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Altered mechanotransduction has been proposed as a putative mechanism for disease pathophysiology, yet evidence remains scarce. Here we introduce a concept we call single cell immuno-mechanical modulation, which links immunology, integrin biology, cellular mechanics, and disease pathophysiology and symptomology. Using a micropatterned hydrogel-laden coverslip compatible with standard fluorescence microscopy, we conduct a clinical mechanobiology study, specifically focusing on immune thrombocytopenia (ITP), an autoantibody-mediated platelet disorder that currently lacks a reliable biomarker for bleeding risk. We discover that in pediatric ITP patients (n = 53), low single platelet contraction force alone is a "physics-based" biomarker of bleeding (92.3% sensitivity, 90% specificity). Mechanistically, autoantibodies and monoclonal antibodies drive increases and decreases of cell force by stabilizing integrins in different conformations depending on the targeted epitope. Hence, immuno-mechanical modulation demonstrates how antibodies may pathologically alter mechanotransduction to cause clinical symptoms and this phenomenon can be leveraged to control cellular mechanics for research, diagnostic, and therapeutic purposes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Abstract: Sickle cell disease (SCD) is canonically characterized by reduced red blood cell (RBC) deformability, leading to microvascular obstruction and inflammation. Although the biophysical properties of sickle RBCs are known to influence SCD vasculopathy, the contribution of poor RBC deformability to endothelial dysfunction has yet to be fully explored. Leveraging interrelated in vitro and in silico approaches, we introduce a new paradigm of SCD vasculopathy in which poorly deformable sickle RBCs directly cause endothelial dysfunction via mechanotransduction, during which endothelial cells sense and pathophysiologically respond to aberrant physical forces independently of microvascular obstruction, adhesion, or hemolysis. We demonstrate that perfusion of sickle RBCs or pharmacologically-dehydrated healthy RBCs into small venule-sized "endothelialized" microfluidics leads to pathologic physical interactions with endothelial cells that directly induce inflammatory pathways. Using a combination of computational simulations and large venule-sized endothelialized microfluidics, we observed that perfusion of heterogeneous sickle RBC subpopulations with varying deformability, as well as suspensions of dehydrated normal RBCs admixed with normal RBCs, leads to aberrant margination of the less-deformable RBC subpopulations toward the vessel walls, causing localized, increased shear stress. Increased wall stress is dependent on the degree of subpopulation heterogeneity and oxygen tension and leads to inflammatory endothelial gene expression via mechanotransductive pathways. Our multifaceted approach demonstrates that the presence of sickle RBCs with reduced deformability leads directly to pathological physical (ie, direct collisions and/or compressive forces) and shear-mediated interactions with endothelial cells and induces an inflammatory response, thereby elucidating the ubiquity of vascular dysfunction in SCD., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The progress of research focused on cholangiocytes and the biliary tree during development and following injury is hindered by limited available quantitative methodologies. Current techniques include two-dimensional standard histological cell-counting approaches, which are rapidly performed, error prone, and lack architectural context or three-dimensional analysis of the biliary tree in opacified livers, which introduce technical issues along with minimal quantitation. The present study aims to fill these quantitative gaps with a supervised machine-learning model (BiliQML) able to quantify biliary forms in the liver of anti-keratin 19 antibody-stained whole slide images. Training utilized 5,019 researcher-labeled biliary forms, which following feature selection, and algorithm optimization, generated an F score of 0.87. Application of BiliQML on seven separate cholangiopathy models [genetic ( Afp -CRE; Pkd1l1 null/Fl , Alb- CRE; Rbp-jk fl/fl , and Albumin- CRE; ROSA NICD ), surgical (bile duct ligation), toxicological (3,5-diethoxycarbonyl-1,4-dihydrocollidine), and therapeutic ( Cyp2c70 -/- with ileal bile acid transporter inhibition)] allowed for a means to validate the capabilities and utility of this platform. The results from BiliQML quantification revealed biological and pathological differences across these seven diverse models, indicating a highly sensitive, robust, and scalable methodology for the quantification of distinct biliary forms. BiliQML is the first comprehensive machine-learning platform for biliary form analysis, adding much-needed morphologic context to standard immunofluorescence-based histology, and provides clinical and basic science researchers with a novel tool for the characterization of cholangiopathies. NEW & NOTEWORTHY BiliQML is the first comprehensive machine-learning platform for biliary form analysis in whole slide histopathological images. This platform provides clinical and basic science researchers with a novel tool for the improved quantification and characterization of biliary tract disorders.

