Effects of long-term exposure to an extremely low frequency magnetic field (15 µT) on selected blood coagulation variables in OF1 mice.

The long-term exposure of OF1 mice to an extremely low frequency magnetic field (ELF-MF; 50 Hz, 15 µT [rms]) has been associated with the appearance of leukaemia. Neoplasms are usually accompanied by changes in haemostatic processes but reports on changes in blood coagulation following exposure to an ELF-MF are scarce and rather fragmentary. The aim of the present work was to determine whether any global or partial coagulation variables are modified after such long-term exposure. A parental generation of six week-old OF1 mice was exposed to an artificial ELF-MF for 14 weeks. Mating was then allowed, and the resulting filial generation raised until the age of 31-35 weeks within the same ELF-MF. Control animals were subjected only to the magnetic field of the Earth. Whole blood samples were extracted from the anesthetised filial generation of mice by cardiac puncture. White blood cells (WBC) were counted, the activated partial thromboplastin time (APTT) and prothrombin time (PT) determined, and plasma fibrinogen, reptilase time (RT), and factor VIII activity examined. The similarity between the results for the present control animals and those recorded in the literature for human blood render OF1 mice a suitable study model. The differences in the studied coagulation variables were largely owed simply to sex. However, the females showed a very significant shortening of the PT time associated with ELF-MF exposure. Exposure also caused significant increases in the female APTT and RT values, and in general reduced the differences between the sexes.

Propionate induces the release of granules from bovine neutrophils.

Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.

Timing and duration of nutrient restriction and its impacts on placental development and umbilical blood flow in adolescent sheep.

We hypothesized that nutrient restriction from day 50-90 of gestation decreases umbilical blood flow and that umbilical blood flow would recover to control values upon realimentation during late gestation (d 90 to 130) or remain reduced in ewes that continued to be nutrient restricted. On d 50 of gestation, young nulliparous whiteface ewes (6-8 mo; n = 41) carrying singletons were randomly assigned to two dietary treatments: 100% of NRC recommendations (CON) or 60% of CON (RES). On d 90 of gestation, ewes either remained on CON or RES until d 130, or CON ewes were RES from d 90 to 130, or RES ewes were realimented to CON from d 90 to 130. This resulted in 4 treatment groups on day 130: CON-CON, CON-RES, RES-RES, RES-CON. Umbilical blood flow and fetal and placental measurements were obtained via ultrasonography every 10 days from day 50-110. Non-survival surgeries were performed on days 50, 90, and 130 (n = 6-7 ewes/group) where uterine artery and umbilical blood flows were measured during surgery via ultrasonography. Conceptus weights were recorded and placentomes collected to determine binucleate cell numbers. The study was conducted as a completely randomized design arrangement with repeated measures. Data were analyzed using the MIXED procedure of SAS. There was a nutritional treatment by day interaction (P < 0.01) with CON ewes having greater umbilical blood flow compared with RES by d 90. Fetal biparietal distance, abdominal width, and kidney area increased (P < 0.05) in CON-RES with all these measurements increasing during late gestation. We partially accept our hypothesis as nutrient restriction during mid gestation decreased umbilical blood flow. However, blood flow did not return to control levels upon realimentation. By d 130, fetal and placental weights were similar between RES-RES and CON-CON. Binucleate cell numbers in the fetal trophoblast were not influenced by nutritional treatments. Our findings suggest that refeeding previously nutrient restricted pregnant adolescent ewes to control levels does not reestablish umbilical blood flow. Adequate placental development during mid gestation could protect the fetus from a decreased umbilical blood flow later in gestation when nutrients were limited by 40%.

Growth variations in OF1 mice following chronic exposure of parental and filial generations to a 15 µT, 50 Hz magnetic field.

The growth of a first filial generation (F1) of OF1 mice was studied following chronic exposure of their mothers and themselves to a magnetic field of 15 µT (rms) and 50 Hz. The parental generation (F0) remained for 98 days in this field, after that time they were mated, went through pregnancy, birth, lactation, and the weaning of their offspring in this field. The latter remained exposed to this field until reaching adulthood (220 days). Control animals were treated in the same way but were exposed only to the Earth's magnetic field. The growth data for the offspring were analyzed using a generalization of Koop's equation. Using this model, four phases were identified: lactation growth acceleration, post-weaning growth acceleration, growth stabilization, and a stationary phase. Exposure to the artificial magnetic field was associated with a marked increase in maximum growth rate in the exposed animals during the post-weaning growth acceleration phase, and with a reduction in mass gain in the F1 mice (especially in males) during the third of these phases. In addition, the growth stabilization phase was more extended in exposed females and shorter in exposed males than in the control animals. Furthermore, statistically significant differences were seen between the mean body masses of exposed and control F1 males from 49-123 days. Exposure to the artificial magnetic field might have been associated with the stimulated growth rate seen over the noticeably shortened second and third growth phases (leaving these animals lighter by the stationary phase compared to controls) and a possible acceleration of aging. Both processes could be responsible for the stationary phase being reached at an earlier age, especially in males.

Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17ß exposure in ovariectomized ewes.

Estradiol-17ß (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

Acute effects of estradiol-17ß on plasma volume and uterine cell proliferation in sheep.

Ovarian steroids play an important role in increasing plasma volume in pregnant females and preparing the uterus for implantation. We hypothesized that a short duration of increased estradiol-17ß (E2) would increase plasma volume and uterine cell proliferation in ovariectomized ewes. Adult non-pregnant Romanov ewes (n = 15) were ovariectomized. After ovariectomy, ewes were individually housed and were offered water at ad libitum intake and were fed a pelleted diet at maintenance once daily according to body weight. After at least 30 days post-ovariectomy ewes were fasted and received an implant placed in the axillary region that contained 100 mg of E2 (E2; n = 8) or a sham implant with no E2 (CON, n = 7). After 24 h, ewes were weighed prior to plasma volume measurement procedures. Plasma volume was determined using the Evans blue dye method. Blood samples were taken at 0 (pre dye injection), 5, 10, 15, 20, 25, 30, 40, 50 and 60 min after dye injection. After the final blood collection, ewes were euthanized with an overdose of sodium pentabarbital and uterine weights were recorded. Uterine cross-sections were fixed in formalin for immunohistochemical localization of Ki67 (a marker of proliferating cells) followed by image generation of luminal epithelium and endometrial stroma (5 areas each/tissue section) and analysis to determine the proportion of proliferating cells. Plasma volume tended to be greater in E2 vs CON (2.75 ± 0.11 vs. 2.54 ± 0.12 L, P = 0.07) and uterine weights were greater in E2 vs CON (27.25 ± 2.35 vs. 17.35 ± 2.51 g, P < 0.01). Water intake after implant placement was similar in E2 and CON (3.85 vs. 4.87 ± 0.67 L; P = 0.28). Cell proliferation in the luminal epithelium was greater in E2 vs CON (6.55 vs. 1.2 ± 1.75%, P = 0.02) and stromal cells tended to be greater in E2 vs CON (0.59 vs 0.37 ± 0.06%, P = 0.07). Our results demonstrate that E2-treatment tends to increase plasma volume acutely and increases uterine cell proliferation in ewes.

New potential targets to modulate neutrophil function in inflammation.

The importance of neutrophils in human disease such as rheumatoid arthritis, asthma, adult respiratory distress syndrome, and COPD has prompted the search for drugs capable to slow down neutrophil-dependent inflammation, without interference with innate immune responses. In this review, we summarize new potential drugs targets against neutrophils mediated inflammatory responses.

Concordance in the reporting of intimate partner violence among male-male couples.

Intimate partner violence (IPV) among male couples is increasingly recognized as a public health concern. Research on IPV in opposite sex couples indicates frequent underreporting of IPV and high levels of discordance in reporting among dyads. Concordance studies inform refinement methods to measure the experience of IPV among dyads; however the lack of dyadic studies of male couples impedes our understanding of the extent to which IPV is differentially reported in male-male dyads. This study utilized baseline data from a randomized controlled trial of a behavioral intervention to optimize antiretroviral therapy (ART) adherence among 160 sero-discordant male couples in three US cities and provides the first analysis of concordance in reporting IPV among male couples. Low degrees of concordance in the reporting of IPV were identified among male dyads, with a greater proportion of men reporting violence perpetration than experiencing violence. The greater reporting of IPV perpetration may be linked to adherence to concepts of masculinity. The results underscore the unique experiences of IPV among male couples and the need to reexamine current IPV measurement and intervention strategies.

Fatty and hydroxycarboxylic acid receptors: The missing link of immune response and metabolism in cattle.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body ß-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body ß-hydroxybutyrate, and GPR81/HCA1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders.

Genomic sequences of five novel HLA class I alleles: A*30:129, B*08:195, B*51:01:62, C*01:147 and C*12:195:02.

