Glutaredoxin 2a overexpression in macrophages promotes mitochondrial dysfunction but has little or no effect on atherogenesis in LDL-receptor null mice.

AIMS: Reactive oxygen species (ROS)-mediated formation of mixed disulfides between critical cysteine residues in proteins and glutathione, a process referred to as protein S-glutathionylation, can lead to loss of enzymatic activity and protein degradation. Since mitochondria are a major source of ROS and a number of their proteins are susceptible to protein-S-glutathionylation, we examined if overexpression of mitochondrial thioltranferase glutaredoxin 2a (Grx2a) in macrophages of dyslipidemic atherosclerosis-prone mice would prevent mitochondrial dysfunction and protect against atherosclerotic lesion formation. METHODS AND RESULTS: We generated transgenic Grx2aMac(LDLR-/-) mice, which overexpress Grx2a as an EGFP fusion protein under the control of the macrophage-specific CD68 promoter. Transgenic mice and wild type siblings were fed a high fat diet for 14 weeks at which time we assessed mitochondrial bioenergetic function in peritoneal macrophages and atherosclerotic lesion formation. Flow cytometry and Western blot analysis demonstrated transgene expression in blood monocytes and peritoneal macrophages isolated from Grx2aMac(LDLR-/-) mice, and fluorescence confocal microscopy studies confirmed that Grx2a expression was restricted to the mitochondria of monocytic cells. Live-cell bioenergetic measurements revealed impaired mitochondrial ATP turnover in macrophages isolated from Grx2aMac(LDLR-/-) mice compared to macrophages isolated from non-transgenic mice. However, despite impaired mitochondrial function in macrophages of Grx2aMac(LDLR-/-) mice, we observed no significant difference in the severity of atherosclerosis between wildtype and Grx2aMac(LDLR-/-) mice. CONCLUSION: Our findings suggest that increasing Grx2a activity in macrophage mitochondria disrupts mitochondrial respiration and ATP production, but without affecting the proatherogenic potential of macrophages. Our data suggest that macrophages are resistant against moderate mitochondrial dysfunction and rely on alternative pathways for ATP synthesis to support the energetic requirements.

Practice effect of the Wingate anaerobic test.

The purpose of this study was to investigate the presence of a practice effect on the Wingate anaerobic test (WAnT). Twenty-five young adult men (mean age = 20 years) performed 2 trials of the WAnT, which were separated by 7 days. Mean peak power (PP) and mean power (MP) for trials I and II were compared using a 1-way repeated measures analysis of variance to determine if a practice effect existed. Mean PP and MP scores were significantly higher (p < 0.025) on trial II (867.64 and 634.68 W for PP and MP, respectively) than on trial I (764.48 and 604.92 W), indicating that a practice effect occurred. Effect size (Cohen's d) for PP and MP was 0.72 and 0.35, indicating a large effect and small effect, respectively. Therefore, at least 1 full administration should be performed prior to a baseline power output measurement.

Apical pressures developed by needles for canal irrigation.

Drying instrumented canals with pressurized air may result in patient morbidity or even fatality. Low pressure and side vent needles have been suggested to lessen the danger. This study observed apical pressures from different needles inserted deeply into small round and ovoid canals as instrumentation progressed. Low-pressure (5 psi) air was injected through the needles, and apical pressures were recorded after each instrument. Pressures varied greatly within each test group. Generalities that can be drawn are that binding the needle within the canal gives higher pressures than with the needle slightly short of binding and that pressures were higher with apexes instrumented to size 30 and higher. With the needle tightly bound, neither needle size, needle design, nor canal shape resulted in statistically significant mean pressure differences. With the needle slightly withdrawn, larger bore needles gave higher pressures than small diameter needles. Caution is advised with the clinical use of pressurized air in the drying of root canals.

Early placental ontogeny in the mouse.

