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Data from the Head Start Impact Study (N = 3,185, age = 3-4 years) were used to determine whether 1 year of Head Start differentially benefited children from homes with high, middle, and low levels of parental preacademic stimulation on three academic outcome domains-early math, early literacy, and receptive vocabulary. Results from residualized growth models showed positive impacts of random assignment to Head Start on all three outcomes, and positive associations between parental preacademic stimulation and academic performance. Two moderated effects were also found. Head start boosted early math skills the most for children receiving low parental preacademic stimulation. Effects of Head Start on early literacy skills were largest for children receiving moderate levels of parental preacademic stimulation. Implications for Head Start are discussed. © 2014 Society for Research in Child Development, Inc.

Introduction: Children and adolescents with elevated internalizing symptoms are at increased risk for depression, anxiety, and other psychopathology later in life. The present study examined the predictive links between two bioecological factors in early childhood-parental hostility and socioeconomic stress-and children's internalizing symptom class outcomes, while considering the effects of child sex assigned at birth on internalizing symptom development from childhood to adolescence., Materials and Methods: The study used a sample of 1,534 children to test the predictive effects of socioeconomic stress at ages 18 and 27 months; hostile parenting measured at child ages 4-5; and sex assigned at birth on children's internalizing symptom latent class outcomes at child ages 7-9, 10-12, 13-15, and 16-19. Analyses also tested the mediating effect of parenting on the relationship between socioeconomic stress and children's symptom classes. Other covariates included parent depressive symptoms at child ages 4-5 and child race and ethnicity., Results: Analyses identified three distinct heterogenous internalizing symptom classes characterized by relative symptom levels and progression: low (35%); moderate and increasing (41%); and higher and increasing (24%). As anticipated, higher levels of parental hostility in early childhood predicted membership in the higher and increasing symptom class, compared with the low symptom class (odds ratio (OR) = .61, 95% confidence interval (CI) [.48,.77]). Higher levels of early childhood socioeconomic stress were also associated with the likelihood of belonging to the higher-increasing symptom class compared to the low and moderate-increasing classes (OR = .46, 95% CI [.35,.60] and OR = .56, 95% CI [.44,.72], respectively). The total ( c = .61) and direct ( c' = .57) effects of socioeconomic stress on children's symptom class membership in the mediation analysis were significant ( p <.001)., Discussion: Study findings suggest that intervening on modifiable bioecological stressors-including parenting behaviors and socioeconomic stressors-may provide important protective influences on children's internalizing symptom trajectories., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Williams, Oro, Blackwell, Liu, Miller, Ganiban, Neiderhiser, DeGarmo, Shaw, Chen, Natsuaki and Leve.)