Five new HLA class I alleles are described, A*30:129, B*08:195, B*51:01:62, C*01:147 and C*12:195:02.

HLA-A*30:125 shows a new antigen binding domain created from HLA-A*30:01 and HLA-A*30:02.

A new HLA-A allele, A*30:125, was characterized in a Spanish individual.

Characterization of a novel HLA-DRB1*07 allele, DRB1*07:83.

A new DRB1*07 allele, DRB1*07:83, was described in a Caucasian Spanish donor.

Propionate induces pH(i) changes through calcium flux, ERK1/2, p38, and PKC in bovine neutrophils.

Propionate is a short-chain fatty acid produced under normal physiological conditions in the rumen of cattle. It is also involved in the inflammatory process and neutrophil function via calcium release, reactive oxygen species and intracellular pH (pH(i)) changes. This study examined the effect of propionate on the pH(i) of bovine neutrophils; specifically if pH(i) changes are controlled by calcium flux, and the mitogen-activated protein kinase (MAPK) pathway. Propionate caused rapid intracellular acidification and sustained alkalinization in bovine neutrophils loaded with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), a fluorescent indicator of pH(i). The acidification phase seems to be controlled by intracellular calcium release and p38 MAPK pathway. The pH recovery phenomenon was mediated by an amiloride-sensitive Na+/H+ exchanger and H+ channel, and was inhibited by UO126 (an ERK1/2 MAPK phosphorylation inhibitor), Gö6850 (a PKC inhibitor) and calcium chelating. Ionomycin, a calcium ionophore, induced intracellular acidification and sustained alkalinization. The intracellular acidification was strongly inhibited by BAPTA-AM (an intracellular calcium chelator) and SB203580 (a p38 MAPK inhibitor). In addition, the intracellular alkalinization was reduced by EGTA (a calcium chelator), UO126, LY294002 (a PI3K inhibitor) and Gö6850. Propionate did not increase superoxide production, however it reduced the superoxide production induced by platelet-activating factor (PAF), and increased the release of superoxide induced by ionomycin. Our results suggest that propionate-induced intracellular acidification is mediated by intracellular calcium release and p38 MAPK activation, and that pH recovery is controlled via ERK1/2 MAPK, PKC and calcium entry in bovine neutrophils.

Amiloride interferes with platelet- activating factor-induced respiratory burst and MMP-9 release in bovine neutrophils independent of Na+/H+ exchanger 1.

Cytoplasmic pH homeostasis is required for an appropriate response in polymorphonuclear neutrophils (PMNs). In these cells, chemotaxis and reactive oxygen species (ROS) production are reduced by the use of Na+/H+ exchanger (NHE-1) inhibitors, but these results are mainly obtained using amiloride, a non-selective NHE-1 inhibitor. In bovine PMNs, the role of NHE-1 in functional responses has not been confirmed yet. The aim of this study was to determine the role of NHE-1 using amiloride and zoniporide in pH regulation, ROS production, matrix metalloproteinase 9 (MMP-9) release and calcium flux in bovine PMNs induced by the platelet activation factor (PAF), additionally we evaluated the presence of NHE-1 and NHE-2 mRNA Our data show the presence only of NHE-1 but not NHE-2 in bovine PMNs. Amiloride or zoniporide inhibited the intracellular alkalization induced by PAF without affecting calcium flux. Amiloride diminished ROS production and MMP-9 release, while zoniporide enhanced ROS production without change the MMP-9 release induced by PAF. Our work led us to conclude that changes in intracellular pH induced by PAF are regulated by NHE-1 in bovine neutrophils, but the effects of amiloride on ROS production and MMP-9 release induced by PAF are not NHE-1 dependent.

Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor.

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants.

An analysis of postnatal growth in OF1 mice within an interval of 1-220 days of age.

The body weight growth of OF1 mice, both females and males, from day of birth to 220 days old is studied herein. After comparison of different theoretical models, an extension of Koops's multiphasic functions, the tetraphasic, is proposed as the best choice in so wide a time interval in mouse development. This function has allowed us to take into account different aspects of growth. Coefficients and phases of this function are characterized. The end of the first phase was established at 17 days of age. The second phase finished at 35 days of age in females and at 39 days in males. This difference increased during the third phase: the end of it was at 173 days of age in males, and at 160 days in females. The end of the fourth phase in OF1 mice would be produced after 220 days of age. Inflexion points in every four phases, in females and males, are situated. The biggest weight increase occurred in the second phase in both sexes, specially in males (1.26 g per day). Biological meanings of every phase are also discussed. The short lifetime of rodents have allowed us to study a great number of growth phases in a short period of time. Thus the present analysis could be a good starting point for the study of growth curves in other species of mammals.

