth in calves offered 8 L/d of milk/d, the ratio of PAR:MFP depth was 40% less at weaning in the front glands (34%) compared with calves offered 4 L of milk/d. Rear glands were less impaired. The ultrasound methods developed here might be useful to monitor the development of mammary glands in response to different nutritional regimens during the preweaning period.

TLR2 Signaling Pathway Combats Streptococcus uberis Infection by Inducing Mitochondrial Reactive Oxygen Species Production.

Mastitis caused by Streptococcus uberis (S. uberis) is a common and difficult-to-cure clinical disease in dairy cows. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2−/− and TLR4−/− mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage, and bacterial count. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host in controlling the secretion of inflammatory factors and the elimination of intracellular S. uberis. Our results demonstrated that TLR2-mediated mROS has a significant effect on S. uberis-induced host defense responses in mammary glands as well as in MECs.

Immunosuppression in Cows following Intramammary Infusion of Mycoplasma bovis

Mycoplasma bovis is a destructive pathogen that causes large economic losses in rearing cattle for beef and dairy worldwide. M. bovis causes suppression of and evades the host immune response; however, the mechanisms of host immune function involved in M. bovis mastitis have not been elucidated. The purpose of this study was to elucidate the characteristics of the bovine immune response to mycoplasmal mastitis. We evaluated the responsiveness of the bovine mammary gland following infusion of M. bovis Somatic cell counts and bacterial counts in milk from the infected quarter were increased. However, the proliferation of peripheral blood mononuclear cells (blood MNCs) and mononuclear cells isolated from M. bovis-stimulated mammary lymph nodes (lymph node MNCs) did not differ from that in the unstimulated cells. Transcriptome analysis revealed that the mRNA levels of innate immune system-related genes in blood MNCs, complement factor D (CFD), ficolin 1 (FCN1), and tumor necrosis factor superfamily member 13 (TNFSF13) decreased following intramammary infusion of M. bovis The mRNA levels of immune exhaustion-related genes, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), lymphocyte activation gene 3 (LAG3), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) of milk mononuclear cells (milk MNCs) in the infected quarter were increased compared with those before infusion. Increase in immune exhaustion-related gene expression and decrease in innate immune response-related genes of MNCs in quarters from cows were newly characterized by M. bovis-induced mastitis. These results suggested that M. bovis-induced mastitis affected the immune function of bovine MNCs, which is associated with prolonged duration of infection with M. bovis.

Effects of Dietary Fat Sources during Late Gestation on Colostrum Quality and Mammary Gland Inflammation in Lipopolysaccharide-Challenged Sows.

Simple SummaryIn the present study, late gestating sows were challenged with lipopolysaccharide (LPS) endotoxin, which can impair the immune system of mammary gland cells and result in an inflammatory response. Additionally, the LPS-treated sows were fed 3% soybean oil (SO), 3% coconut oil (CO) or 3% fish oil (FO) diets and were used to study the effect of fat sources on the colostrum quality and mammary gland inflammation of sows exposed to immune challenge. The results show that FO inclusion exerted anti-inflammatory effects in mammary glands and counteracted the LPS-induced damaged colostrum synthesis and pro-inflammatory response when compared to CO diets. These findings suggest that fatty acid profiles of different oil types in late gestation differentially affect metabolic health in sows, but a longer period of FO supplementation to sows is needed to determine a positive effect on piglets.This study aimed to investigate the effects of maternal lipopolysaccharide (LPS) challenge and dietary fat sources on colostrum quality and inflammatory response in sows. Sixty Landrace × Yorkshire sows were randomly assigned to three dietary treatments supplemented with 3% soybean oil (SO), 3% coconut oil (CO) or 3% fish oil (FO), respectively, from Day 90 of gestation until parturition. On Day 112 of gestation, half the sows from each dietary treatment were challenged with LPS (10 μg/kg BW) or saline. The results showed that maternal LPS challenge decreased colostrum yield and dry matter content. A similar pattern of changes was observed for body weight gain and colostrum intake in piglets from LPS-challenged sows. Maternal LPS challenge increased the levels of tumor necrosis factor α (TNFα), interleukin 1β (IL1β) and IL6 in colostum, and the mRNA abundance of IL6, IL1β and TNFα and the phosphorylation level of p65 in mammary glands. However, the responses of these variables to LPS treatment were lower in sows fed a FO diet. In conclusion, maternal immune challenge reduced the growth performance of piglets by decreasing colostrum yield and intake by piglets, and dietary supplementation with FO in sows attenuates the LPS-induced inflammatory response in mammary glands.