While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods., (© 2023. Springer Nature Limited.)

Integrating microfluidic mixers into lab-on-a-chip devices remains challenging yet important for numerous applications including dilutions, extractions, addition of reagents or drugs, and particle synthesis. High-efficiency mixers utilize large or intricate geometries that are difficult to manufacture and co-implement with lab-on-a-chip processes, leading to cumbersome two-chip solutions. We present a universal dry-film microfluidic mixing sticker that can retrofit pre-existing microfluidics and maintain high mixing performance over a range of Reynolds numbers and input mixing ratios. To attach our pre-mixing sticker module, remove the backing material and press the sticker onto an existing microfluidic/substrate. Our innovation centers around the multilayer use of laser-cut commercially available silicone-adhesive-coated polymer sheets as microfluidic layers to create geometrically complex, easy to assemble designs that can be adhered to a variety of surfaces, namely, existing microfluidic devices. Our approach enabled us to assemble the traditional yet difficult to manufacture "F-mixer" in minutes and conceptually extend this design to create a novel space-saving spiral F-mixer. Computational fluid dynamic simulations and experimental results confirmed that both designs maintained high performance for 0.1 < Re < 10 and disparate input mixing ratios of 1:10. We tested the integration of our system by using the pre-mixer to fluorescently tag proteins encapsulated in an existing microfluidic. When integrated with another microfluidic, our pre-mixing sticker successfully combined primary and secondary antibodies to fluorescently tag micropatterned proteins with high spatial uniformity, unlike a traditional pre-mixing "T-mixer" sticker. Given the ease of this technology, we anticipate numerous applications for point-of-care devices, microphysiological-systems-on-a-chip, and microfluidic-based biomedical research., (© 2023 Author(s).)

The correlation between cardiovascular disease and iron deficiency anemia (IDA) is well documented but poorly understood. Using a multi-disciplinary approach, we explore the hypothesis that the biophysical alterations of red blood cells (RBCs) in IDA, such as variable degrees of microcytosis and decreased deformability may directly induce endothelial dysfunction via mechanobiological mechanisms. Using a combination of atomic force microscopy and microfluidics, we observed that subpopulations of IDA RBCs (idRBCs) are significantly stiffer and smaller than both healthy RBCs and the remaining idRBC population. Furthermore, computational simulations demonstrated that the smaller and stiffer idRBC subpopulations marginate toward the vessel wall causing aberrant shear stresses. This leads to increased vascular inflammation as confirmed with perfusion of idRBCs into our "endothelialized" microfluidic systems. Overall, our multifaceted approach demonstrates that the altered biophysical properties of idRBCs directly lead to vasculopathy, suggesting that the IDA and cardiovascular disease association extends beyond correlation and into causation., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Neutrophil extracellular traps (NETs) is an antimicrobial cobweb-structured material produced by immune cells for clearance of pathogens in the body, but paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs-like material (called 'microwebs', abbreviated as μwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection-relevant conditions. We show that μwebs composed of low-to-intermediate concentrations of DNA-histone complexes successfully trap and inhibit S. aureus growth and biofilm formation. However, with growing concentrations and histone proportions, the resulting microwebs appear gel-like structures accompanied by reduced antimicrobial activity that can even promote formation of S. aureus biofilms. Our simplified model of NETs provides a materials-based evidence on NETs-relevant pathology in the development of biofilms., Competing Interests: Conflict of Interest The authors declare no competing financial interest.

Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration, which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here, we show that deletion of Ptpn21, a protein tyrosine phosphatase highly expressed in HSCs, induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21 -/- HSCs exhibit enhanced mobility, decreased quiescence, increased apoptosis, and defective reconstitution capacity. Ptpn21 deletion also decreased HSC stiffness and increased physical deformability, in part by dephosphorylating Spetin1 (Tyr 246 ), a poorly described component of the cytoskeleton. Elevated phosphorylation of Spetin1 in Ptpn21 -/- cells impaired cytoskeletal remodeling, contributed to cortical instability, and decreased cell rigidity. Collectively, these findings show that Ptpn21 maintains cellular mechanics, which is correlated with its important functions in HSC niche retention and preservation of hematopoietic regeneration capacity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction Patient care in the trauma-surgical intensive care unit (SICU) requires trust and effective communication between nurses and physicians. Our SICU suffered from poor communication and trust between nurses and physicians, negatively impacting the working environment and, potentially, patient care. Methods A SICU Task Force studied communication practices and identified areas for improvement, leading to several interventions. The daily physician rounding was altered to improve communication and to enhance the role of the registered nurses (RN) in rounds. Additionally, a formal night resident rounding time was implemented. Results A post-intervention survey focusing on cooperation, teamwork, and appreciation between nurses and physicians revealed improvement in these domains. Informal feedback from nurses and physicians indicated improved working relationships and satisfaction with the SICU environment. However, results of a national survey performed after the intervention did not show the same level of improvement. Conclusions A Task Force consisting of SICU nurses and physicians can effectively study a widespread communication issue and implement targeted interventions. While informal feedback may indicate improvement, it can be difficult to demonstrate improvement using formal surveys., Competing Interests: The authors have declared that no competing interests exist.

Ultraviolet (UV) spectroscopy is a powerful tool for quantitative (bio)chemical analysis, but its application to molecular imaging and microscopy has been limited. Here we introduce ultraviolet hyperspectral interferometric (UHI) microscopy, which leverages coherent detection of optical fields to overcome significant challenges associated with UV spectroscopy when applied to molecular imaging. We demonstrate that this method enables quantitative spectral analysis of important endogenous biomolecules with subcellular spatial resolution and sensitivity to nanometer-scaled structures for label-free molecular imaging of live cells.

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an "endothelialized" microfluidic system coupled with a microengineered pneumatic valve that induces a vascular "injury". With perfusion of whole blood, hemostatic plug formation is visualized and "in vitro bleeding time" is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.

Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD., (© 2017 by The American Society of Hematology.)

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model., (© 2015 by The American Society of Hematology.)

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium., (© 2015 by The American Society of Hematology.)

Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro- to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro- to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro., (© 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: Contemporary warfare involving civilian populations is a growing public health concern. In addition to the psychological impact, war-related trauma may result in physiological alterations and even broader health effects. Associations were examined between war-related stressors and incident asthma in elderly Kuwaiti civilians following the Iraqi invasion., Methods: A random sample of all Kuwaiti nationals aged 50-69 years on the day prior to the invasion were identified. Among the 7873 meeting eligibility criteria, 5567 (71%) agreed to participate and 5028 completed the questionnaire (91% of those eligible). Of these, 3759 were in Kuwait during the invasion, of whom 2294 were alive at follow-up. After exclusions for prevalent asthma or missingness on covariates, 2066 were available for analysis. War-related experiences were summarised into a continuous score using Rasch modelling. Relative Cox proportional hazard rates (HR) were calculated for asthma adjusting for covariates., Results: Over 13 years of follow-up, physician-diagnosed asthma was reported by 66/996 (6.6%) men and 104/1070 (9.7%) women. In models adjusted for gender, socioeconomic status, smoking, BMI, and air pollution related to burning oil fires, those reporting highest stress exposure were more than twice as likely to report asthma (HR 2.3, 95% CI 1.3, 3.9) compared to civilians reporting no stressors. Experiences were more salient when anchored to fear for loss of life., Conclusions: War-related trauma is associated with increased asthma risk in these elderly civilians. Although prior research has documented the significant and persistent psychological toll of war, these findings implicate even broader health effects.