Fundamental to placental morphogenesis is union between the allantois and the chorion, two tissues initially separated in the conceptus. Results of previous studies in the mouse have suggested that chorio-allantoic union is driven by the developmental maturity of the allantois and involves molecular interactions between Vascular Cell Adhesion Molecule (VCAM-1) in the allantois and alpha4-integrin in the chorion. Little more is known about the cellular and/or molecular control of this important morphogenetic event in any species.Gross, histological, microsurgical and immunohistochemical analyses in the mouse conceptus revealed that placental ontogeny took place in three major steps. The first, chorio-allantoic contact, was not enduring and was mediated by the allantois' mesothelial surface and the mesodermal component of the chorion. Modest amounts of VCAM-1 were found in distal allantoic mesothelium, whilst levels of alpha4-integrin were high throughout chorionic mesoderm. The second step, chorio-allantoic fusion, was more enduring. During this time, the distal allantoic region contained maximal levels of VCAM-1, and all allantoises had expanded far enough to reach the posterior chorion from where they spread toward a central chorionic depression. The last step, breakdown of chorio-allantoic fusing surfaces, was dependent upon chorio-allantoic fusion and resulted in the intimate juxtaposition of allantoic endothelium and chorionic ectoderm, possibly as a result of VCAM-1-mediated interactions. The umbilical connection was thereafter fixed at its perimeter to the chorionic surface by large amounts of VCAM-1 in disto-lateral allantoic mesothelium and alpha4-integrin in the remaining peripheral mesodermal component of the chorion.Thus, chorio-allantoic union is highly regulated, taking place in multiple steps. It is dependent upon the developmental maturity of distal allantoic mesothelium and involves the mesodermal component of the chorion. Breakdown of fusing surfaces enables penetration of the allantoic vasculature into the chorion. These findings provide a secure developmental foundation in which to elucidate the genetic control of early placentation.

Study of the murine allantois by allantoic explants.

The murine allantois will become the umbilical artery and vein of the chorioallantoic placenta. In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos. In this study, the extent to which explanted allantoises grow and differentiate outside of the conceptus was investigated. The explant model was then used to elucidate cell and growth factor requirements in allantoic development. Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes. Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises. DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent. Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF(164). Although VEGF(164) was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelialization of these cells. Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles. On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems.

Methods for evaluating the provision of well child care.

BACKGROUND: Well child visits are important for reducing the incidence of avoidable illness and disease. The Omnibus Reconciliation Act of 1989 (OBRA '89) set goals for well child or Early and Periodic Screening, Diagnosis, and Treatment (EPSDT) visits. Many health plans are evaluated in terms of the number of well child visits provided, yet the method used for collecting the data influences the indicator results and conclusions drawn from them. METHODS: In a retrospective cohort design, medical record review and administrative data were compared as methods for ascertaining the number of well child visits received by Iowa Medicaid-eligible children for the period from July 1, 1997 through December 31, 1998. Compliance with the American Academy of Pediatrics' "Recommendations for Preventive Pediatric Health Care" periodicity guidelines was assessed. RESULTS: Using administrative data, 29.6% (n = 1,489) of children received a well child visit. If medical record review was used, 39.6% (n = 1,003) of children had a visit. The concordance between the rates was quite low (kappa = 0.30). Medical record review supported that an EPSDT visit was provided for only 68% of the children who had a claim or encounter billed as providing well child care (n = 441). DISCUSSION: Administrative data may underestimate the performance of EPSDT visits in comparison to medical record review. In addition, having a claim for an EPSDT visit did not necessarily mean the child received the basic components of a well child exam. The methodology for performance indicators used to evaluate health plans should be carefully validated.

Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain.

We have defined the histone acetylation pattern of the endogenous murine beta-globin domain, which contains the erythroidspecific beta-globin genes. The beta-globin locus control region (LCR) and transcriptionally active promoters were enriched in acetylated histones in fetal liver relative to fetal brain, whereas the inactive promoters were hypoacetylated. In contrast, the LCR and both active and inactive promoters were hyperacetylated in yolk sac. Hypersensitive site two of the LCR was also hyperacetylated in murine embryonic stem cells, whereas beta-globin promoters were hypoacetylated. Thus, the acetylation pattern varied at different developmental stages. Histone deacetylase inhibition selectively increased acetylation at a hypoacetylated promoter in fetal liver, suggesting that active deacetylation contributes to silencing of promoters. We propose that dynamic histone acetylation and deacetylation play an important role in the developmental control of beta-globin gene expression.

Extraordinary infections: a focus on bioterrorism.