Introduction: As paediatric intensive care unit (PICU) mortality declines, there is growing recognition of the morbidity experienced by children surviving critical illness and their families. A comprehensive understanding of the adverse physical, cognitive, emotional and social sequelae common to PICU survivors is limited, however, and the trajectory of recovery and risk factors for morbidity remain unknown., Methods and Analysis: The Post-Intensive Care Syndrome - paediatrics Longitudinal Cohort Study will evaluate child and family outcomes over 2 years following PICU discharge and identify child and clinical factors associated with impaired outcomes. We will enrol 750 children from 30 US PICUs during their first PICU hospitalisation, including 500 case participants experiencing ≥3 days of intensive care that include critical care therapies (eg, mechanical ventilation, vasoactive infusions) and 250 age-matched, sex-matched and medical complexity-matched control participants experiencing a single night in the PICU with no intensive care therapies. Children, parents and siblings will complete surveys about health-related quality of life, physical function, cognitive status, emotional health and peer and family relationships at multiple time points from baseline recall through 2 years post-PICU discharge. We will compare outcomes and recovery trajectories of case participants to control participants, identify risk factors associated with poor outcomes and determine the emotional and social health consequences of paediatric critical illness on parents and siblings., Ethics and Dissemination: This study has received ethical approval from the University of Pennsylvania Institutional Review Board (protocol #843844). Our overall objective is to characterise the ongoing impact of paediatric critical illness to guide development of interventions that optimise outcomes among children surviving critical illness and their families. Findings will be presented at key disciplinary meetings and in peer-reviewed publications at fixed data points. Published manuscripts will be added to our public study website to ensure findings are available to families, clinicians and researchers., Trials Registration Number: NCT04967365., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Reaching population-level impact for families in poverty requires moving beyond a sole focus on individuals, to a wider focus on interactions between individuals and their broader environmental contexts. Place-based initiatives have emerged as a policy response to promote community-level change around these broader interactions between individuals and their local communities through addressing long-standing disparities in housing, employment, education, and health. Together Growing Strong (TGS) is one such place-based initiative focused on transforming the health, wellbeing, and development of young children and their families in Sunset Park, Brooklyn. The Children, Caregivers, and Community (C3) Study is an outcomes-based study designed to assess the trajectories of children and families in Sunset Park along indicators such as family health and wellbeing and child development in relation to TGS program participation. The aims, scope, and protocol of the C3 Study are the subjects of this paper., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Miller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This study evaluated the collateral, or unanticipated, impacts of Smart Beginnings (SB), a two-site, tiered intervention designed to promote responsive parenting and school readiness, on breastfeeding intensity in a low-income sample. Impact analyses for the SB intervention were conducted using an intent-to-treat design leveraging a two-arm random assignment structure. Mothers assigned to the SB intervention group were more than three times more likely to give breastmilk as the only milk source at infant age 6 months than mothers assigned to the control group at one site, an effect not evident at the other study site. As development and growth are the two most salient domains of child health, understanding how interventions impact subsequent parenting practices across both domains is critical to address long-term economic and racial/ethnic disparities. Implications of the findings are discussed for improving the efficacy of interventions based on paediatric primary care., (© 2022 The Authors. Maternal & Child Nutrition published by John Wiley & Sons Ltd.)

Optical coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) are imaging technologies invented in the 1980s that have revolutionized the field of in vivo retinal diagnostics and are now commonly used in ophthalmology clinics as well as in vision science research. Adaptive optics (AO) technology enables high-fidelity correction of ocular aberrations, resulting in improved resolution and sensitivity for both SLO and OCT systems. The potential of gathering multi-modal cellular-resolution information in a single instrument is of great interest to the ophthalmic imaging community. Although similar instruments have been developed for imaging the human retina, developing such a system for mice will benefit basic science research and should help with further dissemination of AO technology. Here, we present our work integrating OCT into an existing mouse retinal AO-SLO system, resulting in a multi-modal AO-enhanced imaging system of the living mouse eye. The new system allows either independent or simultaneous data acquisition of AO-SLO and AO-OCT, depending on the requirements of specific scientific experiments. The system allows a data acquisition speed of 200 kHz A-scans/pixel rate for OCT and SLO, respectively. It offers ∼6 µm axial resolution for AO-OCT and a ∼1 µm lateral resolution for AO-SLO-OCT imaging., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Background: The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging., Methods: We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1 +/GFP ), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1 +/D2 ) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo., Results: Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration., Conclusions: The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation., (© 2021. The Author(s).)