Background: We investigated whether lack of perceived neighborhood safety due to crime, or living in high crime neighborhoods was associated with incident mobility disability in elderly populations. We hypothesized that low-income elders and elders at retirement age (65 - 74) would be at greatest risk of mobility disability onset in the face of perceived or measured crime-related safety hazards., Methods: We conducted the study in the New Haven Established Populations for Epidemiologic Studies of the Elderly (EPESE), a longitudinal cohort study of community-dwelling elders aged 65 and older who were residents of New Haven, Connecticut in 1982. Elders were interviewed beginning in 1982 to assess mobility (ability to climb stairs and walk a half mile), perceptions of their neighborhood safety due to crime, annual household income, lifestyle characteristics (smoking, alcohol use, physical activity), and the presence of chronic co-morbid conditions. Additionally, we collected baseline data on neighborhood crime events from the New Haven Register newspaper in 1982 to measure local area crime rates at the census tract level., Results: At baseline in 1982, 1,884 elders were without mobility disability. After 8 years of follow-up, perceiving safety hazards was associated with increased risk of mobility disability among elders at retirement age whose incomes were below the federal poverty line (HR 1.56, 95% CI 1.02 - 2.37). No effect of perceived safety hazards was found among elders at retirement age whose incomes were above the poverty line. No effect of living in neighborhoods with high crime rates (measured by newspaper reports) was found in any sub-group., Conclusion: Perceiving a safety hazard due to neighborhood crime was associated with increased risk of incident mobility disability among impoverished elders near retirement age. Consistent with prior literature, retirement age appears to be a vulnerable period with respect to the effect of neighborhood conditions on elder health. Community violence prevention activities should address perceived safety among vulnerable populations, such as low-income elders at retirement age, to reduce future risks of mobility disability.

Background/aims: Little is known about the possible effects of social resources on stroke survivors' level and change in cognitive outcomes. Understanding this association may help us identify strategies to improve stroke recovery and help elucidate the etiology of dementia., Methods: We examined the relationship of social ties and social support to cognitive function and cognitive change 6 months after stroke. Participants in the Families in Recovery from Stroke Trial (FIRST) (n = 272) were interviewed approximately 17 days (baseline) and 6 months (follow-up) after stroke. Cognition was assessed with the Mini Mental State Examination (MMSE) and a summary battery of 7 neuropsychological tests. Median-based regression was used to model cognitive outcomes by level of baseline intimate, personal and organizational social ties and received emotional and instrumental support., Results: Baseline social ties and emotional support independently predicted 6-month Cognitive Summary Scores. Emotional support also predicted greater improvements in Cognitive Summary Scores from baseline to the 6-month follow-up. No other social exposures predicted improvements in the MMSE or the Cognitive Summary., Conclusions: Our results suggest that emotional support may promote cognitive resilience while social ties provide cognitive reserve that protects against impaired cognition after stroke. Social ties did not predict cognitive recovery however, so reverse causation cannot be ruled out., ((c) 2008 S. Karger AG, Basel.)

Purpose: To examine the relationship between intervention dose and health behavior change in Healthy Directions-Health Centers, an intervention designed to reduce cancer risk factors. DESIGN. Analysis of intervention condition participant data from a randomized controlled trial., Setting: Community health centers in Massachusetts., Subjects: Patients residing in low-income, working-class, multiethnic neighborhoods., Intervention: Components were clinician endorsement, in-person counseling session and four telephone counseling sessions with a trained health advisor, and social-contextual tailored materials., Measures: Intervention dose was number of six possible intervention components completed by each participant. Changes in fruit and vegetable consumption, red meat consumption, physical activity, and multivitamin intake between baseline (n=1088) and 8-month follow-up (n=967; 89% of baseline sample) were determined., Analysis: Bivariate and multivariate associations between intervention dose and change in health behaviors were examined., Results: In multivariate analysis, the association between intervention dose and increase in multivitamin intake approached significance (p < .07). Seventy percent of participants completed all intervention activities. In bivariate analysis, completion of four telephone counseling calls was associated with decrease in red meat consumption (p < .05)., Conclusion: These findings indicate that future studies should examine the number, content, and length of contacts needed for behavior change. The results also suggest that health centers are a channel for reaching diverse populations, as shown by the high level of intervention implementation.

