Novel Variants in ABCA4-Related Retinopathies with Structural Re-Assessment of Variants of Uncertain Significance.

INTRODUCTION: Conclusive molecular genetic diagnoses in inherited retinal diseases remains a major challenge due to the large number of variants of uncertain significance (VUS) identified in genetic testing. Here, we determined the genotypic and phenotypic spectrum of ABCA4 gene variants in a cohort of Canadian inherited retinal dystrophy subjects. METHODS: This retrospective study evaluated 64 subjects with an inherited retinal dystrophy diagnosis with variants in the ABCA4 gene. Pathogenicity of variants was assessed by comparison to genetic databases and in silico modelling. ABCA4 variants classified as VUS were further evaluated using a cryo-electron structural model of the ABCA4 protein to predict impact on protein function and were also assessed for evolutionary conservation. RESULTS: Conclusive disease-causing biallelic ABCA4 variants were detected in 52 subjects with either Stargardt's disease, cone-rod dystrophy, macular dystrophy, or pattern dystrophy. A further 14 variants were novel comprising 1 nonsense, 1 frameshift, 3 splicing, and 9 missense variants. Based on in silico modelling, protein modelling and evolutionary conservation from human to zebrafish, we re-classified 5 of these as pathogenic and a further 3 as likely pathogenic. We also added to the ABCA4 phenotypic spectrum seen with four known pathogenic variants (c.2161-2A>G; Leu296Cysfs*4; Arg1640Gln; and Pro1380Leu). CONCLUSIONS: This study expands the genotypic and phenotypic spectrum of ABCA4 disease-associated variants. By panel-based genetic testing, we identified 14 novel ABCA4 variants of which 8 were determined to be disease-causing or likely disease-causing. These methodologies could circumvent somewhat the need for labour intensive in vitro and in vivo assessments of novel ABCA4 variants.

Novel pathogenic variants in Tubulin Tyrosine Like 5 (TTLL5) associated with cone-dominant retinal dystrophies and an abnormal optical coherence tomography phenotype.

Purpose: Autosomal recessive cone and cone-rod dystrophies (CD/CRD) are inherited forms of vison loss. Here, we report on and correlate the clinical phenotypes with the underlying genetic mutations. Methods: Clinical information was collected from subjects, including a family history with a chart review. They underwent a full ophthalmic examination, including best-corrected visual acuity, direct and indirect ophthalmoscopy, color vision testing, color fundus photography, contrast sensitivity, autofluorescence, and spectral domain-optical coherence tomography (SD-OCT), and full-field electroretinography. Next-generation panel-based genetic testing was used to identify DNA variants in subject buccal swab samples. Results: Genetic testing in two patients revealed three novel variants in the TTLL5 gene associated with CD/CRD: two missense variants (c.1433G>A;p.(Arg478Gln), c.241C>G;p.(Leu81Val), and one loss-of-function variant (c.2384_2387del;p.(Ala795Valfs*9). Based on in-silico analysis, structural modeling, and comparison to previously reported mutations, these novel variants are very likely to be disease-causing mutations. Combining retinal imaging with SD-OCT analysis, we observed an unusual sheen in the CD/CRD phenotypes. Conclusion: Based on the protein domain location of novel TTLL5 variants and the localization of TTLL5 to the connecting cilium, we conclude that the CD/CRD disease phenotype is characterized as a ciliopathy caused by protein tracking dysfunction. This initially affects cone photoreceptors, where photoreceptor cilia express a high level of TTLL5, but extends to rod photoreceptors over time. Fundus photography correlated with SD-OCT imaging suggests that the macular sheen characteristically seen with TTLL5 mutations derives from the photoreceptor's outer segments at the posterior pole.

Assessing the Potential for Error in Investigating Intraneural Vascularity: A Need for a Standardized Imaging Protocol.

