High concentrations of β-hydroxybutyrate alter the kinetics of bovine spermatozoa

Postpartum cows, mainly with metabolic diseases, such as ketosis, usually experience an increased number of services per conception. During ketosis, high concentrations of β-hydroxybutyrate (BHBA) in follicular, uterine and oviductal fluid have been considered to cause subfertility in cows. However, the effect of sperm exposure to an environment with high BHBA concentration is not known. This study investigated the influence of high levels of BHBA on kinetics, oxidative status and morphology of bovine spermatozoa. To assess the effect of BHBA after sperm selection, bovine spermatozoa were incubated (180 min) with different BHBA concentrations: 0 (Control), 0.8, 2.4 or 5 mM. Sperm kinetics was evaluated after 30, 60, 120 and 180 min, and oxidative status and morphology were analysed at 180 min. Oxidative status was evaluated through the production of reactive oxidative species (ROS), total antioxidant capacity and lipid peroxidation. High concentrations of BHBA decreased the curvilinear velocity, straight line velocity, mean path velocity, linearity, straightness and hyperactivity of spermatozoa. However, there was no effect of BHBA on oxidative and antioxidant capacity as well as on sperm morphology. In conclusion, exposure of bovine spermatozoa to high levels of BHBA impairs sperm kinetics without altering oxidative and antioxidant mechanisms.     

Effect of recombinant β-defensin 1 protein on human sperm motility and viability

The reduction of sperm motility and subsequently reduced ability to undergo capacitation and acrosome reaction are considered as common causes of male infertility. The β-defensin family is a group of well-known secretory proteins with antimicrobial activity that contribute to the process of “sperm maturation” during the passage of spermatozoa in the epididymis when spermatozoa attain its motility. One member of this family is “β-defensin 1” which is present in seminal plasma and spermatozoa. The aim of this study was the incubation of human processed spermatozoa with recombinant β-defensin 1 (500 ng/ml) for 1, 2 and 3 hr at 37°C under 5% CO₂ atmosphere and assessment of sperm viability and motility in 59 semen samples. The analysis of semen samples such as sperm concentration, motility, viability, morphology and semen volume was performed according to the World Health Organization (2010; World health organization laboratory manual for the examination and processing of human semen (p. 287). Geneva, Switzerland: World Health Organization) criteria. The result of the current study shows that the incubation of spermatozoa with recombinant β-defensin significantly maintained percentage of sperm viability and motility compared to processed spermatozoa incubate in the absence of β-defensin in the studied time intervals (p < .05). Therefore, we concluded that recombinant β-defensin 1 protein as an agent with antimicrobial activity can maintain sperm viability and motility in in vitro condition.     

Multimodality imaging of β-cells in mouse models of type 1 and 2 diabetes

OBJECTIVE

β-Cells that express an imaging reporter have provided powerful tools for studying β-cell development, islet transplantation, and β-cell autoimmunity. To further expedite diabetes research, we generated transgenic C57BL/6 “MIP-TF” mice that have a mouse insulin promoter (MIP) driving the expression of a trifusion (TF) protein of three imaging reporters (luciferase/enhanced green fluorescent protein/HSV1-sr39 thymidine kinase) in their β-cells. This should enable the noninvasive imaging of β-cells by charge-coupled device (CCD) and micro-positron emission tomography (PET), as well as the identification of β-cells at the cellular level by fluorescent microscopy.

RESEARCH DESIGN AND METHODS

MIP-TF mouse β-cells were multimodality imaged in models of type 1 and type 2 diabetes.