Introduction: Intervention studies have been designed to change dietary and lifestyle factors associated with chronic diseases, but self-reported behavior change may incorporate intervention-related bias. This study examines plasma nutrient concentration and correlations with self-reports in the Healthy Directions intervention study. The Healthy Directions intervention studies were designed to increase multivitamin use, fruit and vegetable consumption, and physical activity in working-class, multiethnic populations., Methods: Participants in both studies completed interviewer-administered questionnaires that collected information on sociodemographic and health behavior characteristics. Postintervention blood samples were collected from 209 participants and pooled in pairs within study and within intervention group., Results: We found significantly higher plasma concentrations of retinol (P = .01) and alpha-carotene (P = .03) in the intervention than in the usual care group. Self-reported multivitamin users had significantly higher concentrations of retinol (P < .001), beta-carotene (P = .02), and alpha-tocopherol (P < .001). Those who reported four or more fruit and vegetable servings per day had higher lutein and zeaxanthin (P = .05) and beta-cryptoxanthin (P = .05) concentrations than those consuming fewer. Plasma nutrient concentrations were associated with reported multivitamin use and fruit and vegetable intake, but the correlations were generally higher in the usual care group., Conclusion: We found significant postintervention differences in plasma carotenoid and tocopherol concentrations by treatment group, multivitamin use, and fruit and vegetable intake. However, because we only obtained postintervention blood samples, we were unable to assess preintervention-to-postintervention changes in plasma nutrients. Self-reported intakes were significantly correlated with plasma nutrient concentrations, but the strength of the correlations differed by group, suggesting some intervention-related bias in the questionnaire responses.

Introduction: Few papers address the methodological challenges in recruiting participants for studies of cancer prevention interventions designed for multiracial and multiethnic working-class populations. This paper reports the results of the sample selection and survey methods for two group-randomized intervention studies., Methods: The two group-randomized intervention studies, Healthy Directions-Small Business (HD-SB) and Healthy Directions-Health Centers (HD-HC), included a worksite-based study in 26 small manufacturing businesses and a study in 10 outpatient health centers. We used selection and recruitment methods to obtain a multiracial and multiethnic working-class study sample. In 2000 and 2001, we assessed baseline measures of sociodemographic characteristics and behavioral outcomes by self-report. We then computed intraclass correlation coefficients (ICCs)., Results: Of the 1740 participants in the HD-SB study, 68% were non-Hispanic whites, and 76% had working-class occupations. In the HD-HC study, 59% of 2219 participants were non-Hispanic whites. Among those who worked, 51% had working-class occupations. Large percentages of both samples reported not meeting recommended guidelines for the target behaviors. For example, 86% of members of both samples consumed fewer than the recommended five servings of fruits and vegetables per day. The ICCs for the four target behaviors in HD-SB were between 0.006 and 0.02. In the HD-HC study, the ICCs ranged from 0.0004 to 0.003., Conclusion: The two studies were successful in recruiting multiracial and multiethnic working-class participants. Researchers will find the estimates of the primary outcomes and their ICCs useful for planning future studies.

To understand whether neighborhood contexts contribute to the onset or maintenance of mental health problems independently of individual characteristics requires the use of multilevel study designs and analytical strategies. This study used a multilevel analytical framework to examine the relation between neighborhood context and risk of depressive symptoms, using data from the New Haven component of the Established Populations for Epidemiologic Studies of the Elderly, a community-based sample of noninstitutionalized men and women aged 65 years or older and living in the city of New Haven, Connecticut, in 1982. Neighborhoods were characterized by census-based characteristics and also by measures of the neighborhood service environment using data abstracted from the New Haven telephone book Yellow Pages. Living in a poor neighborhood was associated with higher levels of depressive symptoms in older adults, above and beyond individual vulnerabilities. In addition, the presence of more elderly people in the neighborhood was associated with better mental health among older adults. The authors found no evidence that access to services hypothesized to promote social engagement, to provide health services, or to affect the reputation of a neighborhood explained (i.e., mediated) neighborhood variations in depressive symptoms.