Objective: Ischemic stroke (IS) in young patients may differ in etiology and prognosis from later-life IS, which is much more common. A number of single-center and population-based cohorts of affected individuals have been published, but information on the long-term prognosis of these patients is limited., Methods: IS patients (≤55 years), discharged over a 10-year period, were evaluated and prospectively followed. Subgroups were evaluated for type of stroke, antecedent risk factors (RF), long-term outcomes, and occupational status over several years of follow-up., Results: 178 IS individuals from 2001-2010 were divided into older (46-55, n = 118) and younger (18-45, n = 60) age groups. Traditional RF-hypertension, diabetes mellitus, hyperlipidemia-were significantly associated with IS, and increased with age. The distribution and type of IS were similar in both groups, except for an increase in small vessel IS among the older subgroup (p = .003). Of the evaluable patients at 5.1 ± 2.5 years of follow-up (n = 138), a similar proportion of patients in both subgroups had a recurrent IS, but no significant differences were found in most disability indices. Approximately one third of patients suffered from moderate to severe disability, and were unable to return to their prior work., Conclusions: Younger and older IS patients are generally predisposed from the same traditional RF which progress with age. Long-term disabilities tend to worsen over time due to recurrent vascular events. These data emphasize the need for a strategy for early identification of the already well-known stroke RF in younger stroke patients., (© 2021 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Vertebrate retinal photoreceptors signal light by suppressing a circulating "dark current" that maintains their relative depolarization in the dark. This dark current is composed of an inward current through CNG channels and NCKX transporters in the outer segment that is balanced by outward current exiting principally from the inner segment. It has been hypothesized that Kv2.1 channels carry a predominant fraction of the outward current in rods. We examined this hypothesis by comparing whole cell, suction electrode, and electroretinographic recordings from Kv2.1 knockout (Kv2.1-/-) and wild-type (WT) mouse rods. Single cell recordings revealed flash responses with unusual kinetics, and reduced dark currents that were quantitatively consistent with the measured depolarization of the membrane resting potential in the dark. A two-compartment (outer and inner segment) physiological model based on known ionic mechanisms revealed that the abnormal Kv2.1-/- rod photoresponses arise principally from the voltage dependencies of the known conductances and the NCKX exchanger, and a highly elevated fraction of inward current carried by Ca2+ through CNG channels due to the aberrant depolarization. Kv2.1-/- rods had shorter outer segments than WT and dysmorphic mitochondria in their inner segments. Optical coherence tomography of knockout animals demonstrated a slow photoreceptor degeneration over a period of 6 mo. Overall, these findings reveal that Kv2.1 channels carry 70-80% of the non-NKX outward dark current of the mouse rod, and that the depolarization caused by the loss of Kv2.1 results in elevated Ca2+ influx through CNG channels and elevated free intracellular Ca2+, leading to progressive degeneration., (© 2021 Fortenbach et al.)

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SE del /knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

It has been recently demonstrated that structures corresponding to the cell bodies of highly transparent cells in the retinal ganglion cell layer could be visualized noninvasively in the living human eye by optical coherence tomography (OCT) via temporal averaging. Inspired by this development, we explored the application of volumetric temporal averaging in mice, which are important models for studying human retinal diseases and therapeutic interventions. A general framework of temporal speckle-averaging (TSA) of OCT and optical coherence tomography angiography (OCTA) is presented and applied to mouse retinal volumetric data. Based on the image analysis, the eyes of mice under anesthesia exhibit only minor motions, corresponding to lateral displacements of a few micrometers and rotations of a fraction of 1 deg. Moreover, due to reduced eye movements under anesthesia, there is a negligible amount of motion artifacts within the volumes that need to be corrected to achieve volume coregistration. In addition, the relatively good optical quality of the mouse ocular media allows for cellular-resolution imaging without adaptive optics (AO), greatly simplifying the experimental system, making the proposed framework feasible for large studies. The TSA OCT and TSA OCTA results provide rich information about new structures previously not visualized in living mice with non-AO-OCT. The mechanism of TSA relies on improving signal-to-noise ratio as well as efficient suppression of speckle contrast due to temporal decorrelation of the speckle patterns, enabling full utilization of the high volumetric resolution offered by OCT and OCTA.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Neuroinflammation commonly accompanies neurodegeneration, but the specific roles of resident and infiltrating immune cells during degeneration remains controversial. Much of the difficulty in assessing myeloid cell-specific functions during disease progression arises from the inability to clearly distinguish between activated microglia and bone marrow-derived monocytes and macrophages in various stages of differentiation and activation within the central nervous system. Using an inducible model of photoreceptor cell death, we investigated the prevalence of infiltrating monocytes and macrophage subpopulations after the initiation of degeneration in the mouse retina. In vivo retinal imaging revealed infiltration of CCR2 + leukocytes across retinal vessels and into the parenchyma within 48 hours of photoreceptor degeneration. Immunohistochemistry and flow cytometry confirmed and characterized these leukocytes as CD11b + CD45 + cells. Single-cell mRNA sequencing of the entire CD11b + CD45 + population revealed the presence of resting microglia, activated microglia, monocytes, and macrophages as well as 12 distinct subpopulations within these four major cell classes. Our results demonstrate a previously immeasurable degree of molecular heterogeneity in the innate immune response to cell-autonomous degeneration within the central nervous system and highlight the necessity of unbiased high-throughput and high-dimensional molecular techniques like scRNAseq to understand the complex and changing landscape of immune responders during disease progression.

