Digital Whole Slide Imaging Compared With Light Microscopy for Primary Diagnosis in Surgical Pathology.

CONTEXT.­: The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility. OBJECTIVE.­: To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology. DESIGN.­: The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review. RESULTS.­: The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities. CONCLUSIONS.­: WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Atopic Eczema: Genetic Associations and Potential Links to Developmental Exposures.

Atopic eczema (AE), or atopic dermatitis (AD), is a common inflammatory skin disease with a disrupted epidermal barrier and an allergic immune response. AD/AE is prominently characterized by a symptomatic itch and transient skin lesions. Infants compose a significant percentage affected. Two models have been proposed to explain AD/AE skin pathology: the gut microbiome-focused inside-outside model and the outside-inside model concentrating on the disrupted skin barrier/skin microbiome. Gene disruptions contributing to epidermal structure, as well as those in immune system genes, are implicated. Over 30 genes have been linked to AD/AE with Flg and Tmem79/Matt alterations being common. Other linked disruptions are in the interleukin-1 family of cytokines/receptors and the TH2 gene family of cytokines. Inheritable epigenetic modifications of the genes or associated proteins may also be involved. Skin barrier disruption and the allergic immune response have been the main foci in mechanistic studies of AD/AE, but the role of the environment is becoming more apparent. Thus, an examination of in utero exposures could be very helpful in understanding the heterogeneity of AD/AE. Although research is limited, there is evidence that developmental exposure to environmental tobacco smoke or phthalates may impact disease. Management for AD/AE includes topical corticosteroids and calcineurin inhibitors, which safely facilitate improvements in select individuals. Disease heterogeneity warrants continued research not only into elucidating disease mechanism(s), via identification of contributing genetic alterations, but also research to understand how/when these genetic alterations occur. This may lead to the cure that those affected by AD/AE eagerly await.

Cardiac evaluation and management before vascular surgery.

PURPOSE OF REVIEW: The most common and significant morbidity and mortality of vascular surgical procedures remain coronary artery disease, arrhythmia, and heart failure. Cardiac evaluation and medical optimization provide the groundwork for best medical practice in an otherwise high-risk surgical population. The goal of this study is to review the most current literature and guidelines for evaluating patients prior to vascular surgical interventions. From this, we have made our own recommendations regarding both the preoperative and perioperative management of vascular surgical patients. RECENT FINDINGS: Risk stratification using a modified Lee index may be best to assess perioperative cardiac risk. Coronary revascularization should be reserved for those patients with significant coronary disease irrespective of symptoms. ß-blockers, statins, and antiplatelet agents should be considered for all patients with peripheral vascular disease. SUMMARY: The preoperative management of vascular surgical patients requires a complete understanding of the patient's medical history as it relates to their perioperative cardiac risk. Overwhelming data support the use of medical therapy as adjunct to minimize or prevent the risk of future cardiovascular events. As vascular surgery transitions to the outpatient setting, further studies will be required to better elucidate cardiac evaluation in this patient population.

The effects of maternal exposure to bisphenol A on allergic lung inflammation into adulthood.

Bisphenol A (BPA) is a high-production volume chemical classified as an environmental estrogen and used primarily in the plastics industry. BPA's increased usage correlates with rising BPA levels in people and a corresponding increase in the incidence of asthma. Due to limited studies, the contribution of maternal BPA exposure to allergic asthma pathogenesis is unclear. Using two established mouse models of allergic asthma, we examined whether developmental exposure to BPA alters hallmarks of allergic lung inflammation in adult offspring. Pregnant C57BL/6 dams were gavaged with 0, 0.5, 5, 50, or 500 µg BPA/kg/day from gestational day 6 until postnatal day 21. To induce allergic inflammation, adult offspring were mucosally sensitized with inhaled ovalbumin containing low-dose lipopolysaccharide or ip sensitized using ovalbumin with alum followed by ovalbumin aerosol challenge. In the mucosal sensitization model, female offspring that were maternally exposed to ≥ 50 µg BPA/kg/day displayed enhanced airway lymphocytic and lung inflammation, compared with offspring of control dams. Peritoneally sensitized, female offspring exposed to ≤ 50 µg BPA/kg/day presented dampened lung eosinophilia, compared with vehicle controls. Male offspring did not exhibit these differences in either sensitization model. Our data demonstrate that maternal exposure to BPA has subtle and qualitatively different effects on allergic inflammation, which are critically dependent upon route of allergen sensitization and sex. However, these subtle, yet persistent changes due to developmental exposure to BPA did not lead to significant differences in overall airway responsiveness, suggesting that early life exposure to BPA does not exacerbate allergic inflammation into adulthood.