RESULTS

MIP-TF mouse β-cells were readily identified in pancreatic tissue sections using fluorescent microscopy. We show that MIP-TF β-cells can be noninvasively imaged using microPET. There was a correlation between CCD and microPET signals from the pancreas region of individual mice. After low-dose streptozotocin administration to induce type 1 diabetes, we observed a progressive reduction in bioluminescence from the pancreas region before the appearance of hyperglycemia. Although there have been reports of hyperglycemia inducing proinsulin expression in extrapancreatic tissues, we did not observe bioluminescent signals from extrapancreatic tissues of diabetic MIP-TF mice. Because MIP-TF mouse β-cells express a viral thymidine kinase, ganciclovir treatment induced hyperglycemia, providing a new experimental model of type 1 diabetes. Mice fed a high-fat diet to model early type 2 diabetes displayed a progressive increase in their pancreatic bioluminescent signals, which were positively correlated with area under the curve–intraperitoneal glucose tolerance test (AUC-IPGTT).

CONCLUSIONS

MIP-TF mice provide a new tool for monitoring β-cells from the single cell level to noninvasive assessments of β-cells in models of type 1 diabetes and type 2 diabetes.The expression of imaging reporter genes in β-cells has provided powerful tools for studying β-cell development, embryonic and adult stem cell differentiation into β-cells, transdifferentiation, and islet transplantation (1–13). Bioluminescent reporters, such as firefly luciferase, emit photons that can be detected with high sensitivity using a cooled charge-coupled device (CCD). We and others have demonstrated human and rodent islets can be genetically engineered ex vivo to express luciferase so that the islets can be monitored following their transplantation by CCD and that CCD signal corresponded with the implanted β-cell mass (1,2). Several laboratories have generated transgenic mice that express luciferase in their β-cells and shown that the CCD signal from the pancreas is correlated with β-cell mass (3–5). Moreover, the transgenic islets can be monitored after transplantation (3,4,6). Fluorescence imaging and bioluminescence imaging (BLI) are highly useful for research purposes in small animals, but they are not yet applicable to imaging internal organs in large animals because of the limited transparency of tissues to photons.Positron emission tomography (PET) imaging is used clinically to assess various disorders of the heart and brain and for detecting various cancers (7,8). Islets that were engineered ex vivo to express a PET can be noninvasively monitored by microPET after implantation into the liver (9,10). The microPET signal reflects β-cell mass, and islets can be monitored long-term after implantation (11). Radiolabeled ligands of vesicular monoamine transporter (VMAT2) can image β-cells in BB rats, although VMAT2 may not be a sensitive biomarker of β-cell mass in humans (12–14). Mouse pancreatic β-cells are inherently difficult to detect by microPET because of the small size of mice, the long thin shape of the pancreas and its proximity to the probe excretion pathway—which eliminates the vast majority of the tracer and obscures the weaker signals from the pancreas, and the resolution of microPET (1–2 mm³). Recent advances in PET reporter genes and PET probes with better clearance kinetics and biodistribution, however, have allowed progress towards microPET imaging of mouse β-cells in situ (14).Previous imaging studies of β-cells have used animal models in which the β-cells express a single reporter gene. We have developed a series of trifusion imaging reporters consisting of a bioluminescent reporter (e.g., luciferase), linked by a few alanine residues to a fluorescent reporter gene (e.g., enhanced green fluorescent protein [EGFP]), which in turn is linked to a PET reporter (e.g., herpes simplex virus [HSV] thymidine kinase) (15). All three reporters are expressed as a single fusion protein, allowing noninvasive CCD and PET imaging, as well as fluorescent microscopic analysis of tissue sections or fluorescence-activated cell sorter (FACS) isolation of single cells expressing EGFP (15,16). Using an insulin promoter to drive the expression of a trifusion reporter in transgenic mouse β-cells should enable the longitudinal monitoring of β-cells in the same mice by both CCD and microPET. After killing, fluorescent microscopy can identify β-cells at the cellular level and facilitate correlation of β-cell number and mass with the CCD and microPET signals. Because all three reporters are linked together in a fusion protein, the magnitude of signals from one imaging reporter should predict the magnitude of the signals from the other imaging reporters. Here, we provide proof-of principle of the utility of mice expressing a trifusion imaging reporter specifically in their β-cells in mouse models of type 1 diabetes and type 2 diabetes.     

