Heparan Sulfate Regrowth Profiles Under Laminar Shear Flow Following Enzymatic Degradation.

The local hemodynamic shear stress waveforms present in an artery dictate the endothelial cell phenotype. The observed decrease of the apical glycocalyx layer on the endothelium in atheroprone regions of the circulation suggests that the glycocalyx may have a central role in determining atherosclerotic plaque formation. However, the kinetics for the cells' ability to adapt its glycocalyx to the environment have not been quantitatively resolved. Here we report that the heparan sulfate component of the glycocalyx of HUVECs increases by 1.4-fold following the onset of high shear stress, compared to static cultured cells, with a time constant of 19 h. Cell morphology experiments show that 12 h are required for the cells to elongate, but only after 36 h have the cells reached maximal alignment to the flow vector. Our findings demonstrate that following enzymatic degradation, heparan sulfate is restored to the cell surface within 12 h under flow whereas the time required is 20 h under static conditions. We also propose a model describing the contribution of endocytosis and exocytosis to apical heparan sulfate expression. The change in HS regrowth kinetics from static to high-shear EC phenotype implies a differential in the rate of endocytic and exocytic membrane turnover.

Literature search strategies to comply with institutional animal care and use committee review requirements.

Under the US Animal Welfare Act, principal investigators who propose to use animals in their research must demonstrate that they have considered alternatives to potentially painful or distressful procedures when submitting applications to Institutional Animal Care and Use Committees (IACUCs). IACUCs requires that applicants conduct a current literature search to determine if alternatives are available to substitute the proposed animal use and, if the proposed study involves pain or distress, that more humane procedures, as they are described in the literature, be considered. This paper suggests literature search strategies that can be considered for use in order to comply with this IACUC requirement.

The foraging ball as a quick and easy enrichment device for pigs (Sus scrofa).

Providing research pigs with enrichment objects can encourage species-typical behavior such as rooting and foraging. The authors gave pigs hard plastic 'foraging balls' that resembled enrichment devices commonly used for nonhuman primates. Holes were custom-drilled into the balls, and animal caretakers filled them with palatable food items such as jellybeans, unsalted peanuts, cereal, Beggin' Strips, primate biscuits and dog biscuits. Staff members suspended the balls from chains in pigs' enclosures, ensuring that toys did not touch the floor. All pigs manipulated the balls and were able to obtain treats that were supplemental to their standard diet. The simple and effective enrichment device was easily incorporated into the daily routines of research facilities, with little disruption to schedules.

Immunomodulators plus antibiotics delay preterm delivery after experimental intraamniotic infection in a nonhuman primate model.

OBJECTIVE: The purpose of this study was to determine whether treatment with ampicillin together with dexamethasone and indomethacin delays preterm birth that is induced by intraamniotic group B Streptococcus in a nonhuman primate model. STUDY DESIGN: After contraction onset that was induced by group B Streptococcus (10(6) colony-forming units/mL), chronically instrumented rhesus macaques received either no treatment (controls; n = 6); ampicillin (n = 4); or ampicillin + dexamethasone + indomethacin (n = 5). Outcomes included the interval from contraction onset until delivery and concentrations of amniotic fluid inflammatory mediators. RESULTS: Mean interval from contraction onset until delivery was 33 +/- 8.7 hours in controls, 82 +/- 28.0 hours with ampicillin (P = .18, vs controls), and 213 +/- 50.8 hours with ampicillin + dexamethasone + indomethacin (P = .004, vs controls). Ampicillin eradicated group B Streptococcus; however, uterine activity, amniotic fluid cytokines, prostaglandins, and matrix metalloproteinase-9 remained elevated. Ampicillin + dexamethasone + indomethacin suppressed interleukin-1beta, tumor necrosis factor-alpha, and prostaglandins E2 and F2alpha but did not alter matrix metalloproteinase expression or chorioamnionitis. CONCLUSION: The combination of ampicillin + dexamethasone + indomethacin suppressed inflammation and significantly prolonged gestation.

Preterm labor is induced by intraamniotic infusions of interleukin-1beta and tumor necrosis factor-alpha but not by interleukin-6 or interleukin-8 in a nonhuman primate model.