Objective: This study examined the implementation of a Doppler sonography imaging protocol to assess intraneural blood flow, within the median nerve, in healthy individuals. Materials and Methods: A total of 176 participants were examined, and this involved 717 retrospective observations of the images collected. The implemented imaging protocol was assessed, and the data that were collected were cleaned and checked for fidelity and validity. Results: A large percentage of missing evidence (11%-35%) across proximal, mid, and distal carpal tunnel locations. Only a quarter of cases with evidence of intraneural blood flow had the strongest evidence of a power Doppler video clip, of which only three-quarters were valid. The study identified potential areas for improving the imaging protocol to reduce missing data and improve data quality. Conclusion: This study demonstrates the significance of a standardized imaging protocol to guide the sonographic acquisition of Doppler images and provides important insights into potential issues with data quality. The recommendations have the potential to help future studies assess intraneural blood flow in healthy populations in a more rigorous and reliable way. Incorporating the study's recommendations into a standardized protocol, there is potential to enhance the diagnostic accuracy of carpal tunnel syndrome and improve diagnosis and treatment.

Prospects and modalities for the treatment of genetic ocular anomalies.

Over the last three decades, genetic studies have made great strides toward the identification of genes and genetic mechanisms underlying congenital disorders of the eye. However, despite the vast knowledge available this has not translated into treatments to prevent or repair the damage in the clinical setting. Recently, new research in technologies, such as tissue regeneration, next generation designer drugs, and genome editing, have become available for some genetic disorders that might be applicable to congenital ocular diseases in the near future. Here, we provide an overview of the emerging therapeutic modalities and the future prospects they hold for debilitating ocular defects.

An update on the genetics of ocular coloboma.

Ocular coloboma is an uncommon, but often severe, sight-threatening condition that can be identified from birth. This congenital anomaly is thought to be caused by maldevelopment of optic fissure closure during early eye morphogenesis. It has been causally linked to both inherited (genetic) and environmental influences. In particular, as a consequence of work to identify genetic causes of coloboma, new molecular pathways that control optic fissure closure have now been identified. Many more regulatory mechanisms still await better understanding to inform on the development of potential therapies for patients with this malformation. This review provides an update of known coloboma genes, the pathways they influence and how best to manage the condition. In the age of precision medicine, determining the underlying genetic cause in any given patient is of high importance.

Lower patient-reported function at 2 years is associated with elevated knee cartilage T1rho and T2 relaxation times at 5 years in young athletes after ACL reconstruction.

PURPOSE: The purpose was to test the following hypotheses: (1) magnetic resonance imaging (MRI) markers of early knee cartilage degeneration would be present in the involved limb of young athletes after anterior cruciate ligament reconstruction (ACLR) and (2) poor knee function would be associated with MRI markers of cartilage degeneration. METHODS: Twenty-five young athletes after primary, unilateral ACLR (mean age, 16.7 years) were followed to 5-year post-return-to-sport (RTS) clearance, as a part of a larger, prospective cohort study in young athletes post-ACLR. At 2-year post-RTS, patient-reported knee function was evaluated using the Knee injury and Osteoarthritis Outcome Score (KOOS). At 5-year post-RTS, qualitative MRI sequences (3 T) and quantitative T1rho and T2 maps segmented into six regions at the femur and tibia were performed for the involved and uninvolved knee cartilages. Relaxation times were compared between knees using Holm-corrected paired t tests. Linear regression was used to examine the association between KOOS scores at 2 years and relaxation times at 5 years. RESULTS: Elevated T1rho and T2 relaxation times were observed in the involved knee at the anterior medial femoral condyle compared to the uninvolved knee (p = 0.006, p = 0.024, respectively). Lower KOOS-Pain, KOOS-Symptoms, KOOS-ADL, and KOOS-Sport scores at 2-year post-RTS were associated with higher T1rho or T2 relaxation times in various regions of the involved knee at 5-year post-RTS (all p < 0.05). CONCLUSIONS: MRI markers of early cartilage degeneration were identified in the medial compartment of the involved knee in young athletes 5-year post-RTS after ACLR. Lower KOOS scores at 2-year post-RTS were associated with elevated knee cartilage T1rho and T2 relaxation times at 5-year post-RTS. Evaluating patient-reported function over time after ACLR appears to provide insight into future degenerative changes in the knee cartilage matrix.