Developmental exposure to bisphenol A modulates innate but not adaptive immune responses to influenza A virus infection.

Bisphenol A (BPA) is used in numerous products, such as plastic bottles and food containers, from which it frequently leaches out and is consumed by humans. There is a growing public concern that BPA exposure may pose a significant threat to human health. Moreover, due to the widespread and constant nature of BPA exposure, not only adults but fetuses and neonates are also exposed to BPA. There is mounting evidence that developmental exposures to chemicals from our environment, including BPA, contribute to diseases late in life; yet, studies of how early life exposures specifically alter the immune system are limited. Herein we report an examination of how maternal exposure to a low, environmentally relevant dose of BPA affects the immune response to infection with influenza A virus. We exposed female mice during pregnancy and through lactation to the oral reference dose for BPA listed by the US Environmental Protection Agency, and comprehensively examined immune parameters directly linked to disease outcomes in adult offspring following infection with influenza A virus. We found that developmental exposure to BPA did not compromise disease-specific adaptive immunity against virus infection, or reduce the host's ability to clear the virus from the infected lung. However, maternal exposure to BPA transiently reduced the extent of infection-associated pulmonary inflammation and anti-viral gene expression in lung tissue. From these observations, we conclude that maternal exposure to BPA slightly modulates innate immunity in adult offspring, but does not impair the anti-viral adaptive immune response, which is critical for virus clearance and survival following influenza virus infection.

Assistive technology device classification based upon the World Health Organization's, International Classification of Functioning, Disability and Health (ICF).

PURPOSE: To develop an assistive technology device classification (ATDC) consistent with the Assistive Technology Act (ATA2004), Americans with Disabilities Act (ADA2008), International Classification System of Functioning, Disability and Health (ICF), International Classification of Disease, Ninth Revision-Clinical Modification (ICD-9-CM) and American Medical Association's Current Procedural Terminology (CPT). BACKGROUND: Current assistive technology device (ATD) classifications include: the National Classification System for Assistive Technology Devices and ATSs (RTI/NCS) published in 2000; ISO 9999: technical aids for persons with disabilities - classification and terminology (ISO 9999) published in 1992, 1998, 2002 and 2007 and ICF-based AT classification (ICF/AT2007) published in 2009. OBJECTIVES: To derive 'requirements' for ATD classification from the ATA2004, ADA2008, ICF, ICD-9-CM and CPT. Review the ATD classifications and online databases against requirements. Construct the ATDC to be consistent with all requirements and demonstrate with examples. RESULTS: Existing ATD classifications and online databases are inconsistent with requirements. The ATDC is consistent and has inclusion and exclusion criteria, classification rules, employs ICF coding, extendable hierarchy and language and uses standard device naming conventions. Conclusion. The ATDC has broad application to: provision of AT ATSs (ATSs), characterisation and analysis of AT industries, Federally sponsored research pertaining to AT development and commercialisation, and Federal health insurance scope of benefits.

Provision of assistive technology services method (ATSM) according to evidence-based information and knowledge management.