Objective: Some children in the United States continue to be exposed to levels of lead that increase their risk for lowered intellectual functioning and behavior problems. It is unclear whether chelation therapy can prevent or reverse the neurodevelopmental sequelae of lead toxicity. The objective of this study was to determine whether chelation therapy with succimer (dimercaptosuccinic acid) in children with referral blood lead levels between 20 and 44 microg/dL (0.96-2.12 micromol/L) at 12 to 33 months of age has neurodevelopmental benefits at age 7 years., Methods: The Treatment of Lead-Exposed Children (TLC) study is a randomized, double-blind, placebo-controlled trial that was conducted between September 1994 and June 2003 in Philadelphia, PA; Newark, NJ; Cincinnati, OH; and Baltimore, MD. Of 1854 referred children who were between the ages of 12 to 33 months and screened for eligibility, 780 were randomized to the active drug and placebo groups stratified by clinical center, body surface area, blood lead level, and language spoken at home. At 7 years of age, 647 subjects remained in the study. Participants were randomly assigned to receive oral succimer or placebo. Up to 3 26-day courses of succimer or placebo therapy were administered depending on response to treatment in those who were given active drug. Eighty-nine percent had finished treatment by 6 months, with all children finishing by 13 months after randomization. All participants received residential lead hazard control measures before treatment. TLC subjects also received a daily multivitamin supplement before and after treatment(s) with succimer or placebo. Scores on standardized neuropsychological measures that tap cognition, behavior, learning and memory, attention, and neuromotor skills were measured., Results: Chelation therapy with succimer lowered average blood lead levels for approximately 6 months but resulted in no benefit in cognitive, behavioral, and neuromotor endpoints., Conclusion: These new follow-up data confirm our previous finding that the TLC regimen of chelation therapy is not associated with neurodevelopmental benefits in children with blood lead levels between 20 and 44 microg/dL (0.96-2.17 micromol/L). These results emphasize the importance of taking environmental measures to prevent exposure to lead. Chelation therapy with succimer cannot be recommended for children with blood lead levels between 20 and 44 microg/dL (0.96-2.12 micromol/L).

Background: This paper describes the implementation of the Healthy Directions-Health Centers intervention and examines the characteristics of participants associated with completion of intervention activities. Healthy Directions-Health Centers was designed to address social contextual factors relevant to cancer prevention interventions for working class, multi-ethnic populations., Methods: Ten community health centers were paired and randomly assigned to intervention or control. Patients who resided in low income, multi-ethnic neighborhoods were approached for participation. This study targeted fruit and vegetable consumption, red meat consumption, multi-vitamin intake, and physical activity. The intervention components consisted of: (1) a brief study endorsement from a clinician; (2) an in-person counseling session with a health advisor; (3) four follow-up telephone counseling sessions; and (4) multiple distributions of tailored materials., Results: Among the 1,088 intervention group participants, 978 participants (90%) completed at least five out of six intervention activities. Participants who missed clinical appointments were less likely to complete all components of the intervention. Participant characteristics that predicted receipt of clinician endorsement differed from characteristics that predicted completion of health advisor activities. Low acculturation did not present a barrier to delivery of the intervention once the participant was enrolled., Conclusions: Collection and reporting on process evaluation results can help explain variations in program implementation.

New York 1 virus (NY-1) and Sin Nombre virus (SN) are associated with hantavirus pulmonary syndrome (HPS). NY-1 and SN are derived from unique mammalian hosts and geographic locations but have similar G1 and G2 surface proteins (93 and 97% identical, respectively). Focus reduction neutralization assays were used to define the serotypic relationship between NY-1 and SN. Sera from NY-1-positive Peromyscus leucopus neutralized NY-1 and SN at titers of >/=1/3,200 and 16-fold-lower neutralizing titers to NY-1 than to SN. Reference sera to Hantaan, Seoul, and Prospect Hill viruses also failed to neutralize NY-1. These results indicate that SN and NY-1 define unique hantavirus serotypes and implicate the presence of additional HPS-associated hantavirus serotypes in the Americas.

Gastrin plays an important role in regulating gastric acid secretion and gastrointestinal mucosal growth but its cellular sites of action in man have not been determined. Using cryostat sections of gastric mucosal tissue we have identified (125I-gastrin binding followed by fixation-wet emulsion autoradiography) and characterized (125I-gastrin binding followed by counting) a gastrin receptor binding site in the human stomach. This site displayed binding characteristics similar to those observed in isolated cell systems: specifically, 125I-gastrin binding was rapid (t1/2 approximately 10 min at 37 degrees C), temperature-dependent (3.5 fold more radioligand bound at 22 degrees C than at 4 degrees C) and saturable. The binding of the radioligand was also tissue specific and was five-fold greater in the gastric body than in the gastric antrum and duodenum. In the autoradiographs, silver grains were localized only to parietal cells and not to other epithelial cell types. In the presence of 40 nM gastrin grains were no longer present over parietal cells demonstrating that these sites were both saturable and of high affinity. These data provide the first demonstration of gastrin binding sites (putative receptors) on parietal cells in the human stomach and suggest that gastrin acts directly on these cells to help regulate gastric acid secretion and/or mucosal growth.

