Hypometabolism, Alzheimer's Disease, and Possible Therapeutic Targets: An Overview.

The brain is a highly dynamic organ that requires a constant energy source to function normally. This energy is mostly supplied by glucose, a simple sugar that serves as the brain's principal fuel source. Glucose transport across the blood-brain barrier (BBB) is primarily controlled via sodium-independent facilitated glucose transport, such as by glucose transporter 1 (GLUT1) and 3 (GLUT3). However, other glucose transporters, including GLUT4 and the sodium-dependent transporters SGLT1 and SGLT6, have been reported in vitro and in vivo. When the BBB endothelial layer is crossed, neurons and astrocytes can absorb the glucose using their GLUT1 and GLUT3 transporters. Glucose then enters the glycolytic pathway and is metabolized into adenosine triphosphate (ATP), which supplies the energy to support cellular functions. The transport and metabolism of glucose in the brain are impacted by several medical conditions, which can cause neurological and neuropsychiatric symptoms. Alzheimer's disease (AD), Parkinson's disease (PD), epilepsy, traumatic brain injury (TBI), schizophrenia, etc., are a few of the most prevalent disorders, characterized by a decline in brain metabolism or hypometabolism early in the course of the disease. Indeed, AD is considered a metabolic disorder related to decreased brain glucose metabolism, involving brain insulin resistance and age-dependent mitochondrial dysfunction. Although the conventional view is that reduced cerebral metabolism is an effect of neuronal loss and consequent brain atrophy, a growing body of evidence points to the opposite, where hypometabolism is prodromal or at least precedes the onset of brain atrophy and the manifestation of clinical symptoms. The underlying processes responsible for these glucose transport and metabolic abnormalities are complicated and remain poorly understood. This review article provides a comprehensive overview of the current understanding of hypometabolism in AD and potential therapeutic targets.

Establishing a Cystic Fibrosis Learning Network: Interventions to promote collaboration and data-driven improvement at scale.

Introduction: A learning health network is a type of learning health system in which stakeholders use network organization to improve health and health care. Building on existing resources in the cystic fibrosis (CF) community, the Cystic Fibrosis Learning Network (CFLN) was designed to improve medical outcomes and quality of life through an intentional focus on achieving reliable evidence-based chronic care delivery and creating a system for data-driven collaborative learning. Methods: We describe the development and growth of the CFLN considering six domains of a Network Maturity Grid: system leadership; governance and policy management; quality improvement (QI); engagement and community building; data and analytics; and research. We illustrate the impact of the CFLN experience on chronic care processes and indicators of collaborative infrastructure. Results: The CFLN represents 36 accredited care centers in the CF Foundation Care Center Network caring for over 6300 patients. Of 6779 patient clinical care visits/quarter, 77% are entered into the CF Foundation Patient Registry within 30 days, providing timely means to track outcomes. Collaborative visit planning is occurring in 93% of clinical care visits to share agenda setting with patients and families. Almost all CFLN teams (94%, n = 34) have a patient/family partner (PFP), and 74% of PFPs indicate they are actively participating, taking ownership of, or leading QI initiatives with the interdisciplinary care team. In 2022, 97% of centers reported completing 1-13 improvement cycles per month, and 82% contributed to monthly QI progress reports to share learning. Conclusion: The CFLN is a maturing, collaborative infrastructure. CFLN centers practice at an advanced level of coproduction. The CFLN fosters interdisciplinary and PFP leadership and the performance of consistent data-driven improvement cycles. CFLN centers are positioned to respond to rapid changes in evidence-based care and advance the practice of QI and implementation science on a broader scale.

Long-Term Safety and Efficacy of Elexacaftor/Tezacaftor/Ivacaftor in Children Aged ⩾6 Years with Cystic Fibrosis and at Least One F508del Allele: A Phase 3, Open-Label Clinical Trial.

Rationale: A 24-week, phase 3, open-label study showed elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was safe and efficacious in children aged 6-11 years with cystic fibrosis (CF) and one or more F508del-CFTR alleles. Objectives: To assess long-term safety and efficacy of ELX/TEZ/IVA in children who completed the pivotal 24-week phase 3 trial. Methods: In this phase 3, two-part (part A and part B), open-label extension study, children aged ⩾6 years with CF heterozygous for F508del and a minimal function CFTR mutation (F/MF genotypes) or homozygous for F508del (F/F genotype) who completed the 24-week parent study received ELX/TEZ/IVA based on weight. Children weighing <30 kg received ELX 100 mg once daily/TEZ 50 mg once daily/IVA 75 mg every 12 hours, whereas children weighing ⩾30 kg received ELX 200 mg once daily/TEZ 100 mg once daily/IVA 150 mg every 12 hours (adult dose). The 96-week analysis of part A of this extension study is reported here. Measurements and Main Results: Sixty-four children (F/MF genotypes, n = 36; F/F genotype, n = 28) were enrolled and received one or more doses of ELX/TEZ/IVA. Mean (SD) period of exposure to ELX/TEZ/IVA was 93.9 (11.1) weeks. The primary endpoint was safety and tolerability. Adverse events and serious adverse events were consistent with common manifestations of CF disease. Overall, exposure-adjusted rates of adverse events and serious adverse events (407.74 and 4.72 events per 100 patient-years) were lower than in the parent study (987.04 and 8.68 events per 100 patient-years). One child (1.6%) had an adverse event of aggression that was moderate in severity and resolved after study drug discontinuation. From parent study baseline at Week 96 of this extension study, the mean percent predicted FEV1 increased (11.2 [95% confidence interval (CI), 8.3 to 14.2] percentage points), sweat chloride concentration decreased (-62.3 [95% CI, -65.9 to -58.8] mmol/L), Cystic Fibrosis Questionnaire-Revised respiratory domain score increased (13.3 [95% CI, 11.4 to 15.1] points), and lung clearance index 2.5 decreased (-2.00 [95% CI, -2.45 to -1.55] units). Increases in growth parameters were also observed. The estimated pulmonary exacerbation rate per 48 weeks was 0.04. The annualized rate of change in percent predicted FEV1 was 0.51 (95% CI, -0.73 to 1.75) percentage points per year. Conclusions: ELX/TEZ/IVA continued to be generally safe and well tolerated in children aged ⩾6 years through an additional 96 weeks of treatment. Improvements in lung function, respiratory symptoms, and CFTR function observed in the parent study were maintained. These results demonstrate the favorable long-term safety profile and durable clinical benefits of ELX/TEZ/IVA in this pediatric population. Clinical trial registered with www.clinicaltrials.gov (NCT04183790).