Speckle is an inevitable consequence of the use of coherent light in imaging and acts as noise that corrupts image formation in most applications. Optical coherence tomographic imaging, as a technique employing coherence time gating, suffers from speckle. We present here a novel method of suppressing speckle noise intrinsically compatible with adaptive optics (AO) for confocal coherent imaging: modulation of the phase in the system pupil aperture with a segmented deformable mirror (DM) to introduce minor perturbations in the point spread function. This approach creates uncorrelated speckle patterns in a series of images, enabling averaging to suppress speckle noise while maintaining structural detail. A method is presented that efficiently determines the optimal range of modulation of DM segments relative to their AO-optimized position so that speckle noise is reduced while image resolution and signal strength are preserved. The method is active and independent of sample properties. Its effectiveness and efficiency are quantified and demonstrated by both ex vivo non-biological and in vivo biological applications., Competing Interests: R.J.Z, E.N.P and P. Z are listed as inventors on a patent application related to the work presented in this manuscript.

Background: Activation of resident microglia accompanies every known form of neurodegeneration, but the involvement of peripheral monocytes that extravasate and rapidly transform into microglia-like macrophages within the central nervous system during degeneration is far less clear., Methods: Using a combination of in vivo ocular imaging, flow cytometry, and immunohistochemistry, we investigated the response of infiltrating cells in a light-inducible mouse model of photoreceptor degeneration., Results: Within 24 h, resident microglia became activated and began migrating to the site of degeneration. Retinal expression of CCL2 increased just prior to a transient period of CCR2 + cell extravasation from the retinal vasculature. Proliferation of microglia and monocytes occurred concurrently; however, there was no indication of proliferation in either population until 72-96 h after neurodegeneration began. Eliminating CCL2-CCR2 signaling blocked monocyte recruitment, but did not alter the extent of retinal degeneration., Conclusions: These results demonstrate that the immune response to photoreceptor degeneration includes both resident microglia and monocytes, even at very early times. Surprisingly, preventing monocyte infiltration did not block neurodegeneration, suggesting that in this model, degeneration is limited by cell clearance from other phagocytes or by the timing of intrinsic cell death programs. These results show monocyte involvement is not limited to disease states that overwhelm or deplete the resident microglial population and that interventions focused on modulating the peripheral immune system are not universally beneficial for staving off degeneration.

Background: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice., Methods: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor β (gp-130) was injected intravitreally immediately after RD., Results: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade., Conclusions: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

Unlabelled: The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants-S472R, V473D, and N500E-escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface "hot spot" dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering., Importance: The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Adaptive optics scanning laser ophthalmoscopy (AO-SLO) has recently been used to achieve exquisite subcellular resolution imaging of the mouse retina. Wavefront sensing-based AO typically restricts the field of view to a few degrees of visual angle. As a consequence the relationship between AO-SLO data and larger scale retinal structures and cellular patterns can be difficult to assess. The retinal vasculature affords a large-scale 3D map on which cells and structures can be located during in vivo imaging. Phase-variance OCT (pv-OCT) can efficiently image the vasculature with near-infrared light in a label-free manner, allowing 3D vascular reconstruction with high precision. We combined widefield pv-OCT and SLO imaging with AO-SLO reflection and fluorescence imaging to localize two types of fluorescent cells within the retinal layers: GFP-expressing microglia, the resident macrophages of the retina, and GFP-expressing cone photoreceptor cells. We describe in detail a reflective afocal AO-SLO retinal imaging system designed for high resolution retinal imaging in mice. The optical performance of this instrument is compared to other state-of-the-art AO-based mouse retinal imaging systems. The spatial and temporal resolution of the new AO instrumentation was characterized with angiography of retinal capillaries, including blood-flow velocity analysis. Depth-resolved AO-SLO fluorescent images of microglia and cone photoreceptors are visualized in parallel with 469 nm and 663 nm reflectance images of the microvasculature and other structures. Additional applications of the new instrumentation are discussed.