Nitrite and S-Nitrosohemoglobin Exchange Across the Human Cerebral and Femoral Circulation: Relationship to Basal and Exercise Blood Flow Responses to Hypoxia.

BACKGROUND: The mechanisms underlying red blood cell (RBC)-mediated hypoxic vasodilation remain controversial, with separate roles for nitrite () and S-nitrosohemoglobin (SNO-Hb) widely contested given their ability to transduce nitric oxide bioactivity within the microcirculation. To establish their relative contribution in vivo, we quantified arterial-venous concentration gradients across the human cerebral and femoral circulation at rest and during exercise, an ideal model system characterized by physiological extremes of O2 tension and blood flow. METHODS: Ten healthy participants (5 men, 5 women) aged 24±4 (mean±SD) years old were randomly assigned to a normoxic (21% O2) and hypoxic (10% O2) trial with measurements performed at rest and after 30 minutes of cycling at 70% of maximal power output in hypoxia and equivalent relative and absolute intensities in normoxia. Blood was sampled simultaneously from the brachial artery and internal jugular and femoral veins with plasma and RBC nitric oxide metabolites measured by tri-iodide reductive chemiluminescence. Blood flow was determined by transcranial Doppler ultrasound (cerebral blood flow) and constant infusion thermodilution (femoral blood flow) with net exchange calculated via the Fick principle. RESULTS: Hypoxia was associated with a mild increase in both cerebral blood flow and femoral blood flow (P<0.05 versus normoxia) with further, more pronounced increases observed in femoral blood flow during exercise (P<0.05 versus rest) in proportion to the reduction in RBC oxygenation (r=0.680-0.769, P<0.001). Plasma gradients reflecting consumption (arterial>venous; P<0.05) were accompanied by RBC iron nitrosylhemoglobin formation (venous>arterial; P<0.05) at rest in normoxia, during hypoxia (P<0.05 versus normoxia), and especially during exercise (P<0.05 versus rest), with the most pronounced gradients observed across the bioenergetically more active, hypoxemic, and acidotic femoral circulation (P<0.05 versus cerebral). In contrast, we failed to observe any gradients consistent with RBC SNO-Hb consumption and corresponding delivery of plasma S-nitrosothiols (P>0.05). CONCLUSIONS: These findings suggest that hypoxia and, to a far greater extent, exercise independently promote arterial-venous delivery gradients of intravascular nitric oxide, with deoxyhemoglobin-mediated reduction identified as the dominant mechanism underlying hypoxic vasodilation.

NLRP3 inflammasome activation drives bystander cone photoreceptor cell death in a P23H rhodopsin model of retinal degeneration.

The molecular signaling leading to cell death in hereditary neurological diseases such as retinal degeneration is incompletely understood. Previous neuroprotective studies have focused on apoptotic pathways; however, incomplete suppression of cell death with apoptosis inhibitors suggests that other mechanisms are at play. Here, we report that different signaling pathways are activated in rod and cone photoreceptors in the P23H rhodopsin mutant rat, a model representing one of the commonest forms of retinal degeneration. Up-regulation of the RIP1/RIP3/DRP1 axis and markedly improved survival with necrostatin-1 treatment highlighted necroptosis as a major cell-death pathway in degenerating rod photoreceptors. Conversely, up-regulation of NLRP3 and caspase-1, expression of mature IL-1ß and IL-18 and improved cell survival with N-acetylcysteine treatment suggested that inflammasome activation and pyroptosis was the major cause of cone cell death. This was confirmed by generation of the P23H mutation on an Nlrp3-deficient background, which preserved cone viability. Furthermore, Brilliant Blue G treatment inhibited inflammasome activation, indicating that the 'bystander cell death' phenomenon was mediated through the P2RX7 cell-surface receptor. Here, we identify a new pathway in cones for bystander cell death, a phenomenon important in development and disease in many biological systems. In other retinal degeneration models different cell-death pathways are activated, which suggests that the particular pathways that are triggered are to some extent genotype-specific. This also implies that neuroprotective strategies to limit retinal degeneration need to be customized; thus, different combinations of inhibitors will be needed to target the specific pathways in any given disease.