PURPOSE. This article develops a standardised method for assistive technology service (ATS) provision and a logical basis for research to improve health care quality. The method is 'interoperable' across disabilities, disciplines, assistive technology devices and ATSs. BACKGROUND. Absence of a standardised and interoperable method for ATS provision results in ineffective communication between providers, manufacturers, researchers, policy-makers and individuals with disabilities (IWD), a fragmented service delivery system, inefficient resource allocation and sub-optimal outcomes. OBJECTIVES. Synthesise a standardised, interoperable AT service method (ATSM) fully consistent with key guidelines, systems, models and Federal legislation. Express the ATSM using common and unambiguous language. RESULTS. Guidelines, systems, models and Federal legislation relevant to ATS provision are reviewed. These include the RESNA Guidelines for Knowledge and Skills for Provision of Assistive Technology Products and Services (RESNA Guidelines), IMPACT2 model, international classification of functioning, disability and health (ICF) and AT device classification (ATDC). Federal legislation includes the Assistive Technology Act of 2004, Americans with Disabilities Act of 2008 and Social Security Act. Based on these findings, the ATSM is synthesised and translated into common and accessible language. CONCLUSION. ATSM usage will improve communication between stakeholders, service delivery coherence, resource allocation and intervention outcomes.

Activation of pulmonary dendritic cells and Th2-type inflammatory responses on instillation of engineered, environmental diesel emission source or ambient air pollutant particles in vivo.

The biological effects of acute particulate air pollution exposure in host innate immunity remain obscure and have relied largely on in vitro models. We hypothesized that single acute exposure to ambient or engineered particulate matter (PM) in the absence of other secondary stimuli would activate lung dendritic cells (DC) in vivo and provide information on the early immunological events of PM exposure and DC activation in a mouse model naïve to prior PM exposure. Activation of purified lung DC was studied following oropharyngeal instillation of ambient particulate matter (APM). We compared the effects of APM exposure with that of diesel-enriched PM (DEP), carbon black particles (CBP) and silver nanoparticles (AgP). We found that PM species induced variable cellular infiltration in the lungs and only APM exposure induced eosinophilic infiltration. Both APM and DEP activated pulmonary DC and promoted a Th2-type cytokine response from naïve CD4+ T cells ex vivo. Cultures of primary peribronchial lymph node cells from mice exposed to APM and DEP also displayed a Th2-type immune response ex vivo. We conclude that exposure of the lower airway to various PM species induces differential immunological responses and immunomodulation of DC subsets. Environmental APM and DEP activated DC in vivo and provoked a Th2 response ex vivo. By contrast, CBP and AgP induced altered lung tissue barrier integrity but failed to stimulate CD4+ T cells as effectively. Our work suggests that respirable pollutants activate the innate immune response with enhanced DC activation, pulmonary inflammation and Th2-immune responsiveness.

New developments in the preoperative evaluation and perioperative management of coronary artery disease in patients undergoing vascular surgery.

BACKGROUND: Preoperative evaluation and perioperative management of cardiac disease in patients undergoing vascular surgery (VS) is important for patients and vascular surgeons. Recent evidence has emerged that has allowed us to develop contemporary paradigms for evaluating and managing coronary artery disease in VS patients perioperatively. METHODS: The utility of stress testing, the role of preoperative coronary revascularization, the optimal use of beta-blockers and statins, and the role of antiplatelet therapy in VS patients were reviewed in the literature. RESULTS: The revised Lee cardiac risk index, based on the number of risk factors (high-risk surgery, ischemic heart disease, congestive heart failure, cerebrovascular disease, insulin-dependent diabetes mellitus, renal failure, hypertension, and age >75) quantitates cardiac risk. Stress testing is not predictive of myocardial ischemia/infarction (MI) or death and is only recommended in patients with unstable angina or an active arrhythmia. Stress testing for patients with 0 to 2 risk factors delays VS up to 3 weeks. In high-risk patients (>or=3 risk factors), it helps to identify patients who may develop myocardial ischemia and would benefit from a 30-day period to optimize medical therapy before VS. Stress testing and coronary catheterization do not predict which coronary artery to revascularize to prevent MI or death. Revascularization does not decrease MI or death rates at 1 month or 6 years. Although beta-blocker treatment decreases cardiac risk with VS, timing and dosage (titration) influence outcomes, improper usage may increase stroke and death rate, and not all VS patients should be taking these drugs. Patients with >or=1 risk factor should be considered to begin a low dose beta-blocker 1 month before VS. Preoperative statin use sharply decreases MI, stroke, and death perioperatively and long-term postoperatively. CONCLUSION: Routine stress testing should not be performed before VS. The Lee index should be used to stratify risk in patients undergoing VS. Patients with >or=3 risk factors or active cardiac conditions should undergo stress testing, if VS can be delayed. All VS patients, except those with 0 risk factors, should be considered for a beta-blocker (bisoprolol, 2.5-5 mg/d started 1 month before VS, titrated to a pulse <70 beats/min and a systolic blood pressure >or=120 mm Hg). Intermediate risk factors may not require aggressive heart rate control but simply maintenance on a low-dose beta-blocker. Statins should be started (ideally 30 days) before all VS using long-acting formulations such as fluvastatin (80 mg/d) for patients unable to take oral medication.