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells

Objective: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC)stimulated by lipopolysaccharide (LPS). Methods : Two-directional ( forward and backward)suppression subtractive hybridization ( SSH ) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. Results: These analyses have identified both novel and known genes whose expression is influenced by LPS.The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. Conclusions: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.     

Inhalation of corticosteroid and β-agonist for persistent cough following pulmonary resection

Background  

Patients undergoing pulmonary resection often suffer from a dry, hacking cough, which is usually refractory to opioid cough suppressors such as codeine. The cough is often painful and impairs the quality of life of the patients. The efficacy of an inhaled corticosteroid plus β2-agonist against the persistent cough after pulmonary resection was evaluated in this study.     

Comparison of the effects of human β-defensin 3, vancomycin, and clindamycin on Staphylococcus aureus biofilm formation

Despite improvements in surgical techniques and implant design in orthopedic surgery, implantation-associated infections are still a challenging problem for surgeons. In 2006, trace quantities of human β-defensin 3 (hBD-3) were found in human bone tissue and bone cells. Human β-defensin 3 is a 45-amino-acid peptide that is considered the most promising class of defensin antimicrobial peptides and may help in the prevention and treatment of implantation-associated infections. Studies of the effectiveness of hBD-3 against Staphylococcus aureus showed that hBD-3 was more potent at low concentrations than other antibiotics. The effect of hBD-3 on S aureus biofilms has not been reported. We studied the effect of hBD-3, vancomycin, and clindamycin on S aureus biofilms and on the survival of the bacteria in the biofilms.Staphylococcus aureus biofilms were examined with confocal scanning laser microscopy. Staining with LIVE/DEAD BacLight viability stain (Molecular Probes Europe BV, Leiden, The Netherlands) differentiated between live and dead bacteria within the biofilms, and extracellular polymeric substances (slime) from the biofilms was evaluated after staining with calcofluor white (Sigma Chemical Company, Rocky Hill, New Jersey). Human β-defensin 3 and clindamycin reduced the S aureus biofilm area. Human β-defensin 3 was significantly more effective against bacteria from the S aureus biofilms than was clindamycin. Vancomycin did not reduce the S aureus biofilm area.     

Progressive erosion of β-cell function precedes the onset of hyperglycemia in the NOD mouse model of type 1 diabetes

OBJECTIVE

A progressive decline in insulin responses to glucose was noted in individuals before the onset of type 1 diabetes. We determined whether such abnormalities occurred in prediabetic NOD mice—the prototypic model for human type 1 diabetes.

RESEARCH DESIGN AND METHODS

Morning blood glucose was measured every other day in a cohort of NOD females. Glucose tolerance and insulin secretion were measured longitudinally by intraperitoneal glucose tolerance tests in NOD/ShiLtJ and BALB/cJ mice 6 to 14 weeks of age. Arginine-stimulated insulin secretion and insulin sensitivity were assessed during intraperitoneal arginine or intraperitoneal insulin tolerance tests.

RESULTS

During prediabetes, NOD females displayed a progressive increase in glucose levels followed by an acute onset of hyperglycemia. First-phase insulin responses (FPIRs) during the intraperitoneal glucose tolerance test (IPGTT) declined before loss of glucose tolerance in NOD. The failure of FPIR could be detected, with a decline in peak insulin secretion during IPGTT. Arginine-stimulated insulin secretion remained unchanged during the study period. The decline in insulin secretion in NOD mice could not be explained by changes in insulin sensitivity.