The possibility of biological or chemical terrorist attacks presents a very real threat. Current preparedness efforts are ongoing at federal, state, and local levels. A National Pharmaceutical Stockpile containing antibiotics, antidotes, and other pharmaceutical supplies has been established and continues to be developed. To provide enhanced local response capabilities, Metropolitan Medical Response Systems are being formed. Pharmacists are needed by their communities to become involved with preparedness efforts.

Growth in the pre-fusion murine allantois.

Prior to fusion with the chorion, the extraembryonic mesoderm of the murine (Mus musculus) allantois differentiates with distal-to-proximal polarity into at least two cell lineages: a chorio-adhesive cell lineage called mesothelium, and the endothelium of the umbilical vasculature. How the allantois grows is less clear, but cell proliferation and addition of mesoderm from the underlying primitive streak appear to play important roles. The aim of this study was to analyze growth in the murine allantois. Techniques of histology and microsurgery were used to examine pre-fusion allantoises at nine developmental timepoints that differed by approximately 2 h. Cell counts revealed that allantoic size increased over time. Two hours of exposure to colcemid enhanced mitotic figures, which were used to calculate the relative number of proliferating cells (mitotic index, MI) in pre-fusion allantoises at each developmental timepoint. Cell proliferation was highest in nascent allantoises and showed signs of slowing by two somite pairs. By five to six-somite pairs, when most allantoises are attaching to the chorion, the overall MI decreased significantly. No regional differences in the mitotic index were observed at any developmental stage. Total cell numbers and the mitotic index were then used to discover the extent of streak contribution to pre-fusion allantoises. Cell proliferation and streak activity were highest in nascent allantoises, after which growth occurred predominantly by cell proliferation. Formation of allantoic regenerates by microsurgical removal and culture in intact conceptuses provided independent confirmation that, as the allantois matured, the primitive streak ceased to be a major contributor to its growth. Thus, the allantois grows by both mitosis and addition of mesoderm from the streak. That the periods of highest cell proliferation and streak activity coincided raises intriguing questions concerning their interplay in the control of growth in the murine allantois.

Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells.

The human beta-globin locus control region (LCR) confers high-level, tissue-specific expression to the beta-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for beta-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in alpha- and beta-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of beta-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate beta-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of beta-globin gene expression through activation of the LCR.

Inhaled corticosteroids for asthma therapy: patient compliance, devices, and inhalation technique.

BACKGROUND: Patient compliance, inhalation devices, and inhalation techniques influence the effectiveness of inhaled medications. METHODS: This article presents the results of a systematic literature review of studies measuring compliance with inhaled corticosteroids, measuring inhalation technique with different inhalation devices, and estimating the proportion of inhaled drug that is deposited in the lung. RESULTS: Overall, patients took the recommended doses of inhaled medication on 20 to 73% of days. Frequency of efficient inhalation technique ranged from 46 to 59% of patients. Education programs have been shown to improve compliance and inhalation techniques. The lung deposition achieved with different inhalers depends on particle size as well as inhaler technique. CONCLUSION: This review demonstrates that multiple factors may come between a prescription of an inhaled corticosteroid and the arrival of that medicine at its target organ, the lung.

Healthcare use among U.S. women aged 45 and older: total costs and costs for selected postmenopausal health risks.

The purpose of this study is to estimate the level of healthcare use and costs incurred by postmenopausal women overall and for these selected conditions: cardiovascular disease, osteoporosis, breast cancer, and gynecological cancers. National healthcare survey and discharge data were used to estimate healthcare use by women aged 45 and older. Clinical Classification for Health Policy Research (CCHPR) codes were used to identify patients whose primary diagnosis or procedure corresponded with the selected conditions. National weights were used to estimate resource use. Treatment costs were estimated using cost/charge ratios or the Medicare fee schedule to calculate costs for each individual procedure. Estimated total annual medical care treatment costs for women 45 and older were about $186 billion in 1997 dollars, including about $60.4 billion for cardiovascular disease, $12.9 billion for osteoporosis, and $5.0 billion for breast and gynecological cancers. For each condition, estimated resource use and costs are reported for hospitalization, outpatient, nursing home, and home healthcare services. Resource use and costs are also reported by age and expected source of payment. The economic burden of disease for conditions commonly affecting postmenopausal women is substantial. Prior research establishes that hormone replacement therapy (HRT) may be effective in reducing the burden of disease among women who continue preventive therapy for many years, but few at-risk women do so. New alternatives for prevention, such as selective estrogen receptor modulators (SERMs), may be effective in reducing the burden of disease among postmenopausal women.