Measuring usability of assistive technology from a multicontextual perspective: the case of power wheelchairs.

Assistive technology (AT) devices enable people with disabilities to function in multiple contexts and activities. The usability of such devices is fundamentally indicative of the user's level of participation in multiple roles and occupations. Seventy people who used power wheelchairs were interviewed using a novel tool, the Usability Scale for Assistive Technology (USAT). The USAT uses a human factors science framework to investigate the wheelchair user's perceived independence in mobility-related activities within home, workplace, community, and outdoors in accordance with the characteristics of the wheelchair, environmental factors, and abilities and skills of the user to operate the wheelchair. Descriptive analysis of the data revealed usability issues with the use of power wheelchairs in all contexts. Users confronted far more significant issues within the community and outdoor environment compared with those at home and in the workplace. These issues have been elucidated and applied to an intervention framework with relevance to a multitude of AT stakeholders.

Maximizing immune responses: the effects of covalent peptide linkage to beta-2-microglobulin.

Major histocompatability molecules (MHC) are involved in presentation of peptide antigens for recognition by the immune system. The density and stability of presented peptides is a critical parameter in determining the magnitude of the immune response. Increasing the half-life and density of an MHC class I-peptide complex should promote a stronger cytotoxic T lymphocyte (CTL) response to clinically important peptides, including those that exhibit low or suboptimal MHC class I binding affinity. We hypothesized that the covalent linkage of a known tumor antigen peptide to beta-2-microglobulin (beta2m) would increase peptide immunogenicity and, therefore, in vivo effectiveness as an antitumor vaccine in BALB/c mice. The iL3 peptide fusion protein (iL3-L12-hbeta2m) was developed based on the mutant iL3 peptide, derived from the L3 ribosomal protein, and expressed in the mutagenized murine fibroblastic tumor cell line, BCA34. The iL3-L12-beta2m and a negative control fusion protein utilizing the H-2K(d)-restricted NP(147-155) influenza peptide (NP-L12-hbeta2m) were both produced in E. coli for exogenous antigen presentation by dendritic cells. In vitro, the iL3-L12-hbeta2m protein was found to stabilize H-2K(d) over time on the surface of H-2K(d)-expressing target cells and sensitized them to peptide-specific CTL-mediated lysis. Furthermore, mice immunized with dendritic cells pulsed with the iL3-L12-hbeta2m protein rejected a challenge with BCA34 cells significantly more so than mice immunized with dendritic cells pulsed with free peptide and hbeta2m. We conclude that vaccines incorporating peptides covalently linked to beta2m may have future potential in the specific targeting of human malignancy.

Disruption of the transcription factor Nrf2 promotes pro-oxidative dendritic cells that stimulate Th2-like immunoresponsiveness upon activation by ambient particulate matter.