Early response of the parathyroid gland to withdrawal of a calcimimetic compound in uremic rats.

Little is known about changes in parathyroid cells when calcimimetics are withdrawn. We examined the response of parathyroid glands to cinacalcet (Cina) withdrawal in uremic Sprague-Dawley rats fed a high-phosphate diet to develop secondary hyperparathyroidism and divided into groups treated with vehicle (UC), Cina, and Cina and maxacalcitol (Maxa), a vitamin D receptor activator (CiNa + Maxa). After 2 wk of treatment, vehicle and Cina were withdrawn and Maxa was continued. Rats were analyzed immediately (day 0) and 7 days (day 7) after withdrawal. The Cina and CiNa + Maxa groups had significantly lower parathyroid hormone (PTH) than the UC group on day 0, although PTH in the Cina group reached UC levels on day 7. On day 0, there were significantly more proliferating cell nuclear antigen-positive cells in the UC group compared with normal controls, and this increase was significantly suppressed in the Cina and CiNa + Maxa groups. On day 7, the Cina group, but not the CiNa + Maxa group, showed a significant increase in proliferating cell nuclear antigen-positive cells compared with the UC group. This increase was related to parathyroid cell diameter regression to UC levels, whereas combination treatment maintained diameter suppression. These results indicate that parathyroid growth activity is stimulated by Cina withdrawal, although the PTH level was not further increased. Continuous administration of Cina may be required for optimal control of secondary hyperparathyroidism, and simultaneous use of a vitamin D receptor activator may be advisable during Cina withdrawal.

Innate immune response of mammary gland induced by intramammary infusion of Bifidobacterium breve in lactating dairy cows.

This study aimed to evaluate innate immune responses of mammary glands induced by intramammary infusion of Bifidobacterium breve in dairy cows. Somatic cell counts in quarters of cows showed a marked increase following B. breve infusion on days 1 and 2. Opsonized-stimulated chemiluminescence response in quarter milk was significantly (P<0.05) increased by B. breve infusion on days 1 to 3 compared to that of pre-infusion. Lactoferrin concentrations in B. breve-infused quarter milk increased significantly (P<0.05) on days 2 to 4 and 6 compared to those of pre-infusion. IgG and IgA concentrations in B. breve-infused quarters significantly (P<0.05) increased on days 2 to 4 for IgG and days 3, 4, 6 and 8 for IgA compared to those of pre-infusion. Interleukin (IL)-1β and IL-8 mRNA levels in somatic cells from B. breve-infused quarters were significantly (P<0.05) upregulated on day 1 compared to those on days 0 and 14. Conversely, IL-6 mRNA levels in somatic cells from B. breve-infused quarters on days 0, 1 and 14 and NF-κB mRNA levels on day 0 were significantly (P<0.05) down-regulated compared to those of control. IL-1β, tumor necrosis factor (TNF)-α and IL-6 concentrations increased on days 1, 3 and 7 after B. breve infusion in quarters. Intramammary infusion of B. breve (3 × 109 cfu) induces a massive influx of leukocytes and enhances innate immune response in mammary glands. This event may contribute to the enhancing host defense in the mammary gland.

The Relation between Role of Serum Cortisol Level and Response to Various Respiratory Support Strategies among Preterm Infants

Introduction: Preterm infants are liable to various health problems including respiratory distress syndrome (RDS). There is variation in response to respiratory support. In preterm infants, cortisol hormone is secreted by the adrenocortical gland in response to stress. Objectives: To compare the serum cortisol levels in blood among preterm infants who needed different respiratory support strategies e.g. headbox, continuous positive airway pressure (CPAP), intubation surfactant extubation (INSURE) and mechanical ventilation (MV) and to correlate the cortisol levels to the severity of respiratory distress syndrome (RDS). Material and Methods: Observational prospective study that assessed the serum cortisol levels in preterm infants with RDS after initial respiratory support aged 28 - 34 gestational weeks that were admitted to the neonatal intensive care unit of Al Zahraa hospital of Al-Azhar University and Al-Estekama hospital between February 2019 and November 2019. Infants were classified into three groups, Group 1: 29 infants with severe RDS who needed a mechanical ventilator. Ten of them needed surfactant therapy. Group 2: 33 infants with moderate RDS who needed CPAP. Three of them needed surfactant therapy. Group 3: 28 infants with symptoms of mild RDS who needed headbox. None of them needed surfactant therapy. Blood samples were collected on the first day of life and were processed using the colorimetric ELISA method. Demographic and medical information was collected. Results: A total of 90 preterm infants were included. The serum cortisol reference was 4.3 - 22.4 mg/dl. Group 1 showed significantly higher serum cortisol levels compared to group 2 and group 3 (39.22 ± 9.91 mg/dl vs 28.96 ± 6.48 mg/dl vs 25.88 ± 5.42 mg/dl) respectively, (P-value = 0.001). Infants in group 2 who needed surfactant therapy had higher serum cortisol levels than those who did not need surfactant therapy (32.30 ± 5.92 mg/dl vs 28.33 ± 6.27 mg/dl). The serum cortisol levels were observed having a negative correlation with gestational age and birth weight. No significant differences were observed in terms of gender or type of delivery. Conclusion: Cortisol levels had a positive correlation with the severity of RDS who needed various respiratory support strategies.