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.

Unlabelled:  Background. The diabetic foot is a common site of infections that frequently result in significant patient morbidity and mortality. Antibiotic treatment is an important part of therapy with selection of the specific agent depending on the stage of ulceration. Recently, ertapenem has entered use as first line therapy for moderate to severe diabetic foot infections. The following prospective study recounts the experience of using ertapenem as tertiary salvage therapy following failure of first and second-line antibiotics., Methods: Forty consecutive patients with diabetic foot ulcers (IDSA classification moderate-severe), 28 with soft tissue infections alone, 12 with concomitant osteomyelitis, were included in the study. Patients were referred from the diabetic foot clinic at the authors' institution after failure of treatment with cephalexin and ciprofloxacin/clindamycin combination, and were treated with ertapenem 1-g daily intravenously (IV). Endpoints were significant clinical improvement (resolution of fever, leukocytosis, surrounding erythema, and purulent discharge)., Results: Clinical improvement was noted in all 28 individuals with soft tissue infections-all of these individuals had complete ulcer closure after a mean of 30 ± 8 treatment days. Of the 12 patients with concomitant osteomyelitis, only 25% required surgical intervention. Nine individuals were cured following a mean of 60 ± 15 treatment days of home IV antibiotic therapy., Conclusion: Ertapenem is an efficacious, easy to use, and cost saving antibiotic for the treatment of diabetic foot infections that have failed therapy with traditional antibiotics.

The energies and wave functions of several lowest-lying vibrational states of FHF(-), ClHCl(-), and BrHBr(-) have been computed by a finite-difference method with and without the Born-Oppenheimer (BO) separation between the heavy (halogen) and light (hydrogen) particle motion. The so-called diagonal BO correction (DBOC), which includes the effect of the heavy particles' kinetic energy operator acting on the light particles' wave functions, has also been made to the energies. The errors caused by the BO approximation are found to be remarkably small (ca. 10(-5) au) and can be systematically and effectively reduced by the DBOC except for states excited in the heavy particle motion. When the bare mass of the light particle is used instead of the reduced mass in the BO approximation and, therefore, the translational degrees of freedom are not correctly decoupled, the errors in the BO treatment become greater by a factor of 2-7. However, these additional errors are almost completely erased by the DBOC. Analytical and numerical results suggest that the remaining errors in the BO and DBOC treatments be proportional to epsilon(1) and epsilon(2), where epsilon is the mass ratio of the light to heavy particles, when the corrections are made to the potential energy surfaces and the wave functions for these surfaces are determined variationally. When the DBOC is applied in the first-order perturbation approximation, the remaining errors scale as epsilon(3/2).

The generality of a multilevel factorial model of social competence (SC) for preschool children was tested in a 5-group, multinational sample (N = 1,540) using confirmatory factor analysis. The model fits the observed data well, and tests constraining paths for measured variables to their respective first-order factors across samples also fit well. Equivalence of measurement models was found at sample and sex within-sample levels but not for age within sample. In 2 groups, teachers' ratings were examined as correlates of SC indicators. Composites of SC indicators were significantly associated with both positive and negative child attributes from the teachers' ratings. The findings contribute to understanding of both methodological and substantive issues concerning SC in young children.