OBJECTIVES: The purpose of this study was to determine the relative contributions of individual proinflammatory cytokines and chemokines to the triggering of preterm labor. STUDY DESIGN: Eighteen chronically instrumented pregnant rhesus monkeys at 135 +/- 3 days gestation (term = 167 days) received 1 of 5 intraamniotic infusions: (1) interleukin-1beta (IL-1beta) (10 microg; n = 5), (2) tumor necrosis factor-alpha (TNF-alpha) (10-100 microg; n = 5), (3) IL-6 (20 microg twice a day; n = 2), (4) IL-8 (20 microg twice a day; n = 2), and (5) saline control (n = 4). Primary study outcomes were the mean uterine hourly contraction area (mm Hg x s/h) in 24 hours during peak response to cytokine infusion (all groups) and the interval from cytokine infusion until labor onset (IL-1beta, IL-6, and IL-8 groups). Secondary outcomes were quantities of amniotic fluid cytokines and chemokines (IL-1beta, TNF-alpha, IL-6, and IL-8), prostaglandins E2 and F2alpha, leukocytes, and matrix metalloproteinase-9 (MMP-9). Histopathology of fetal lungs and placental membranes was assessed. RESULTS: IL-1beta stimulated the most intense contraction patterns, resulting in preterm labor in all cases. TNF-alpha induced a variable degree of uterine activity among individual animals stimulating either preterm labor (n = 2) or a uterine contraction pattern of moderate intensity (n = 3). Despite prolonged elevations in amniotic fluid levels, neither IL-6 nor IL-8 induced preterm labor or an increase in uterine activity until near term. The mean interval from the initiation of IL-6 and IL-8 infusion to the onset of labor was significantly longer than after IL-1beta (21.9 vs 1.1 days; P < .01), and did not differ from the saline control group (27.6 days; P = NS). Intraamniotic infusion of IL-1beta or TNF-alpha was associated with significant elevations in all tested amniotic fluid cytokines, IL-8, prostaglandins, MMP-9 and leukocytes compared with gestational age-matched saline controls. IL-6 and IL-8 infusions were not associated with increases in IL-1beta or TNF-alpha and only produced a moderate increase in amniotic fluid prostaglandins. All cytokine infusions induced histologic chorioamnionitis and an accumulation of neutrophils in fetal lungs. CONCLUSION: Preterm labor was induced by intraamniotic infusions of IL-1beta and TNF-alpha, but not by IL-6 or IL-8 although inflammatory changes in fetal membranes and lungs were uniformly present. Our results indicate a primary role for IL-1beta and TNF-alpha in the triggering of preterm labor associated with inflammation or infection.

Prospective study of fetal DNA in serum and disease activity during pregnancy in women with inflammatory arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) usually improves during pregnancy and recurs postpartum. Fetal cells and cell-free DNA reach the maternal circulation during normal pregnancy. The present study investigated dynamic changes in levels of fetal DNA in serum from women with RA and inflammatory arthritis during and after pregnancy to test the hypothesis that the levels of circulating fetal DNA correlate with arthritis improvement. METHODS: Twenty-five pregnant patients were prospectively studied. A real-time quantitative polymerase chain reaction panel targeting unshared, paternally transmitted HLA sequences, a Y chromosome-specific sequence, or an insertion sequence within the glutathione S-transferase M1 gene was used to measure cell-free fetal DNA. Results were expressed as fetal genomic equivalents per milliliter (gE/ml) of maternal serum. Physical examinations were conducted during and after pregnancy. RESULTS: Levels of fetal DNA in women with improvement in or remission of arthritis were higher than those in women with active disease, especially in the third trimester. Overall, an inverse relationship between serum fetal DNA levels and disease activity was observed (P < 0.001). Serum fetal DNA increased with advancing gestation, reaching median levels of 24 gE/ml (range 0-334), 61 gE/ml (range 0-689), and 199 gE/ml (range 0-2,576) in the first, second, and third trimesters, respectively, with fetal DNA clearance observed postpartum. Arthritis improvement was initially noted in the first trimester for most patients, increased further or was sustained with advancing gestation, and was active postpartum. CONCLUSION: Changes in serum fetal DNA levels correlated with arthritis improvement during pregnancy and recurrence postpartum. Immunologic mechanisms by which pregnancy might modulate RA activity are described.

Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome.