Anolis carolinensis as a model to understand the molecular and cellular basis of foveal development.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

Conceptual frameworks for the workplace change adoption process: elements integration from decision making and learning cycle process.

Sound workplace ergonomics and safety-related interventions may be resisted by employees, and this may be detrimental to multiple stakeholders. Understanding fundamental aspects of decision-making, behavioural change, and learning cycles may provide insights into pathways influencing employees' acceptance of interventions. This manuscript reviews published literature on thinking processes and other topics relevant to decision making and incorporates the findings into two new conceptual frameworks of the workplace change adoption process. Such frameworks are useful for thinking about adoption in different ways and testing changes to traditional intervention implementation processes. Moving forward, it is recommended that future research focuses on systematic exploration of implementation process activities that integrate principles from the research literature on sense-making, decision-making, and learning processes. Such exploration may provide the groundwork for development of specific implementation strategies that are theoretically grounded and provide a revised understanding of how successful intervention adoption processes work. Practitioner summary: Adoption and acceptance of workplace changes may be facilitated through sound implementation strategies. This manuscript explores several principles of sense-making and decision-making processes that can potentially be used by industrial practitioners to inform the design and development of implementation strategies for interventions that improve workplace ergonomics and safety. ABBREVIATIONS:  Musculoskeletal Disorders (MSDs); National Institute for Occupational Safety and Health (NIOSH); National Occupational Research Agenda (NORA); Health and Safety Executive (HSE).

An ex vivo gene therapy approach in X-linked retinoschisis.

PURPOSE: X-linked retinoschisis (XLRS) is juvenile-onset macular degeneration caused by haploinsufficiency of the extracellular cell adhesion protein retinoschisin (RS1). RS1 mutations can lead to either a non-functional protein or the absence of protein secretion, and it has been established that extracellular deficiency of RS1 is the underlying cause of the phenotype. Therefore, we hypothesized that an ex vivo gene therapy strategy could be used to deliver sufficient extracellular RS1 to reverse the phenotype seen in XLRS. Here, we used adipose-derived, syngeneic mesenchymal stem cells (MSCs) that were genetically modified to secrete human RS1 and then delivered these cells by intravitreal injection to the retina of the Rs1h knockout mouse model of XLRS. METHODS: MSCs were electroporated with two transgene expression systems (cytomegalovirus (CMV)-controlled constitutive and doxycycline-induced Tet-On controlled inducible), both driving expression of human RS1 cDNA. The stably transfected cells, using either constitutive mesenchymal stem cell (MSC) or inducible MSC cassettes, were assayed for their RS1 secretion profile. For single injection studies, 100,000 genetically modified MSCs were injected into the vitreous cavity of the Rs1h knockout mouse eye at P21, and data were recorded at 2, 4, and 8 weeks post-injection. The control groups received either unmodified MSCs or vehicle injection. For the multiple injection studies, the mice received intravitreal MSC injections at P21, P60, and P90 with data collection at P120. For the single- and multiple-injection studies, the outcomes were measured with electroretinography, optokinetic tracking responses (OKT), histology, and immunohistochemistry. RESULTS: Two lines of genetically modified MSCs were established and found to secrete RS1 at a rate of 8 ng/million cells/day. Following intravitreal injection, RS1-expressing MSCs were found mainly in the inner retinal layers. Two weeks after a single injection of MSCs, the area of the schisis cavities was reduced by 65% with constitutive MSCs and by 83% with inducible MSCs, demonstrating improved inner nuclear layer architecture. This benefit was maintained up to 8 weeks post-injection and corresponded to a significant improvement in the electroretinogram (ERG) b-/a-wave ratio at 8 weeks (2.6 inducible MSCs; 1.4 untreated eyes, p<0.05). At 4 months after multiple injections, the schisis cavity areas were reduced by 78% for inducible MSCs and constitutive MSCs, more photoreceptor nuclei were present (700/µm constitutive MSC; 750/µm inducible MSC; 383/µm untreated), and the ERG b-wave was significantly improved (threefold higher with constitutive MSCs and twofold higher with inducible MSCs) compared to the untreated control group. CONCLUSIONS: These results establish that extracellular delivery of RS1 rescues the structural and functional deficits in the Rs1h knockout mouse model and that this ex vivo gene therapy approach can inhibit progression of disease. This proof-of-principle work suggests that other inherited retinal degenerations caused by a deficiency of extracellular matrix proteins could be targeted by this strategy.