Experiential learning in the animal sciences: effect of 13 years of a beef cattle management practicum.

As the source of students shifts from rural to urban and suburban communities, students entering agricultural programs have less practical livestock experience. The career goals indicated by most of these students require knowledge of and experience with practical applications of their course work. The objective of this study was to examine the profile of students enrolled in an experiential beef cattle course 1) to describe the demographic and occupational characteristics of students enrolled and 2) to assess the perceived value of course activities to graduates completing the course as related to their skill attainment and career development. The questionnaire was sent to all 312 students who were enrolled in the course from 1983 to 1996. Over 61% of the respondents indicated they had enrolled in the course to gain experience working with beef cattle. Over 39% took the course to enhance their application to the College of Veterinary Medicine. When asked to rate the value of the course, as it related to skill development, they noted it was most helpful in teaching cattle handling skills, growth performance measurement, live animal evaluation, nutritional management, carcass and meat product value determination, and breed identification.

Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis.

The aim of this study was to determine whether the blood vessels of the murine allantois are formed by vasculogenesis or angiogenesis. Morphological analysis revealed that differentiation of allantoic mesoderm into an outer layer of mesothelium and an inner vascular network begins in the distal region of the allantois, which is most remote from other tissues, as early as the late neural plate stage (approximately 7.75 days postcoitum). Nascent blood vessels were not found in the base of the allantois until 4-somite pairs had formed in the fetus (approximately 8.25 days postcoitum), and vascular continuity with the yolk sac and fetus was not present until the 6-somite-pair stage (approximately 8.5 days postcoitum). Immunohistochemical analysis demonstrated that flk-1, a molecular marker of early endothelial cells, is expressed in significantly more distal than basal core cells in the early allantois and never in mesothelium. Furthermore, synchronous grafting of donor yolk sac containing blood islands into blood islands of headfold-stage host conceptuses provided no evidence that the yolk sac contributes endothelial cells to the allantois. Finally, when removed from conceptuses and cultured in isolation, neural plate and headfold-stage allantoises formed a conspicuous vascular network that was positive for Flk-1. Hence, the vasculature of the allantois is formed intrinsically by vasculogenesis rather than extrinsically via angiogenesis from the adjacent yolk sac or fetus. Whether allantoic vasculogenesis is associated with erythropoiesis was also investigated. Benzidine-staining in situ revealed that primitive erythroid cells were not identified in the allantois until 6-somite pairs when continuity between its vasculature and that of the yolk sac was first evident. Nevertheless, a small number of allantoises removed from conceptuses at a considerably earlier stage were found to contain erythroid precursor cells following culture in isolation. To determine whether such erythroid cells could be of allantoic origin, host allantoises were made chimeric with lacZ-expressing donor allantoises that were additionally labeled with [3H]methyl thymidine. Following culture and autoradiography, many lacZ-expressing benzidine-stained cells were observed in donor allantoises, but none contained silver grains above background. Moreover, no cells of donor allantoic origin were found in the fetus or yolk sac. Hence, vasculogenesis seems to be independent of erythropoiesis in the allantois and to involve a distal-to-proximal gradient in differentiation of allantoic mesoderm into the endothelial cell lineage. Furthermore, this gradient is established earlier than reported previously, being present at the neural plate stage.

A comparison of the pattern of interstitial pneumonitis following allogeneic bone marrow transplantation before and after the introduction of prophylactic ganciclovir therapy in 1989.

A comparison was made of the pattern of interstitial pneumonitis (IP) following allogeneic bone marrow transplantation before and after the introduction of ganciclovir prophylaxis to minimize the risk of cytomegalovirus (CMV) disease in the St Vincent's Hospital bone marrow transplant program in 1989. A total of 456 recipients of allogeneic transplants were included. 280 received no prophylactic ganciclovir while 176 received prophylactic ganciclovir. The overall incidence of interstitial pneumonitis dropped from 19.6 to 12.5% (P = 0.03) and this was primarily due to a reduction in the incidence of CMV-IP which fell from 12.9 to 1.7% (P < 0.0005). The incidence of idiopathic IP was not different between the two groups (6.3 vs 3.2%), nor was the incidence of Pneumocystis carinii pneumonia (2.9 and 0.6%). Prophylactic ganciclovir has thus had a significant impact in reducing both the overall incidence of IP and specifically cytomegalovirus IP in allogeneic marrow transplant recipients. The most common form of IP in patients given prophylactic ganciclovir is now idiopathic interstitial pneumonitis.