Graft-versus-host disease (GVHD) is seen in skin, intestinal mucosa, and liver after autologous stem cell transplantation. We reviewed 681 consecutive patients to estimate the probability of gastrointestinal (GI) GVHD, response to treatment, risk factors for development, and effect on survival. GI GVHD was defined by persistent symptoms, mucosal abnormalities at endoscopy, and histology showing apoptotic crypt cells with or without lymphoid infiltrates. The proportion of patients with GI GVHD was 90/681 (13%). Nausea and vomiting occurred in 90% and diarrhea in 40%. The mean time to developing symptoms was day +15, that to histologically proven diagnosis was day +42, and that to starting prednisone treatment was day +45 after stem cell infusion. Treatment with a short course of prednisone effected durable responses in 79% of patients, and an additional 18% responded to a second course of prednisone. A multivariable logistic regression model demonstrated that the combined factor of a diagnosis of breast cancer or hematologic malignancy and female sex was statistically significantly associated with the probability of GI GVHD (P = .003). Survival in patients with GI GVHD was not statistically different than that in those without GVHD. We conclude that women with breast cancer or hematologic malignancy are more likely to develop GI GVHD after autologous transplantation, and that treatment with prednisone was effective.

USDA perspective on environmental enrichment for animals.

This article provides a brief historical background of the events and circumstances that led to the 1985 Animal Welfare Act (AWA) amendments. It describes the development of the regulations promulgated by the US Department of Agriculture (USDA) in 1991 as a result of these amendments, the reasoning given for the proposals, and the revisions that were made during the process. Information is included on USDA implementation of the regulations regarding exercise for dogs and environmental enhancement for nonhuman primates. Also mentioned briefly are the requirements for socialization of marine mammals and space requirements for certain other regulated warm-blooded species. These requirements apply to animal dealers (breeders and brokers), exhibitors, commercial transporters, and research facilities. The standards for exercise and environmental enhancement were different from any others previously contained in the AWA regulations, and required more research and understanding of species-specific needs by the regulated community. Finally, this article describes some of the initiatives being undertaken by the research community and USDA-Animal and Plant Health Inspection Services (APHIS)-Animal Care to provide the necessary education and guidance indicated by the violation history data.

Adverse outcomes after preterm labor are associated with tumor necrosis factor-alpha polymorphism -863, but not -308, in mother-infant pairs.

OBJECTIVE: Two single-base polymorphisms of the tumor necrosis factor-alpha gene (TNF-alpha) at positions -863 and -308 are associated with variation in production of TNF-alpha (TNF-alpha). TNF-alpha genotypes were tested for association with adverse outcomes in mother-infant pairs with preterm labor. STUDY DESIGN: We analyzed a cohort of 118 mother-infant pairs with preterm labor before 34 weeks' gestation. Polymerase chain reaction was used on extracted deoxyribonucleic acid for polymorphism assay. Outcomes included amniotic fluid TNF-alpha concentration, histologic chorioamnionitis, delivery gestational age, and composite neonatal morbidity. Statistical significance was determined by chi 2 and Kruskal-Wallis analysis of variance. RESULTS: Mothers homozygous for the -863 polymorphism (AA) had significantly earlier deliveries ( P = .02), more chorioamnionitis ( P = .03), and greater composite neonatal morbidity ( P = .03). Neither maternal nor fetal carriage of the -308 polymorphism was associated with adverse outcome. CONCLUSION: In women with preterm labor before 34 weeks' gestation, maternal homozygous carriage of the -863 polymorphism may be associated with preterm delivery and adverse neonatal outcome.

Risk factors for syngeneic graft-versus-host disease after adult hematopoietic cell transplantation.

Syngeneic graft-versus-host disease (sGVHD) has been described after hematopoietic cell transplantation (HCT) but remains poorly defined. We retrospectively reviewed adult syngeneic HCTs at our center (1980-2002) for sGVHD to investigate incidence, morbidity, and risk factors with a primary focus on parity. Among 119 transplantations, there were 21 cases of biopsy-proven sGVHD. The cumulative incidence was 18%, with multiorgan involvement in 6 cases and 1 death. sGVHD was more frequent when the donor was parous (32%) than nulliparous (9%) or male (13%; P =.03) and when the recipient was parous (31%) than nulliparous (7%) or male (13%; P =.02). Other univariable risk factors included older age (P <.01), busulfan/melphalan/thiotepa conditioning (P <.01), interleukin-2 (P =.02), HLA-A26 (P =.03), and more recent transplantation year (P <.01). Overall, risk factors were similar to those described in GVHD. Although an independent effect of parity could not be completely separated from other factors, donor and recipient pregnancy history merits further investigation.

Microchimerism: an investigative frontier in autoimmunity and transplantation.