Detection of Intraneural Median Nerve Microvascularity Using Contrast-Enhanced Sonography: A Pilot Study.

OBJECTIVES: Demonstrating vascularity within the human median nerve may be difficult using power Doppler sonography. To this end, a pilot study documenting contrast-enhanced vascularity of the median nerve was conducted. METHODS: Patients undergoing contrast-enhanced transthoracic echocardiography were recruited for this study (n = 24). During echocardiography, a simultaneous contrast-enhanced sonographic examination of the median nerve was conducted. The study and study protocol were built from preclinical evidence. Image analysis was based on the power Doppler pixel intensity within a defined region of interest to obtain quantitative data representing the average pixel intensity, maximum pixel intensity, and power Doppler pixel dot count. Semiquantitative data representing the power Doppler dot count grading were also obtained. RESULTS: Spearman correlations between analytical methods showed strong positive, statistically significant (P< .05) correlations between the average pixel intensity and maximum pixel intensity and between the power Doppler dot count and dot count grading. Statistically significant increases in the average pixel intensity and power Doppler dot count were seen at all but 1 time point throughout the contrast-enhanced sonographic examination when compared to precontrast administration. Statistically significant increases in the maximum pixel intensity were seen at all but 4 time points. CONCLUSIONS: These pilot results represent early evidence that contrast-enhanced sonography can be used to image median nerve vascularity. In this convenience sample, median nerve contrast-enhanced sonographic data collection was feasible, safe, and consistent.

Klippel-Trénaunay Syndrome: Need for Careful Clinical Classification.

Klippel-Trénaunay syndrome (KTS) is a rare congenital malformation characterized by a triad of clinical presentations: (1) capillary malformations manifesting as a "port wine stain"; (2) limb hypertrophy; and (3) venous varicosities. It is distinguished from Parkes-Weber syndrome by the absence of substantial arteriovenous shunting. Due to the clinical implications of an arteriovenous fistula, differentiation between the two syndromes is important, as the prognosis and treatment greatly differ. We present a series of 5 cases of suspected KTS, while emphasizing the difficulties in distinguishing KTS from Parkes-Weber syndrome without diagnostic imaging and underscoring the importance of accurately classifying patients with the appropriate syndrome.

Collaborating with mammographers to address their work-related musculoskeletal discomfort.

Mammographers are an understudied group of health care workers, yet the prevalence of musculoskeletal (MSK) symptoms in mammographers appears to be elevated, similar to many occupations in health care. In this study, we used a participatory approach to identify needs and opportunities for developing interventions to reduce mammographers' exposures to risk factors that lead to the development of MSK symptoms. In this paper, we present a number of those needs and several intervention concepts along with evaluations of those concepts from experienced mammographers. We include findings from a preliminary field test of a novel intervention concept to reduce the need to adopt awkward postures while positioning patients for a screening or diagnostic mammogram. Practitioner Summary: This paper discusses needs, opportunities and methods for working with mammographers in order to develop interventions to reduce their exposure to risk factors for work-related musculoskeletal discomfort. Results from a field test of a novel intervention to reduce mammographers' awkward work postures while positioning patients are presented.

Collaborating with cardiac sonographers to develop work-related musculoskeletal disorder interventions.

For more than two decades, surveys of imaging technologists, including cardiac sonographers, diagnostic medical sonographers and vascular technologists, have consistently reported high prevalence of work-related musculoskeletal discomfort (WRMSD). Yet, intervention research involving sonographers is limited. In this study, we used a participatory approach to identifying needs and opportunities for developing interventions to reduce sonographers' exposures to WRMSD risk factors. In this paper, we present some of those needs. We include descriptions of two interventions, targeted for cardiac sonographers, that were developed, through an iterative process, into functional prototypes that were evaluated in pilot tests by practicing sonographers. One of these interventions is now in daily use. We would like other engineers and ergonomists to recognise this area of opportunity to apply their knowledge of biomechanics and design in order to begin to address the high prevalence of WRMSDs in sonographers, by working with sonographers to develop useful and usable interventions. Practitioner Summary: This paper discusses needs, opportunities and methods for working with sonographers in order to develop interventions to reduce their exposure to risk factors for work-related musculoskeletal discomfort. Results from field tests of two novel interventions targeting cardiac sonographers are also presented.