Cold ischemia-induced damage to vascular endothelium results in permeability alterations in transplanted lungs.

UNLABELLED: Despite suggestions of a connection between endothelial damage and permeability alterations after ischemia and reperfusion in pulmonary tissue undergoing transplantation, no direct correlation between vascular endothelial discontinuity and parenchymal edema has yet been shown. METHODS: Forty-two rat lungs were harvested and stored for 48 or 72 hours under hypothermic and ischemic conditions. Stored pulmonary tissue was studied before transplantation and 5 minutes or 24 hours after transplantation by light microscopy and scanning electron microscopy of arterial vascular endothelium. RESULTS: Stored lungs not subjected to revascularization showed moderate perivascular edema, with small intercellular gaps in endothelial monolayers. Five minutes after transplantation, pulmonary tissue appeared congested, with perivascular and alveolar edema. Examination of vascular endothelium by scanning electron microscopy showed detachment of endothelial cells. Twenty-four hours after transplantation, edema, hemorrhage, and vascular congestion were found in all specimens. Arterial vascular endothelium showed weak intercellular connections, numerous intercellular gaps, and widespread cell detachment. Bronchial epithelial cells appeared damaged after storage, with loss of cilia, blebbing of apical cytoplasm, and cellular rounding. These changes were maintained 5 minutes after transplantation but appeared totally reversed after 24 hours in specimens stored 48 hours, whereas bronchial denudation was observed in 72-hour stored lungs. Statistically significant positive correlations (Kendall p < 0.001) between revascularization time and alveolar edema and hemorrhage were found for both storage periods. CONCLUSION: The results from this study demonstrate correlation between loss of endothelial monolayer continuity and histologic evidence of vascular permeability increases in pulmonary tissue before and after lung transplantation.

Ultraviolet exposure of thymocytes: selective inhibition of apoptosis.

PURPOSE: To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. MATERIALS AND METHODS: Cells were exposed to UV radiation with emission peaks of 365 nm (UVA) exposures of 1620-10200 J m(-2), of 312 nm (UVB) exposures of 34-1620 J m(-2) or of 254 nm (UVC) exposures of 1.5-1620 J m(-2), and incubated for 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate or anti-Fas antibody. Additionally, cells were irradiated with gamma-rays (5 Gy) before UVB exposure (408 J m(-2)) at different times. Apoptosis was quantified by DNA fragmentation. RESULTS: Up to an irradiation of 5000 J m(-2), UVA exposure did not show any effect on thymocyte apoptosis, while at 10200 J m(-2) irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone-induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol-12-myristate-13-acetate and gamma-irradiation, but not by anti-Fas antibody. CONCLUSIONS: The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase in the apoptotic pathway is discussed.

Effects of hypothermic storage on the vascular endothelium: a scanning electron microscope study of morphological change in human vein.

OBJECTIVE: The aims of this study were: i) to identify morphological changes occurring in the endothelium of human umbilical veins subjected to the typical storage procedures used in transplantation and ii) to determine the relative efficacy of preservation solutions containing intra and extracellular levels of sodium and potassium. EXPERIMENTAL DESIGN: Prospective. PROCEDURE: Scanning electron micrographs were taken pre and post cold hypoxic storage of human umbilical veins for 3 or 16 hours. RESULTS: Cold preservation resulted in severe cell detachment with subsequent loss of monolayer continuity and exposure of thrombogenic basal membrane components (highly significant after only 3 hours of cold storage, Kruskal-Wallis, p < 0.01). The morphological alterations culminated in EC with spherical shapes. Cytoplasmic membranes presented an increased number of microvilli and intercellular processes, followed by microvillous swelling and surface blebbing as damage increased. Bleb detachment was seen in severely damaged specimens. However, morphological preservation was not significantly affected by the duration of hypoxia or the ionic balance of the solution tested. CONCLUSIONS: The present results demonstrate that even short periods (3 hours) of cold storage without revascularization cause significant morphological damage to the endothelium. The ionic composition of the preservation solution did not significantly affect the process. The morphological changes seen in this study could explain storage-related problems such as loss of normal vascular permeability and increased thrombogenicity, problems often associated with transplantation procedures.