CONCLUSIONS

There was an impressive decline in FPIR before changes in glucose tolerance, suggesting that impairment of FPIR is an early in vivo marker of progressive β-cell failure in NOD mice and human type 1 diabetes. We portend that these phenotypes in NOD mice follow a similar pattern to those seen in humans with type 1 diabetes and validate, in a novel way, the importance of this animal model for studies of this disease.Type 1 diabetes is an autoimmune disease resulting from the destruction of pancreatic insulin-producing β-cells. The NOD mouse is the most commonly used rodent model of the disease. Studies in this mouse strain have led to interventions that have been translated to clinical investigations with human type 1 diabetic patients (1–5). However, intervening at the right therapeutic window is critical to the efficacy of certain therapies. For example, anti-mouse thymocyte globulin can delay or reverse diabetes in NOD only when used in the late prediabetes stage or at onset of the disease (6).Islet autoimmunity can be identified in the prediabetic stage. Specifically, in NOD mice, insulin autoantibodies can be detected as early as at 3 weeks of age (7). However, as in humans, autoantibody positivity can be transient as well as observed in nonprogressors (8,9). Therefore, markers of β-cell mass and function are needed to identify progression from the onset of islet autoimmunity to and through the stages of prediabetes.Previous cross-sectional studies have demonstrated decreased first-phase insulin secretion in NOD females at different ages. Using pancreatic perfusion, Kano et al. (10) documented that NOD mice maintain normal fasting glucose even though a significant decrease was measured in first-phase insulin response (FPIR) to glucose. In these studies, increased fasting glucose and loss of FPIR was associated with the degree of insulitis. In cross-sectional studies using intravenous glucose tolerance test, Reddy et al. (11) showed an age-related progressive decline in FPIR to glucose. In another cross-sectional study using in situ pancreas perfusion, Sreenan et al. (12) reported progressive decreases in glucose- and arginine-stimulated insulin secretion in NOD females at 8, 13, and 18 weeks of age. Although single-point analyses were performed, there have not been reports involving sequential analysis of single mice over time to confirm this progression. In this study, we sought to determine whether similar metabolic signatures exist in both humans and in prediabetic NOD mice.     

Genome‐wide association of an integrated osteoporosis‐related phenotype: Is there evidence for pleiotropic genes?

Multiple musculoskeletal traits assessed by various methods at different skeletal sites serve as surrogates for osteoporosis risk. However, it is a challenge to select the most relevant phenotypes for genetic study of fractures. Principal component analyses (PCA) were conducted in participants of the Framingham Osteoporosis Study on 17 measures including bond mineral density (BMD) (hip and spine), heel ultrasound, leg lean mass (LLM), and hip geometric indices, adjusting for covariates (age, height, body mass index [BMI]), in a combined sample of 1180 men and 1758 women, as well as in each sex. Four principal components (PCs) jointly explained ～69% of the total variability of musculoskeletal traits. PC1, explaining ～33% of the total variance, was referred to as the component of “Bone strength,” because it included the hip and spine BMD as well as several hip cross‐sectional properties. PC2 (20.5% variance) was labeled as “Femoral cross‐sectional geometry;” PC3 (～8% variance) captured only ultrasound measures; PC4, explaining ～7% variance, was correlated with LLM and hip geometry. We then evaluated ～2.5 mil SNPs for association with PCs 1, 2, and 4. There were genome‐wide significant associations (p < 5 × 10^?8) between PC2 and HTR1E (that codes for one of the serotonin receptors) and PC4 with COL4A2 in women. In the sexes‐combined sample, AKAP6 was associated with PC2 (p = 1.40 × 10^?7). A single nucleotide polymorphism (SNP) in HTR1E was also associated with the risk of nonvertebral fractures in women (p = 0.005). Functions of top associated genes were enriched for the skeletal and muscular system development (p < 0.05). In conclusion, multivariate combination provides genetic associations not identified in the analysis of primary phenotypes. Genome‐wide screening for the linear combinations of multiple osteoporosis‐related phenotypes suggests that there are variants with potentially pleiotropic effects in established and novel pathways to be followed up to provide further evidence of their functions. © 2012 American Society for Bone and Mineral Research     

Analysis of genes encoding laminin β2 and related proteins in patients with Galloway–Mowat syndrome