RD3 gene delivery restores guanylate cyclase localization and rescues photoreceptors in the Rd3 mouse model of Leber congenital amaurosis 12.

RD3 is a 23 kDa protein implicated in the stable expression of guanylate cyclase in photoreceptor cells. Truncation mutations are responsible for photoreceptor degeneration and severe early-onset vision loss in Leber congenital amaurosis 12 (LCA12) patients, the rd3 mouse and the rcd2 collie. To further investigate the role of RD3 in photoreceptors and explore gene therapy as a potential treatment for LCA12, we delivered adeno-associated viral vector (AAV8) with a Y733F capsid mutation and containing the mouse Rd3 complementary DNA (cDNA) under the control of the human rhodopsin kinase promoter to photoreceptors of 14-day-old Rb(11.13)4Bnr/J and In (5)30Rk/J strains of rd3 mice by subretinal injections. Strong RD3 transgene expression led to the translocation of guanylate cyclase from the endoplasmic reticulum (ER) to rod and cone outer segments (OSs) as visualized by immunofluorescence microscopy. Guanylate cyclase expression and localization coincided with the survival of rod and cone photoreceptors for at least 7 months. Rod and cone visual function was restored in the In (5)30Rk/J strain of rd3 mice as measured by electroretinography (ERG), but only rod function was recovered in the Rb(11.13)4Bnr/J strain, suggesting that the latter may have another defect in cone phototransduction. These studies indicate that RD3 plays an essential role in the exit of guanylate cyclase from the ER and its trafficking to photoreceptor OSs and provide a 'proof of concept' for AAV-mediated gene therapy as a potential therapeutic treatment for LCA12.

Carpal tunnel syndrome severity staging using sonographic and clinical measures.

INTRODUCTION: Ultrasonography may be valuable in staging carpal tunnel syndrome severity, especially by combining multiple measures. This study aimed to develop a preliminary severity staging model using multiple sonographic and clinical measures. METHODS: Measures were obtained in 104 participants. Multiple categorization structures for each variable were correlated to diagnostic severity based on nerve conduction. Goodness-of-fit was evaluated for models using iterative combinations of highly correlated variables. Using the best-fit model, a preliminary scoring system was developed, and frequency of misclassification was calculated. RESULTS: The severity staging model with best fit (rho 0.90) included patient-reported symptoms, functional deficits, provocative testing, nerve cross-sectional area, and nerve longitudinal appearance. An 8-point scoring scale classified severity accurately for 79.8% of participants. CONCLUSIONS: This severity staging model is a novel approach to carpal tunnel syndrome evaluation. Including more sensitive measures of nerve vascularity, nerve excursion, or other emerging techniques may refine this preliminary model.

Pax2 regulates a fadd-dependent molecular switch that drives tissue fusion during eye development.

Tissue fusion is an essential morphogenetic mechanism in development, playing a fundamental role in developing neural tube, palate and the optic fissure. Disruption of genes associated with the tissue fusion can lead to congenital malformations, such as spina bifida, cleft lip/palate and ocular coloboma. For instance, the Pax2 transcription factor is required for optic fissure closure, although the mechanism of Pax2 action leading to tissue fusion remains elusive. This lack of information defining how transcription factors drive tissue morphogenesis at the cellular level is hampering new treatments options. Through loss- and gain-of-function analysis, we now establish that pax2 in combination with vax2 directly regulate the fas-associated death domain (fadd) gene. In the presence of fadd, cell proliferation is restricted in the developing eye through a caspase-dependent pathway. However, the loss of fadd results in a proliferation defect and concomitant activation of the necroptosis pathway through RIP1/RIP3 activity, leading to an abnormal open fissure. Inhibition of RIP1 with the small molecule drug necrostatin-1 rescues the pax2 eye fusion defect, thereby overcoming the underlying genetic defect. Thus, fadd has an essential physiological function in protecting the developing optic fissure neuroepithelium from RIP3-dependent necroptosis. This study demonstrates the molecular hierarchies that regulate a cellular switch between proliferation and the apoptotic and necroptotic cell death pathways, which in combination drive tissue morphogenesis. Furthermore, our data suggest that future therapeutic strategies may be based on small molecule drugs that can bypass the gene defects causing common congenital tissue fusion defects.