Oxidative stress is important in dendritic cell (DC) activation. Environmental particulate matter (PM) directs pro-oxidant activities that may alter DC function. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates expression of antioxidant and detoxification genes. Oxidative stress and defective antioxidant responses may contribute to the exacerbations of asthma. We hypothesized that PM would impart differential responses by Nrf2 wild-type DCs as compared with Nrf2(-/-) DCs. We found that the deletion of Nrf2 affected important constitutive functions of both bone marrow-derived and highly purified myeloid lung DCs such as the secretion of inflammatory cytokines and their ability to take up exogenous Ag. Stimulation of Nrf2(-/-) DCs with PM augmented oxidative stress and cytokine production as compared with resting or Nrf2(+/+) DCs. This was associated with the enhanced induction of Nrf2-regulated antioxidant genes. In contrast to Nrf2(+/+) DCs, coincubation of Nrf2(-/-) DCs with PM and the antioxidant N-acetyl cysteine attenuated PM-induced up-regulation of CD80 and CD86. Our studies indicate a previously underappreciated role of Nrf2 in innate immunity and suggest that deficiency in Nrf2-dependent pathways may be involved in susceptibility to the adverse health effects of air pollution in part by promoting Th2 cytokine responses in the absence of functional Nrf2. Moreover, our studies have uncovered a hierarchal response to oxidative stress in terms of costimulatory molecule expression and cytokine secretion in DCs and suggest an important role of heightened oxidative stress in proallergic Th2-mediated immune responses orchestrated by DCs.

Diesel-enriched particulate matter functionally activates human dendritic cells.

Epidemiologic studies have associated exposure to airborne particulate matter (PM) with exacerbations of asthma. It is unknown how different sources of PM affect innate immunity. We sought to determine how car- and diesel exhaust-derived PM affects dendritic cell (DC) activation. DC development was modeled using CD34+ hematopoietic progenitors. Airborne PM was collected from exhaust plenums of Fort McHenry Tunnel providing car-enriched particles (CEP) and diesel-enriched particles (DEP). DC were stimulated for 48 hours with CEP, DEP, CD40-ligand, or lipopolysaccharide. DC activation was assessed by flow cytometry, enzyme-linked immunosorbent assay, and standard culture techniques. DEP increased uptake of fluorescein isothiocyanate-dextran (a model antigen) by DC. Diesel particles enhanced cell-surface expression of co-stimulatory molecules (e.g., CD40 [P < 0.01] and MHC class II [P < 0.01]). By contrast, CEP poorly affected antigen uptake and expression of cell surface molecules, and did not greatly affect cytokine secretion by DC. However, DEP increased production of TNF, IL-6, and IFN-gamma (P < 0.01), IL-12 (P < 0.05), and vascular endothelial growth factor (P < 0.001). In co-stimulation assays of PM-exposed DC and alloreactive CD4+ T cells, both CEP and DEP directed a Th2-like pattern of cytokine production (e.g., enhanced IL-13 and IL-18 and suppressed IFN-gamma production). CD4+ T cells were not functionally activated on exposure to either DEP or CEP. Car- and diesel-enriched particles exert a differential effect on DC activation. Our data support the hypothesis that DEP (and to a lesser extent CEP) regulate important functional aspects of human DC, supporting an adjuvant role for this material.

Conceptualization and measurement of assistive technology usability.

UNLABELLED: The concept of product usability has been discussed in several areas of product research and development. Usability, within the realm of assistive technology (AT) devices, determines how effectively and efficiently AT users with disabilities can function in different contexts and environments. OBJECTIVE: This article conceptualizes and proposes the significance of AT usability and its measurement in entirety from a human factors perspective. Conceptual models that characterize the interaction of the AT user, the AT device, the context and the involved activity is considered to be the hallmark of measurement of AT usability. On that basis, the article highlights the methodology and the initial progress of the development of an AT outcome tool, the Usability Scale for Assistive Technology (USAT), to measure self reported degree of AT usability. METHODOLOGY: In order to identify usability indicators for measurement, a qualitative study was conducted by exploring the experiences of AT users. Ten participants who used either wheeled mobility or computer based AT devices were interviewed with questions based on the theme of a usability framework. RESULTS: The coded interview data generated more than 800 usability indicators specific to the two categories of AT devices. These indicators were mapped to a generic usability criteria list for construction of the USAT-Wheeled Mobility and the USAT-Computer Access. IMPLICATIONS: The USAT, when developed, is projected to be valid and useful for AT outcomes research as well as clinical practice. The use of the USAT will enable researchers and clinicians to comprehensively identify factors that underlie effectiveness and efficiency in AT device use and establish intervention protocols to optimize user-AT interaction.