Effect of subclinical mastitis on the yield and cheese-making properties of ewe’s milk

Abstract This review covers an update of scientific knowledge about productive and technological consequences of subclinical mastitis in sheep milk. The literature reports individual milk yield losses of 2.6–43.1 %, being modulated by several factors as infection severity, production level, causal agents, and unilateral or bilateral IMI (1 or 2 infected glands, respectively). A compensatory increase of milk production from the uninfected gland when only one half was infected has been quantified in 6.6 %, compared with healthy halves of control sheep. This compensatory adaptation highlights the risk of underestimating subclinical mastitis in sheep. The mammary gland response is quick and milk yield losses in absolute terms remained constant within the following weeks, both when infection appear during lactation and when it is present from lambing. With respect to the changes on main components in milk due to subclinical mastitis it has been clearly established a decrease in the concentration of lactose and an increase of that of whey proteins. The role played by lactose as an osmotic regulator results in a more accentuated decrease of its concentration in milk. This is why lactose is considered at present as a reliable potential indicator of subclinical mastitis. Whey proteins increase as a result of the increase of the blood-milk barrier permeability and the increased proteolysis of caseins. However, the content in milk of fat and casein are modified depending on the magnitude of milk yield reduction, being affected by a concentration or dilution effect. In any case, the ratio casein to protein (parameter independent of the milk volume) decreases as a result of infection. The impairment of physical and chemical characteristics due to decreased udder health status is the responsible of the negative effect of increased SCC on the coagulation properties of milk, the curd yield and the quality of cheese. Low ratio of casein to protein in high bulk tank SCC milk enhances the extension of the rennet coagulation time (RCT) and curd firming time (k20) because there are more serum proteins and the stability of casein micelles are reduced as a result of hydrolysis. Those changes in turn led to poor syneresis, lower cheese yield, increased moisture content and lower fat and protein content in cheese. Finally, there is a favourable relationship between lactose and milk technological properties because the decrease of this component in case of mastitis is associated with an increase of milk pH. Thus, the three parameters, SCC, pH, and lactose affect, contemporarily and independently, milk quality and coagulation properties, and this is why have been highlighted as potential indicators traits for improving cheese-making ability of sheep milk.

Effects of milking frequency and domperidone injections on milk production and prolactin signaling in the mammary gland of dairy cows

Eleven mid-lactation Holstein cows were milked twice daily during the first 2 experimental weeks. During wk 3 to 10, the cows were differentially milked: right quarters were milked thrice daily (3×) and left quarters were milked once daily (1×). During wk 11 to 14, all quarters were milked twice daily. After 4 wk of differential milking, the cows received daily i.m. injections of the dopamine antagonist domperidone (DOMP; 300 mg; n = 6) or of dimethyl sulfoxide as the control (CTL; n = 5) for 8 wk (wk 7-14). During the differential milking period (wk 3-6), milk production was greater for quarters milked 3× than for those milked 1× but did not differ between DOMP and CTL cows. During the differential milking + injection period (wk 7-10), milk production continued to differ according to milking frequency. However, DOMP injection did not have an effect or interact with milking frequency on milk production. During the injection period (wk 11-14), milk production remained greater in the quarters previously milked 3× and milk production increased in DOMP injected cows but not in CTL cows. Injections of DOMP increased prolactin concentration, which was greater in the serum of DOMP cows than in that of CTL cows during the differential milking + injection and the injection periods. The expression of genes that are directly related to milk synthesis (CSN2, LALBA, and ACACA) was greater in the 3× quarters than in the 1× quarters. In addition, DOMP increased CSN2 expression during the injection period. The expression of both isoforms of the PRLR gene was greater in the 3× quarters during the differential milking + injection and the injection periods. At the protein level, injections of DOMP tended to increase the number of long PRLR isoform during the differential milking + injection period. The number of short PRLR isoform was greater in the 1× quarters than in the 3× quarters during the differential milking, the differential milking + injection, and the injection periods. The total amount of STAT3 protein was greater in the 1× quarters during the differential milking and the differential milking + injection periods. The amount of phosphorylated STAT3 protein was greater in the 1× quarters during the differential milking period. The total amount of phosphorylated STAT5 protein was greater in the 3× quarters during the differential milking and the differential milking + injection periods. The results of this experiment support the hypothesis that the responsiveness of the mammary gland to PRL is modulated by milking frequency, although the underlying mechanism remains to be determined.