Transcompartmental inflammatory responses in humans: IV versus endobronchial administration of endotoxin*.

OBJECTIVES: Transcompartmental signaling during early inflammation may lead to propagation of disease to other organs. The time course and the mechanisms involved are still poorly understood. We aimed at comparing acute transcompartmental inflammatory responses in humans following lipopolysaccharide-induced pulmonary and systemic inflammation. DESIGN: Randomized, double-blind, placebo-controlled, crossover study. SETTING ICU SUBJECTS: Healthy male volunteers. INTERVENTIONS: Fifteen volunteers (mean age, 23; SD, 2 yr) received Escherichia coli endotoxin (lipopolysaccharide, 4 ng/kg) IV or endobronchially on two different study days. Groups were evaluated by bronchoalveolar lavage at baseline (0 hr) and 2, 4, 6, 8, or 24 hours postchallenge. Cardiorespiratory variables were continuously recorded throughout the study day, and plasma and bronchoalveolar lavage fluid markers of inflammation were measured. MEASUREMENTS AND MAIN RESULTS: IV endotoxin elicited a systemic inflammatory response with a time-dependent increase and peak in tumor necrosis factor-α, interleukin-6, and leukocyte counts (all p < 0.001). Furthermore, a delayed (6-8 hr) increase in bronchoalveolar lavage fluid interleukin-6 concentration (p < 0.001) and alveolar leukocyte count (p = 0.03) and a minor increase in bronchoalveolar lavage fluid tumor necrosis factor-α were observed (p = 0.06). Endobronchial endotoxin was followed by progressive alveolar neutrocytosis and increased bronchoalveolar lavage fluid tumor necrosis factor-α, interleukin-6, and albumin (all p < 0.001); a systemic inflammatory response was observed after 2-4 hours, with no change in plasma tumor necrosis factor-α. CONCLUSIONS: Acute lung or systemic inflammation in humans is followed by a transcompartmental proinflammatory response, the degree and differential kinetics of which suggests that the propagation of inflammation may depend on the primary site of injury.

Use of contrast-enhanced sonography to investigate intraneural vascularity in a cohort of Macaca fascicularis with suspected median mononeuropathy.

OBJECTIVES: The purpose of this study was to provide clinical evidence of the use of contrast-enhanced sonography in detecting and quantifying changes in intraneural vascularity due to median mononeuropathy. METHODS: Five Macaca fascicularis monkeys were exposed to 20 weeks of repetitive work to increase their risk of developing median mononeuropathy. Contrast-enhanced sonograms were obtained in 30-second increments for 7 minutes while a contrast agent was being delivered. Data were collected immediately at the conclusion of the 20-week work exposure and then again during a recovery phase approximately 3 months after the completion of work. Quantitative analysis and trend graphs were used to analyze median nerve perfusion intensity. This study also compared the use of both manual counting of pixels and semiautomatic measurement using specialized software. RESULTS: Based on the average data, maximum intensity values were identified as the best indicators of nerve hyperemia. Paired t tests demonstrated significantly higher maximum intensities in the working stage for 4 of the 5 subjects (P < .01). CONCLUSIONS: This study provides preliminary evidence that (1) in a controlled exposure model, a change in intraneural vascularity of the median nerve between working and recovery can be observed; (2) this vascular change can be measured using an objective technique that quantifies the intensity of vascularity; and (3) contrast-enhanced sonography may improve the ability to reliably capture and measure low-flow microvascularity.