Endothelial progenitor cell release into circulation is triggered by hyperoxia-induced increases in bone marrow nitric oxide.

Endothelial progenitor cells (EPC) are known to contribute to wound healing, but the physiologic triggers for their mobilization are often insufficient to induce complete wound healing in the presence of severe ischemia. EPC trafficking is known to be regulated by hypoxic gradients and induced by vascular endothelial growth factor-mediated increases in bone marrow nitric oxide (NO). Hyperbaric oxygen (HBO) enhances wound healing, although the mechanisms for its therapeutic effects are incompletely understood. It is known that HBO increases nitric oxide levels in perivascular tissues via stimulation of nitric oxide synthase (NOS). Here we show that HBO increases bone marrow NO in vivo thereby increasing release of EPC into circulation. These effects are inhibited by pretreatment with the NOS inhibitor l-nitroarginine methyl ester (l-NAME). HBO-mediated mobilization of EPC is associated with increased lower limb spontaneous circulatory recovery after femoral ligation and enhanced closure of ischemic wounds, and these effects on limb perfusion and wound healing are also inhibited by l-NAME pretreatment. These data show that EPC mobilization into circulation is triggered by hyperoxia through induction of bone marrow NO with resulting enhancement in ischemic limb perfusion and wound healing.

The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia.

OBJECTIVE: Vasculogenesis relies on the recruitment of bone marrow-derived endothelial progenitor cells (BMD EPCs) and is stimulated by tissue-level ischemia. We hypothesized that the BMD EPC response is impaired in ischemic wounds and studied the relationship between BMD EPCs and wound healing. METHODS: We used transgenic Tie-2/LacZ mice, which carry the beta-galactosidase (beta-gal) reporter gene under Tie-2 promoter control. Wild-type mice were lethally irradiated and reconstituted with Tie-2/LacZ bone marrow. Four weeks later, the mice underwent unilateral femoral artery ligation/excision and bilateral wounding of the hindlimbs. Ischemia was confirmed and monitored with laser Doppler imaging. A subset of mice received incisional vs excisional nonischemic bilateral hindlimb wounds, without femoral ligation. Excisional wound closure was measured by using daily digital imaging and software-assisted calculation of surface area. RESULTS: Ischemia resulted in significantly delayed wound healing and differentially affected the number of BMD EPCs recruited to wound granulation tissue and muscle underlying the wounds. At 3 days postwounding, the granulation tissue of the wound base contained significantly fewer numbers of BMD EPCs in ischemic wounds compared with the nonischemic wounds (P < .05). In contrast, significantly more BMD EPCs were present in the muscle underlying the ischemic wounds at this same time point compared with the muscle under the nonischemic wounds (P < .05). In ischemic wounds, eventual wound closure significantly correlated with a delayed rise in BMD EPCs within the wound granulation tissue (Kendall's correlation, -.811, P = .0005) and was significantly associated with a gradual recovery of hindlimb perfusion (P < .0001). By 7 days postwounding, BMD EPCs were incorporated into the neovessels in the granulation tissue. At 14 days and 75 days, BMD EPCs were rarely observed within the wounds. CONCLUSIONS: Granulation tissue of excisional ischemic wounds showed significantly less BMD EPCs 3 days postwounding, in association with significantly delayed wound closure. However, the number of BMD EPCs were increased in ischemic hindlimb skeletal muscle, consistent with the notion that ischemia is a powerful signal for vasculogenesis. To our knowledge, this is the first report identifying a deficit in BMD EPCs in the granulation tissue of ischemic skin wounds and reporting the key role for these cells in both ischemic and nonischemic wound healing.