Infectious Bronchitis Virus Immune Responses in the Harderian Gland upon Initial Vaccination.

In recent years, Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis (IB) virus (IBV) vaccines have been used to characterize the immune responses of chickens subsequent to vaccination on day of hatch or beyond. Perhaps because ArkDPI vaccines display increased heterogeneity, the results on cell immune responses have shown ambiguity. In the current study, we investigated the effects of vaccination with a highly stable and homogeneous Massachusetts (Mass)-type vaccine on days 1 or 7 of age on Harderian gland (HG) responses. Confirming previous studies, both IBV serum antibodies and lachrymal IgA levels were greater upon vaccination on day 7 compared with vaccination on day 1 of age. Unlike results with ArkDPI viruses, a clear trend was detected for both B and T cells in the HG after Mass-type vaccination. Consistent with antibody responses, B- and T-helper (CD3+CD4+) cell frequencies were higher in birds vaccinated on day 7 of age. Cytotoxic T cells (CD3+CD8+) were also increased compared with chickens vaccinated on day 1 of age. Depending on the most likely age of IB outbreaks to occur in a particular region, postponing the first IBV vaccination may optimize immune responses.

Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

Subclinical mastitis in Lacaune sheep: Causative agents, impacts on milk production, milk quality , oxidative profiles and treatment efficacy of ceftiofur

Mastitis is a major disease affecting dairy sheep. It is caused by microorganisms that generate inflammation of the mammary gland in response to tissue invasion. This syndrome affects the welfare of ewes, as well as the production and quality of the milk, thereby reducing its productive efficiency. Because mastitis causes inflammation process, it also increases the production of free radicals that cause lesions via lipoperoxidation, causing damage to proteins, cells and tissues. One way to minimize the impact of the disease is antimicrobial treatment. Nevertheless, the continuous use of antimicrobials contributes to microbial resistance, in addition to producing residues in the milk and derivatives if not given during the grace period. Therefore, the objective of this study was to evaluate the consequences of subclinical mastitis on ewe health, milk production, milk composition and quality. We also evaluated the susceptibility of the bacteria in vitro using disk diffusion antibiograms. Finally, we performed two-way testing of efficacy of treatment in Lacaune ewes using the same agents. In the first stage of the study, 30 lactating ewes (±90 days) were used, 10 of which were negative on the CMT (California Mastitis Test) used as control group (CG) and 20 sheep with subclinical mastitis diagnosed by CMT (MG). Samples were collected and several analyses were performed on the milk and blood. We found that ewes in the MG had higher lipid peroxidation in serum and milk, as well as lower production, with reduction of the total dry extract in milk. There were 15 isolates of Staphylococcus hyicus, four isolates of each S. epidermidis and S. intermedius, and two isolates of Corynebacterium spp. The primary hematological result was leukocytosis in ewes with mastitis. Based on the antibiogram, we chose ceftiofur for in vivo tests. In this stage, we divided the sheep with subclinical mastitis into two subgroups of 10 ewes each, to receive drug by two routes: intramuscular (IM) and intramammary (IMM). In the IMM group, of the 10 CMT-positive ewes at the beginning of the experiment, seven were already negative by the racket test 120 h after the last application (70% efficacy). In the IM group, of the 10 positive ewes, only four were negative after 120 h of the final application, a low efficacy treatment (40%). We evaluated antimicrobial residues in the milk of treated animals. We found this material within 5 days after treatment in the two forms used; despite the fact that the product's stated withholding period is 3 days. We conclude that ewes with mastitis produce less milk of lower quality. We also conclude that, although ceftiofur is 100% effective in vitro, when used in ewes with mastitis, the efficacy did not exceed 70%, and was more efficient when administered via the intramammary route.