Adult diarrheal rotavirus, ADRV, is a noncultivable human group B rotavirus. A complete cDNA copy of ADRV gene segment 2 has been cloned and sequenced. Gene segment 2 contains 2844 bases and encodes one long open reading frame beginning at base 14 and terminating at base 2812. Gene 2 encodes a protein containing 933 amino acids with a calculated molecular weight of 105.6 kDa and a pl of 5.5. The gene 2 polypeptide contains significant homology with the VP2 protein which comprises the core of group A rotavirus strains. The gene 2 protein has been expressed in a rabbit reticulocyte lysate in vitro and is identical in molecular mass with a protein previously demonstrated to be present on iodinated EDTA-treated virions and from in vitro translations of total ADRV mRNA. A recombinant baculovirus containing gene segment 2 has been constructed and used to express the encoded VP2 equivalent protein. The expressed VP2 protein is immunoprecipitable by hyperimmune anti-ADRV serum, porcine group B infection serum, and human convalescent serum but not by hyperimmune serum to group A rotavirus. Our results suggest that ADRV gene segment 2 encodes the VP2 protein equivalent to group A rotavirus strains present in the core of the group B virion.

Background: Recent studies have reported associations between particulate air pollution and daily mortality rates. Population-based, cross-sectional studies of metropolitan areas in the United States have also found associations between particulate air pollution and annual mortality rates, but these studies have been criticized, in part because they did not directly control for cigarette smoking and other health risks., Methods: In this prospective cohort study, we estimated the effects of air pollution on mortality, while controlling for individual risk factors. Survival analysis, including Cox proportional-hazards regression modeling, was conducted with data from a 14-to-16-year mortality follow-up of 8111 adults in six U.S. cities., Results: Mortality rates were most strongly associated with cigarette smoking. After adjusting for smoking and other risk factors, we observed statistically significant and robust associations between air pollution and mortality. The adjusted mortality-rate ratio for the most polluted of the cities as compared with the least polluted was 1.26 (95 percent confidence interval, 1.08 to 1.47). Air pollution was positively associated with death from lung cancer and cardiopulmonary disease but not with death from other causes considered together. Mortality was most strongly associated with air pollution with fine particulates, including sulfates., Conclusions: Although the effects of other, unmeasured risk factors cannot be excluded with certainty, these results suggest that fine-particulate air pollution, or a more complex pollution mixture associated with fine particulate matter, contributes to excess mortality in certain U.S. cities.

A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization.

Adult diarrheal rotavirus (ADRV) is a currently noncultivatable group B human rotavirus responsible for epidemic outbreaks of gastroenteritis in China. Gene segment 5 of ADRV encodes the major inner capsid protein, VP6. ADRV gene 5 was inserted into a recombinant baculovirus by homologous recombination between baculovirus shuttle plasmid pACYM1-AD5 and AcNPV genomic DNA. Baculovirus recombinants were selected visually and plaque purified and VP6 expression was detected by Coomassie staining of PAGE-separated proteins. The baculovirus-expressed gene 5 polypeptide is 44 kDa, the same as for the major inner capsid protein present on EDTA-treated ADRV virions and in vitro-expressed VP6 protein. The expressed protein is oligomeric and in the absence of reducing agents multimerizes to apparent trimer, hexamer, and greater molecular mass as assayed by SDS-PAGE. The VP6 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum, by a group B-specific monoclonal antibody and by porcine group B rotavirus infection serum. The baculovirus-expressed protein is immunogenic and antibodies to the expressed protein recognize ADRV virions. The ADRV VP6 protein should be useful for developing diagnostic assays for serum antibodies to group B rotavirus as well as for generating hyperimmune serum and monoclonal antibodies for detecting viral antigen from ADRV and other group B rotaviruses.