Normal human fibroblasts enable melanoma cells to induce angiogenesis in type I collagen.

BACKGROUND: We previously reported that fibroblasts induce human microvascular endothelial cells (HMVECs) to differentiate from monolayer to capillarylike morphology. We now test the hypothesis that fibroblasts modulate angiogenesis in melanoma cells. METHODS: We tested 12 human melanoma lines (2 radial growth phase (RGP), 3 vertical growth phase (VGP), and 7 metastatic (MM)) for ability to induce HMVECs to invade/migrate into collagen and form capillarylike networks. HMVEC monolayers were overlaid with 3-dimensional collagen gels embedded with melanoma cells alone (M), fibroblasts alone (F), or a 1:1 mixture of the 2 cells (M+F). After 5 days, gels were removed, fixed, and HMVEC networks were quantified by von Willebrand's factor (vWF) immunofluorescence. The influence of soluble factors on HMVEC invasion/migration into collagen was assessed with the use of acellular 3-D collagen gels overlaid on HMVEC monolayers, cultured with conditioned media (CM) derived from monolayers of M, F, or M+F. Angiogenic growth factors involved in the observed invasion/migration were identified with the use of a RayBio Cytokine Antibody Array (RayBiotech, Norcross, Ga). RESULTS: Cell line-specific variability in melanoma-supported angiogenesis was observed only when in combination with fibroblasts (analysis of variance [ANOVA], P < .01). Melanoma plus fibroblasts uniformly resulted in a significantly higher angiogenic response than melanoma alone (P < .05). One vertical growth phase and one metastatic melanoma line, while weakly angiogenic alone, induced significantly higher angiogenesis than either fibroblast or melanoma alone (P < .05) when combined with fibroblasts. CM from M or M+F induced significantly less HMVEC invasion/migration into collagen than CM from fibroblasts alone. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 were identified as significantly elevated in the media derived from M+F cultures, compared with either cell type alone. CONCLUSION: To our knowledge, this is the first report demonstrating that melanoma-supported angiogenesis in collagen is more significantly influenced by normal skin-derived fibroblasts than by the intrinsic biology of the melanoma cell type. Interleukin 8, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-2 are implicated as potential paracrine factors regulating this observed effect.

Angiogenesis, vasculogenesis, and induction of healing in chronic wounds.

A key central stage of wound healing requires neovascularization of the wound base granulation tissue. In the adult, neovascularization is now known to occur by both angiogenesis and vasculogenesis. Understanding the biology of these 2 processes offers promising new therapeutic options for patients who suffer from chronic, nonhealing ischemic wounds. The authors review the current literature on the processes of angiogenesis and vasculogenesis and how it relates to wound healing.

Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels.