Biomechanical modeling of neck flexion for deformable alignment of the salivary glands in head and neck cancer images

During head and neck (HN) cancer radiation therapy, analysis of the dose-response relationship for the parotid glands (PG) relies on the ability to accurately align soft tissue organs between longitudinal images. In order to isolate the response of the salivary glands to delivered dose, from deformation due to patient position, it is important to resolve the patient postural changes, mainly due to neck flexion. In this study we evaluate the use of a biomechanical model-based deformable image registration (DIR) algorithm to estimate the displacements and deformations of the salivary glands due to postural changes.A total of 82 pairs of CT images of HN cancer patients with varying angles of neck flexion were retrospectively obtained. The pairs of CTs of each patient were aligned using bone-based rigid registration. The images were then deformed using biomechanical model-based DIR method that focused on the mandible, C1 vertebrae, C3 vertebrae, and external contour. For comparison, an intensity-based DIR was also performed. The accuracy of the biomechanical model-based DIR was assessed using Dice similarity coefficient (DSC) for all images and for the subset of images where the PGs had a volume change within 20%. The accuracy was compared to the intensity-based DIR.The PG mean ± STD DSC were 0.63 ± 0.18, 0.80 ± 0.08, and 0.82 ± 0.15 for the rigid registration, biomechanical model-based DIR, and intensity based DIR, respectively, for patients with a PG volume change up to 20%. For the entire cohort of patients, where the PG volume change was up to 57%, the PG mean ± STD DSC were 0.60 ± 0.18, 0.78 ± 0.09, and 0.81 ± 0.14 for the rigid registration, biomechanical model-based DIR, and intensity based DIR, respectively. The difference in DSC of the intensity and biomechanical model-based DIR methods was not statistically significant when the volume change was less than 20% (two-sided paired t-test, p = 0.12). When all volume changes were considered, there was a significant difference between the two registration approaches, although the magnitude was small.These results demonstrate that the proposed biomechanical model with boundary conditions on the bony anatomy can serve to describe the varying angles of neck flexion appearing in images during radiation treatment and to align the salivary glands for proper analysis of dose-response relationships. It also motivates the need for dose response modeling following neck flexion for cases where parotid gland response is noted.

Delta-radiomics features during radiotherapy improve the prediction of late xerostomia

The response of the major salivary glands, the parotid glands, to radiation dose is patient-specific. This study was designed to investigate whether parotid gland changes seen in weekly CT during treatment, quantified by delta-radiomics features (Δfeatures), could improve the prediction of moderate-to-severe xerostomia at 12 months after radiotherapy (Xer12m). Parotid gland Δfeatures were extracted from in total 68 planning and 340 weekly CTs, representing geometric, intensity and texture characteristics. Bootstrapped forward variable selection was performed to identify the best predictors of Xer12m. The predictive contribution of the resulting Δfeatures to a pre-treatment reference model, based on contralateral parotid gland mean dose and baseline xerostomia scores (Xerbaseline) only, was evaluated. Xer12m was reported by 26 (38%) of the 68 patients included. The most predictive Δfeature was the contralateral parotid gland surface change, which was significantly associated with Xer12m for all weeks (p < 0.04), but performed best for week 3 (ΔPG-surfacew3; p < 0.001). Moreover, ∆PG-surfacew3 showed a significant predictive contribution in addition to the pre-treatment reference model (likelihood-ratio test; p = 0.003), resulting in a significantly better model performance (AUCtrain = 0.92; AUCtest = 0.93) compared to that of the pre-treatment model (AUCtrain = 0.82; AUCtest = 0.82). These results suggest that mid-treatment parotid gland changes substantially improve the prediction of late radiation-induced xerostomia.

The genomic landscape of estrogen receptor α binding sites in mouse mammary gland.

Estrogen receptor α (ERα) is the major driving transcription factor in the mammary gland development as well as breast cancer initiation and progression. However, the genomic landscape of ERα binding sites in the normal mouse mammary gland has not been completely elucidated. Here, we mapped genome-wide ERα binding events by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in the mouse mammary gland in response to estradiol. We identified 6237 high confidence ERα binding sites in two biological replicates and showed that many of these were located at distal enhancer regions. Furthermore, we discovered 3686 unique genes in the mouse genome that recruit ER in response to estradiol. Interrogation of ER-DNA binding sites in ER-positive luminal epithelial cells showed that the ERE, PAX2, SF1, and AP1 motifs were highly enriched at distal enhancer regions. In addition, comprehensive transcriptome analysis by RNA-seq revealed that 493 genes are differentially regulated by acute treatment with estradiol in the mouse mammary gland in vivo. Through integration of RNA-seq and ERα ChIP-seq data, we uncovered a novel ERα targetome in mouse mammary epithelial cells. Taken together, our study has identified the genomic landscape of ERα binding events in mouse mammary epithelial cells. Furthermore, our study also highlights the cis-regulatory elements and cofactors that are involved in estrogen signaling and may contribute to ductal elongation in the normal mouse mammary gland.