Recent studies indicate cells transfer between fetus and mother during pregnancy and can persist in both decades later. The presence within one individual of a small population of cells from another genetically distinct individual is referred to as microchimerism. Naturally acquired microchimerism has recently been investigated in autoimmune diseases, including scleroderma, thyroiditis, primary biliary cirrhosis, Sjögren syndrome, systemic lupus, dermatomyositis, and neonatal lupus. Iatrogenic chimerism has been investigated in transplantation and following blood transfusion. Considering findings of naturally acquired microchimerism along with iatrogenic microchimerism suggests microchimerism can have detrimental and/or beneficial effects in both settings. Recent identification of tissue-specific microchimerism either from naturally acquired or iatrogenic microchimerism (eg, cardiac myocytes) raises the possibility that microchimerism can be a target of autoimmunity or alternatively contribute to tissue repair. Advances in this new frontier of research with varied and numerous implications for human health are summarized.

A canine socialization and training program at the National Institutes of Health.

Well-socialized and obedient dogs are easier to handle and may make better research models. The authors describe the program they have implemented at the NIH, which has benefited both the animals and their caretakers.

The genetic contribution towards preterm delivery.

Indirect evidence supports a possible genetic predisposition towards preterm birth. The recurrence of spontaneous preterm delivery in individual women, families and ethnic groups suggests a long-acting aetiology, consistent with a genetic factor. Genetic contributions from both mother and fetus probably play a role in determining gestational length. Preliminary genetic association studies implicate gene variants of tumor necrosis factor-alpha (TNF-alpha) in preterm birth. Further understanding of a genetic predisposition begins with investigation of the pathogenesis of preterm delivery. Technological advances in the study of the human genome (genomics) and protein complement (proteomics) will allow identification of novel genes and proteins involved in preterm delivery. Insight into the complex gene regulation and protein production in preterm delivery may contribute to an understanding of a genetic basis. A discovered genetic factor may lead to medical breakthroughs and reductions in prematurity, neonatal morbidity and mortality.

Male DNA in female donor apheresis and CD34-enriched products.

Increased risk of graft-versus-host disease (GVHD) has been described in recipients of hematopoietic stem cell transplantations when the donor is a parous woman. Cells from prior pregnancies are now known to persist in women and could contribute to GVHD. We asked whether male DNA (presumed fetal microchimerism) is present in apheresis products of female donors. A total of 50 samples were studied by using real-time quantitative polymerase chain reaction (PCR) for the Y chromosome-specific sequence DYS14. Among 29 growth factor-mobilized peripheral blood mononuclear cell (G-PBMC) products, 34% were positive for male DNA. Quantitative results, expressed as DNA genome equivalent of male cells per million host cells (gEq/mil), ranged from 0 to 35 gEq/mil. Among 21 CD34-enriched cell fractions, 48% were positive with a range of 0 to 357 gEq/mil. In summary, male DNA was frequently detected in G-PBMC and CD34-enriched products from female donors. Whether fetal microchimerism contributes to GVHD merits further investigation.

Interleukin-18 in the plasma of women with preeclampsia.

OBJECTIVE: Interleukin-18 (IL-18) is a proinflammatory cytokine capable of stimulating interferon gamma and tumor necrosis factor-alpha production. Our purpose was to determine whether abnormal levels of IL-18 in maternal plasma correlate with the diagnosis of preeclampsia. STUDY DESIGN: A case control study design was used to enroll 61 patients: controls (n = 31) and preeclamptic women (n = 30). A standard enzyme-linked immunosorbent assay measured plasma IL-18. Statistical methods included Student t tests and chi(2) tests. RESULTS: Mean IL-18 levels were lower in preeclampsia than in controls (185 +/- 74 pg/mL vs 224 +/- 75 pg/mL, P =.053). Administration of betamethasone (BMZ) and/or hydralazine (HYD) was significantly associated with a lower IL-18 compared with controls (159 +/- 50 pg/mL vs 224 +/- 75 pg/mL, P =.002). After women who received BMZ or HYD were excluded, levels of IL-18 in preeclampsia were similar to those of controls (213 +/- 87 pg/mL, P =.69). There was no association between gestational age and IL-18. CONCLUSION: Lower IL-18 was associated with administration of either BMZ or HYD. After patients receiving these medications were excluded, IL-18 levels were similar in controls and preeclamptic women. IL-18 was not associated with gestational age.