Galloway-Mowat syndrome (GMS) is a rare autosomal recessive disorder characterized by early onset nephrotic syndrome and microcephaly with various anomalies of the central nervous system. GMS likely represents a heterogeneous group of disorders with hitherto unknown genetic etiology. The clinical phenotype to some extent overlaps that of Pierson syndrome (PS), which comprises congenital nephrotic syndrome and distinct ocular abnormalities but which may also include neurodevelopmental deficits and microcephaly. PS is caused by mutations of LAMB2, the gene encoding laminin beta2. We hypothesized that GMS might be allelic to PS or be caused by defects in proteins that interact with laminin beta2. In a cohort of 18 patients with GMS or a GMS-like phenotype we therefore analyzed the genes encoding laminin beta2 (LAMB2), laminin alpha5 (LAMA5), alpha3-integrin (ITGA3), beta1-integrin (ITGB1) and alpha-actinin-4 (ACTN4), but we failed to find causative mutations in these genes. We inferred that LAMA5, ITGA3, ITGB1, and ACTN4 are not directly involved in the pathogenesis of GMS. We excluded LAMB2 as a candidate gene for GMS. Further studies are required, including linkage analysis in families with GMS to identify genes underlying this disease.     

Mechanical and Biological Evaluations of β-Tricalcium Phosphate/Silicone Rubber Composite as a Novel Soft-Tissue Implant

Background  

Although silicone rubber (SR) implants are most commonly used and effective for soft-tissue augmentation, they still have been implicated in many adverse reactions. To overcome this problem, a novel composite β-tricalcium phosphate/silicone rubber (β-TCP/SR) was prepared by adding β-TCP into a SR matrix. This study was to evaluate its application potential by investigating the mechanical properties and biocompatibility of β-TCP/SR.     

A Novel Effect of β-Adrenergic Receptor on Mammary Branching Morphogenesis and its Possible Implications in Breast Cancer

Understanding the mechanisms that govern normal mammary gland development is crucial to the comprehension of breast cancer etiology. β-adrenergic receptors (β-AR) are targets of endogenous catecholamines such as epinephrine that have gained importance in the context of cancer biology. Differences in β₂-AR expression levels may be responsible for the effects of epinephrine on tumor vs non-tumorigenic breast cell lines, the latter expressing higher levels of β₂-AR. To study regulation of the breast cell phenotype by β₂-AR, we over-expressed β₂-AR in MCF-7 breast cancer cells and knocked-down the receptor in non-tumorigenic MCF-10A breast cells. In MCF-10A cells having knocked-down β₂-AR, epinephrine increased cell proliferation and migration, similar to the response by tumor cells. In contrast, in MCF-7 cells overexpressing the β₂-AR, epinephrine decreased cell proliferation and migration and increased adhesion, mimicking the response of the non-tumorigenic MCF-10A cells, thus underscoring that β₂-AR expression level is a key player in cell behavior. β-adrenergic stimulation with isoproterenol induced differentiation of breast cells growing in 3-dimension cell culture, and also the branching of murine mammary epithelium in vivo. Branching induced by isoproterenol was abolished in fulvestrant or tamoxifen-treated mice, demonstrating that the effect of β-adrenergic stimulation on branching is dependent on the estrogen receptor (ER). An ER-independent effect of isoproterenol on lumen architecture was nonetheless found. Isoproterenol significantly increased the expression of ERα, Ephrine-B1 and fibroblast growth factors in the mammary glands of mice, and in MCF-10A cells. In a poorly differentiated murine ductal carcinoma, isoproterenol also decreased tumor growth and induced tumor differentiation. This study highlights that catecholamines, through β-AR activation, seem to be involved in mammary gland development, inducing mature duct formation. Additionally, this differentiating effect could be resourceful in a breast tumor context.     

Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model

Type 1 diabetes is preceded by islet β-cell dysfunction, but the mechanisms leading to β-cell dysfunction have not been rigorously studied. Because immune cell infiltration occurs prior to overt diabetes, we hypothesized that activation of inflammatory cascades and appearance of endoplasmic reticulum (ER) stress in β-cells contributes to insulin secretory defects. Prediabetic nonobese diabetic (NOD) mice and control diabetes-resistant NOD-SCID and CD1 strains were studied for metabolic control and islet function and gene regulation. Prediabetic NOD mice were relatively glucose intolerant and had defective insulin secretion with elevated proinsulin:insulin ratios compared with control strains. Isolated islets from NOD mice displayed age-dependent increases in parameters of ER stress, morphologic alterations in ER structure by electron microscopy, and activation of nuclear factor-κB (NF-κB) target genes. Upon exposure to a mixture of proinflammatory cytokines that mimics the microenvironment of type 1 diabetes, MIN6 β-cells displayed evidence for polyribosomal runoff, a finding consistent with the translational initiation blockade characteristic of ER stress. We conclude that β-cells of prediabetic NOD mice display dysfunction and overt ER stress that may be driven by NF-κB signaling, and strategies that attenuate pathways leading to ER stress may preserve β-cell function in type 1 diabetes.     

Murine β-defensin-2 may regulate the effect of bacillus Calmette-Guérin (BCG) in normal mouse bladder

PurposeWe investigated whether bacillus Calmette-Guérin (BCG)–induced secretion of murine β-defensin-2 (mBD2) and determined whether mBD2 regulated BCG effects in the normal mouse bladder.Materials and methodsA total of 140 C57BL/6 female mice were divided into 28 groups, and the experiment was performed over 3 steps. In the first step (20 groups), mice bladders were stimulated with different doses of BCG (multiplicity of infection [MOI] 0, 1, 10, 30, and 100) and histological analysis was conducted in bladder specimens isolated at different times (0, 4, 8, and 24 h after instillation) to determine optimal dose and time point of BCG internalization and urine mBD2 and cytokine concentration. In the second step (4 groups), BCG internalization and urine cytokine levels were measured after pretreatment of different recombinant mBD2 (rmBD2) (0, 1, 2.5, and 5 ng/ml) at optimal dose and time point. In the third step (4 groups), BCG internalization and urine cytokine levels were compared between pretreatment conditions (control, rmBD2, anti-mBD2 Ab, and rmBD2+anti-mBD2 Ab). Urine was collected for estimating mBD2 levels and a multiplex analysis for 9 cytokines. Real-time polymerase chain reaction assay was used for estimating the relative BCG cell number in mice bladder tissue.ResultsBladder edema was induced by BCG (MOI 30 and 100), which progressed to an inflammatory infiltrate composed primarily of neutrophils and increased mBD2 secretion at 4 hours after instillation. Relative BCG cell number and urinary cytokine levels (interferon-γ and interleukins [IL]-2, -4, -6, and -10) response pattern was characterized by a peak at 4 hours after instillation followed by rapid decline. The levels of interferon-γ, and IL-1β, -2, -4, -6, and -10 and relative BCG cell numbers decreased in a dose-dependent manner according to pretreatment with rmBD2 protein, and the responses were potentiated in the anti-mBD2 pretreatment group at 4 hours after BCG (MOI 30) instillation.ConclusionThe present results suggest that the mouse urothelium produces mBD2 in response to intravesicular BCG as a defense mechanism against BCG, and blocking mBD2 by an anti-mBD2 antibody increased the effectiveness of BCG.     

Novel biodegradable α-TCP/poly(amino acid) composite artificial lamina following spinal surgery for prevention of intraspinal scar adhesion

Biodegradable copolymer α-TCP/poly(amino acid) composite artificial lamina was prepared and used in goat cervical vertebra resection repair. Cervical 4 was removed by laminectomy, and a vertebra defect of 27 × 9 mm was made. α-TCP/poly(amino acid) composite artificial lamina was inserted in the test group. The efficiency of the copolymer during repair and reconstruction of the goats’ vertebra was tested by using X-ray, CT scanning, and histological and biomechanical measurements. In the 24 weeks following the operation, the artificial lamina refrained from shifting, and no dural adhesion pressure was observed. In contrast, the control group suffered from infiltration of soft tissue in the spinal canal, dural pressure and α-TCP/poly(amino acid) degradation. In conclusion, α-TCP/poly(amino acid) composite artificial lamina can significantly prevent scar tissue from infiltrating the spinal canal.