Crystals of baculovirus-expressed adeno-associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit-cell parameters a = 254.7 A, alpha = 62.3 degrees) and shown to diffract X-rays to at least 2.5 A resolution. The diffraction data were subsequently processed and reduced with an overall R(sym) of 12.3% and a completeness of 89.0%. Based on the unit-cell volume, rotation-function and translation-function results and packing considerations, there is one virus capsid (60 viral proteins) per unit cell and there are ten viral proteins per crystallographic asymmetric unit. The AAV1 capsid shares both the twofold and threefold crystallographic symmetry operators. The AAV1 data have been initially phased using a polyalanine model (based on the crystal structure of AAV4) to 4.0 A resolution and the structure determination and refinement is in progress using tenfold noncrystallographic symmetry electron-density averaging.

Objective: To determine the spectrum and prevalence of the varied manifestations, associated conditions and laboratory abnormalities of patients with primary Sjögren's syndrome in Israel and compare them between individuals of Sephardic and Ashkenazi descent and with data from the literature., Methods: A retrospective study of a cohort of 201 consecutive patients diagnosed and followed at a single academic medical center. All cases were diagnosed using stringent criteria according to the American European Concensus Group including a labial minor salivary gland biopsy in all cases., Results: Patients' mean age was 57 years and 84% were women. Overall, more than 98% of patients had sicca symptoms of dry eyes and mouth. About 35% of the cohort had hematological manifestations--primarily immune cytopenias, protein immunoelectrophoresis abnormalities and lymphoma. About 20% had associated neurological conditions (not only peripheral but often central nervous system) and 15% had pulmonary involvement. In addition, thyroid disease, liver disease, vascular or cutaneous manifestations, synovitis, ocular and renal disease could be found. In fact, the presenting manifestation was extraglandular or an abnormal test result in 39% of the patients., Conclusion: No significant differences were found in glandular or extraglandular manifestations or laboratory test results between Ashkenazi and Sephardic patients, despite their genetic differences. A negative history of sicca symptoms effectively rules out primary Sjögren's syndrome in this cohort. These symptoms may not be volunteered by patients and the large variety of extraglandular involvement patterns and associated conditions observed may dominate the patient's presentation, and mandate physicians' awareness and a high index of suspicion for a timely diagnosis.

Clinical predictions alone are insufficiently accurate to identify patients with specific types of bloodstream infection; laboratory assays might improve such predictions. Therefore, we performed a prospective cohort study of 356 episodes of sepsis syndrome and did Limulus amebocyte lysate (LAL) assays for endotoxin. The main outcome measures were bacteremia and infection due to gram-negative organisms; other types of infection were secondary outcomes. Assays were defined as positive if the result was > or = 0.4 enzyme-linked immunosorbent assay units per milliliter. There were positive assays in 119 (33%) of 356 episodes. Assay positivity correlated with the presence of fungal bloodstream infection (P < .003) but correlated negatively with the presence of gram-negative organisms in the bloodstream (P = .04). A trend toward higher rates of mortality in the LAL assay-positive episodes was no longer present after adjusting for severity. Thus, results of LAL assay did not correlate with the presence of bacteremia due to gram-negative organisms or with mortality after adjusting for severity but did correlate with the presence of fungal bloodstream infection.

Natural killer (NK) cell number and function were determined in 69 systemic sclerosis (SSc) patients (41 with diffuse cutaneous SSc, 24 with limited cutaneous SSc, and 4 with scleroderma in an overlap syndrome). The results were compared with those obtained from 5 patients with Raynaud's disease and from 27 normal controls. Natural and antibody-dependent killing was reduced in the total group of SSc patients compared with controls, but these differences were primarily attributable to patients with the diffuse form of the disease who were seen early in their illness (less than 5 years after onset). NK cell numbers were not significantly reduced in patients compared with controls, although lower numbers were observed in individuals with early diffuse disease. Other clinical parameters, such as treatment with D-penicillamine or the presence of scleroderma-specific autoantibodies, did not exert an independent effect on NK cell function. These findings suggest a possible central role for NK cells in the pathogenesis of SSc.