OBJECTIVE: Neovascularization, angiogenesis, and collagen constriction are essential for wound healing. We tested whether vascular endothelial growth factor-C (VEGF-C) can promote collagen constriction, capillary sprouting (angiogenesis), and invasion/migration of bone marrow-derived endothelial progenitor cells into collagen (vasculogenesis). METHODS: We used a recently characterized three-dimensional collagen matrix assay with either monolayers of human dermal microvascular endothelial cells (HMVECs) or bone marrow-derived endothelial progenitor cells (BMD EPCs), obtained from Tie-2 LacZ transgenic mice, overlaid with an acellular layer and then a cellular layer of collagen embedded with fibroblasts, that were nontransduced or transduced with either LacZ adenoviral vector (Ad5) or VEGF-C/Ad5. The ability of VEGF-C to enhance fibroblast-mediated collagen constriction was measured, and gels overlying HMVECs or BMD EPCs were co-cultured, harvested, and assayed for HMVEC migration, sprouting, and capillary-like formation; gels containing BMD EPCs were assayed for EPC invasion/migration into the collagen extracellular matrix. RESULTS: VEGF-C significantly increased collagen constriction and formation of capillary-like structures with true lumina (P < .05) assessed by von Willebrand factor and VEGF receptor-2 immunoassaying. VEGF-C induced a significant increase in HMVEC migration, tubular polarization, and branching sprouts associated with a significant up-regulation of membrane type 1 matrix metalloproteinase (MT1-MMP) ( P < .05). Fibroblasts were necessary to support BMD-EPC invasion/migration from the monolayer into the collagen. Moreover, fibroblasts overexpressing VEGF-C significantly enhanced EPC invasion/migration ( P < .05) into the extracellular matrix by two-fold, and this effect could not be achieved with equivalent levels of exogenous VEGF-C in the absence of fibroblasts. The addition of a soluble VEGF-C competitor protein only partially inhibited these responses, reducing the EPCs by three-fold, but significant numbers of EPCs still invaded/migrated into the extracellular matrix, suggesting that other fibroblast-specific signals also contribute to the vasculogenic response. CONCLUSION: Fibroblast-specific expression of VEGF-C promotes collagen constriction by fibroblasts and enhances microvascular endothelial cell migration, branching, and capillary sprouting in association with up-regulating MT1-MMP expression. Fibroblasts are necessary for BMD EPC invasion/migration into collagen, and their overexpression of VEGF-C enhances this fibroblast-mediated vasculogenic effect. Collectively, these findings suggest a role for VEGF-C in multiple biologic steps required for wound healing (angiogenesis, vasculogenesis, and collagen constriction). CLINICAL RELEVANCE: Ischemic wound healing remains an unsolved problem with no previously identified molecular target for therapeutic intervention. This study demonstrates that VEGF-C overexpression by fibroblasts stimulates multiple biologic processes known to impact wound healing, such as collagen constriction, capillary sprouting, and EPC invasion and migration through extracellular matrix. Most ischemic wounds fail to heal and frequently lead to major limb amputation. Available cytokine ointments are ineffective, and revascularization is often not technically feasible. Even when these procedures are accomplished, many ischemic wounds frequently still do not heal because of multifactorial tissue level impairments in the fibroblastic and neovascularization responses at the wound base. Our findings identify an important role for two novel tissue level targets, dermis-derived fibroblasts and VEGF-C, in collagen constriction, angiogenesis, and postnatal vasculogenesis from BMD EPCs. Thus the findings are particularly relevant to the unsolved clinical problem of ischemic wound healing.

Stability of meropenem in polyvinyl chloride bags and an elastomeric infusion device.

PURPOSE: The stability of meropenem in i.v. solutions stored in polyvinyl chloride (PVC) bags and an elastomeric infusion device at concentrations commonly used in home care was studied. METHODS: Vials of meropenem were reconstituted with sterile water for injection and mixed with 0.9% sodium chloride injection (NS) to yield concentrations of 4, 10, and 20 mg/mL. Six replicate solutions were prepared in PVC containers and six in the Homepump ECLIPSE elastomeric infusion device. All solutions were stored at an average temperature of 5 degrees C and sampled immediately after preparation and at intervals up to 120 hours (five days); the 4-mg/mL solution was also sampled at 144 and 168 hours (seven days). Samples were assayed for meropenem concentration by stability-indicating high-performance liquid chromatography. RESULTS: All solutions of meropenem retained over 90% of the initial drug concentration at five days. The 4-mg/mL solutions retained over 93% of the initial concentration at seven days. The rate of meropenem decay did not differ significantly between PVC and elastomeric infusion containers for the 4- and 20-mg/mL solutions; however, there was a difference for the 10-mg/mL solutions. CONCLUSION: Meropenem 4 mg/mL in NS was stable for at least seven days in PVC bags and elastomeric infusion containers when stored at 5 degrees C, and meropenem 10 and 20 mg/mL in NS was stable for at least five days in both containers at 5 degrees C.