Developmental potency of the murine allantois.

The murine allantois is the future umbilical component of the placenta. The base of the allantois is also thought to contain the future germ line. We have examined the fate and developmental potency of cells within the murine allantois during gastrulation. lacZ-expressing headfold-stage allantoises (approximately 8.0 days postcoitum; dpc) were subdivided into three proximodistal regions and transplanted into three sites in synchronous non-transgenic host embryos: the primitive streak at the level of prospective paraxial mesoderm, the primitive streak at the level of lateral plate mesoderm, and the base of the allantois. After 23 hours in culture, operated conceptuses were examined histologically for contribution of donor allantoic cells to the conceptus. None of the allantoic regions contributed to paraxial mesoderm when placed into the fetus, but all three colonized the endothelium and adjacent mesenchyme of the dorsal aorta. The mid-region was most efficient at colonizing endothelium, whereas the base was the only allantoic region to exhibit relative pluripotency, colonizing several derivatives of all three primary germ layers. Differences in the state of differentiation along the proximodistal axis of the allantois were further borne out when the three allantoic regions were placed into the base of the allantois of host conceptuses. Striking differences were observed in final position along the proximodistal axis of the host allantois. Most grafted cells translocated distally from the base; however, basal donor allantoic cells translocated typically only as far as the host's mid-region, whereas donor allantoic tip cells typically returned to the tip, often colonizing the chorioallantoic fusion junction. Together, our data reveal that the headfold-stage allantois may contain a proximodistal gradient of differentiation, and raise intriguing questions about how this gradient was established and the role it plays in umbilical vasculogenesis.

The role of blame, distress, and anger in the hypermasculine man.

Research has demonstrated an association between the hypermasculine personality pattern and a history of sexually aggressive behavior. This study was conducted to examine emotions experienced by hypermasculine or macho men when prevented from attaining a goal relevant to their sense of attractiveness and sexuality by a woman. It was hypothesized that macho males would respond to high and moderate threats to their masculine identity with greater blame and anger than nonmacho males. Macho men's blame was hypothesized to mediate the transformation of negative emotions such as distress into anger. After screening with the Hypermasculinity Inventory, 34 high hypermasculine and 36 low hypermasculine men were assigned to one of three experimental conditions in which the feedback received from a female partner was either highly threatening, moderately threatening, or neutral in nature. Measures of emotion and blame were collected after the men received their feedback. Results of the study indicated that macho and nonmacho men differed only in the moderate threat condition. Macho men in this condition reported greater anger yet less blame than the nonmacho men. The pattern of results is most consistent with Berkowitz's cognitive-neoassociationistic model of emotion, which does not require blame for anger to occur, as does Lazaru's cognitive-motivational-relational theory of emotion. Results of the study suggest that anger in macho men is associated with the level of surprise in a situation.

Lenograstim administration to HLA-identical donor-recipient pairs to accelerate marrow recovery post-transplant.

In an attempt to accelerate marrow recovery after HLA-identical sibling bone marrow transplantation, the donors of 12 patients with haematological malignancy were given recombinant human granulocyte colony-stimulating factor (rHuG-CSF; lenograstim; Granocyte) 5 micrograms/kg/day for seven doses prior to marrow harvest. All 12 recipients also received lenograstim 5 micrograms/kg/day from the day of transplant until their neutrophil count was 1.0 x 10(9)/1. In addition to lenograstim post-transplant and lenograstim-stimulated donor bone marrow the first six recipients also received donor peripheral blood stem cells that had been enriched for CD34+ stem/progenitor cells and T cell depleted on an immune absorption column (cohort 1). The second six patients (cohort 2) received lenograstim post-transplant and lenograstim-stimulated donor marrow only. All 12 patients showed a marked elevation of their circulating white blood cell count (predominantly neutrophils) on day 1 post-transplant. Compared to carefully matched historical control patients the rate of neutrophil engraftment was significantly accelerated in both patient cohorts and platelet engraftment was accelerated in cohort 2.