Pulmonary function of children aged 6-18 years is described based on 82,462 annual measurements of forced vital capacity (FVC), forced expired volume in 1 second (FEV1), and forced expiratory flow between 25% and 75% of FVC (FEF25-75%) from 11,630 white children and 989 black children. Median height, FVC, FEV1, FEV1/FVC, and FEF25-75% for each 3 months of age are compared among race and sex subgroups. Race- and sex-specific percentile distributions of FVC, FEV1, FEV1/FVC, and FEF25-75% are presented for each centimeter of height (growth curves). For the same height, boys have greater lung function values than girls, and whites have greater ones than blacks. Lung function increases linearly with age until the adolescent growth spurt at about age 10 years in girls and 12 in boys. The pulmonary function vs. height relationship shifts with age during adolescence. Thus, a single equation or the pulmonary function-height growth chart alone does not completely describe growth during the complex adolescent period. Nevertheless, race- and sex-specific growth curves of pulmonary function vs. height make it easy to display and evaluate repeated measures of pulmonary function for an individual child. Race-, sex-, and age-specific regression equations based on height are provided, which permit the evaluation of growth during adolescence with improved accuracy and, more importantly, in comparison with previous observations for the same child.

The relationship between 6 chronic respiratory symptoms and the performance of an excessively variable FEV1 (test failure) was examined among 8,522 white adults in 6 U.S. cities. A total of 747 (8.9%) performed an excessively variable FEV1 according to the American Thoracic Society criterion. After adjusting for smoking, age, and city of residence in 6 separate logistic regression models, the odds ratios for FEV1 failure among men were 2.32, 1.39, 1.40, 1.82, 2.61, 1.92 for moderate breathlessness, chronic cough, phlegm, wheeze, asthma, and recurrent chest illness, respectively. Among women, FEV1 failure was significantly associated with moderate breathlessness, chronic phlegm, wheeze, and asthma with odds ratios of 1.55, 1.45, 1.62, and 1.95, respectively. When all symptoms were evaluated simultaneously in a single logistic regression model, only breathlessness and asthma remained associated with FEV1 failure; odds ratio = 1.97 for asthma and 2.03 for breathlessness among men and 1.53 for both asthma and breathlessness among women. The 11-yr mortality experience of subjects with test failure, as defined by 2 different criteria, was compared to that of the quartile of the cohort with the highest cross-sectional test results. After adjusting for age, gender, and smoking, the relative risks of mortality were 1.62 and 1.98 for subjects with an FEV1 failure as defined by the ATS and 6-Cities criteria, respectively, and 1.99 and 1.90 for the groups with FVC failure as defined by the 2 criteria. Thus test failure is almost as strong a predictor of mortality as poor FEV1.

As part of a longitudinal study of the respiratory health effects of air pollution, we measured the lung function of 2,454 white adults 25 to 74 yr of age who had never smoked and who reported no respiratory symptoms. These measurements were analyzed to develop a simple model for the cross-sectional dependence of pulmonary function on height, sex, and age. Both forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) can be effectively standardized for body size by dividing each pulmonary function measurement by the square of the standing height (HT2). The age-specific distribution of these standardized measurements is approximately Gaussian, with variance that is independent of age. Plots of FEV1/HT2 and FVC/HT2 against age showed a nonlinear relationship consistent with an increase in the rate of pulmonary function loss with age. On the basis of these graphic analyses, both pulmonary function measurements were fitted to a four-parameter normative model including sex and linear and quadratic terms in age as dependent variables. This model gave predictions that were very close to those from more complicated models currently in use. Predicted percentile levels were calculated for each sex and age, and shown to describe the observations well. The estimated annual change in height-standardized lung function based on the cross-sectional model was compared with the observed change between the first and second examinations of these adults 3 yr later. The observed changes were close to predicted values, except for subjects younger than 35 yr of age at their first examination. The observed change was larger for men than for women. Such simple longitudinal comparisons are subject to selection bias. In this study, subjects in the lowest quartile of FEV1/HT2 for their age and sex at the first examination had a lower probability of providing a lung function measurement 3 yr later.

Results are presented from a 9- to 12-yr mortality follow-up of 8,427 white adults in six U.S. cities between 25 and 74 yr of age at enrollment. Survival analyses were performed for all causes on 941 confirmed deaths, and for specific primary causes for the subset of 851 death with death certificates. Relative level of FEV1 compared with predicted was a strong predictor of sex-specific chronic obstructive pulmonary disease (COPD), cardiovascular (CV), and all-cause mortality, even after adjusting for age, respiratory symptoms, and smoking. Even in this relatively large cohort, the total number of respiratory deaths was small, and no trend in COPD mortality could be determined over the period of study.