Selenite Inhibits Notch Signaling in Cells and Mice.

Selenium is an essential micronutrient with a wide range of biological effects in mammals. The inorganic form of selenium, selenite, is supplemented to relieve individuals with selenium deficiency and to alleviate associated symptoms. Additionally, physiological and supranutritional selenite have shown selectively higher affinity and toxicity towards cancer cells, highlighting their potential to serve as chemotherapeutic agents or adjuvants. At varying doses, selenite extensively regulates cellular signaling and modulates many cellular processes. In this study, we report the identification of Delta-Notch signaling as a previously uncharacterized selenite inhibited target. Our transcriptomic results in selenite treated primary mouse hepatocytes revealed that the transcription of Notch1, Notch2, Hes1, Maml1, Furin and c-Myc were all decreased following selenite treatment. We further showed that selenite can inhibit Notch1 expression in cultured MCF7 breast adenocarcinoma cells and HEPG2 liver carcinoma cells. In mice acutely treated with 2.5 mg/kg selenite via intraperitoneal injection, we found that Notch1 expression was drastically lowered in liver and kidney tissues by 90% and 70%, respectively. Combined, these results support selenite as a novel inhibitor of Notch signaling, and a plausible mechanism of inhibition has been proposed. This discovery highlights the potential value of selenite applied in a pathological context where Notch is a key drug target in diseases such as cancer, fibrosis, and neurodegenerative disorders.

Autism Detection in Children by Combined Use of Gaze Preference and the M-CHAT-R in a Resource-Scarce Setting.

Most children with autism spectrum disorder (ASD), in resource-limited settings (RLS), are diagnosed after the age of four. Our work confirmed and extended results of Pierce that eye tracking could discriminate between typically developing (TD) children and those with ASD. We demonstrated the initial 15 s was at least as discriminating as the entire video. We evaluated the GP-MCHAT-R, which combines the first 15 s of manually-coded gaze preference (GP) video with M-CHAT-R results on 73 TD children and 28 children with ASD, 36-99 months of age. The GP-MCHAT-R (AUC = 0.89 (95%CI: 0.82-0.95)), performed significantly better than the MCHAT-R (AUC = 0.78 (95%CI: 0.71-0.85)) and gaze preference (AUC = 0.76 (95%CI: 0.64-0.88)) alone. This tool may enable early screening for ASD in RLS.

High-Throughput Screening Identifies Genes Required for Candida albicans Induction of Macrophage Pyroptosis.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

The Skin Cancer Objective Structured Clinical Examination (SCOSCE): A multi-institutional collaboration to develop and validate a clinical skills assessment for melanoma.

BACKGROUND: Assessing medical students on core skills related to melanoma detection is challenging in the absence of a well-developed instrument. OBJECTIVE: We sought to develop an objective structured clinical examination for the detection and evaluation of melanoma among medical students. METHODS: This was a prospective cohort analysis of student and objective rater agreement on performance of clinical skills and assessment of differences in performance across 3 schools. RESULTS: Kappa coefficients indicated excellent agreement for 3 of 5 core skills including commenting on the presence of the moulage (k = 0.87, 95% confidence interval 0.77-0.96), obtaining a history for the moulage (k = 0.84, 95% confidence interval 0.74-0.94), and making a clinical impression (k = 0.80, 95% confidence interval 0.68-0.92). There were no differences in performance across schools with respect to 3 of 5 core skills: commenting on the presence of the moulage (P = .15), initiating a history (P = .53), and managing the suspicious lesion (P value range .07-.17). Overall, 54.2% and 44.7% of students commented on the presence of the moulage and achieved maximum performance of core skills, respectively, with no difference in performance across schools. LIMITATIONS: Limitations include overall sample size of students and schools. CONCLUSION: The Skin Cancer Objective Structured Clinical Examination represents a potentially important instrument to measure students' performance on the optimal step-by-step evaluation of a melanoma.

Curricular factors associated with medical students' practice of the skin cancer examination: an educational enhancement initiative by the integrated skin exam consortium.

IMPORTANCE: As medical school curricula become progressively integrated, a need exists to optimize education related to the skin cancer examination (SCE) for melanoma, a relevant competency gap that influences secondary prevention efforts. OBJECTIVES: To identify curricular factors associated with medical students' confidence, intent, and performance regarding the SCE. DESIGN, SETTING, AND PARTICIPANTS: Survey-based cross-sectional study from the Integrated Skin Exam Consortium at accredited US medical schools among a volunteer sample of second-year students representing 8 geographically varied public and private institutions. Students were administered a questionnaire to assess characteristics, curricular exposures, and educational and practical experiences related to skin cancer, as well as knowledge of melanoma risk and a detection method. MAIN OUTCOMES AND MEASURES: Primary outcomes were confidence in performing the SCE, intent to perform an integrated skin examination, and actual performance of the SCE. RESULTS: Physical diagnosis session and clinical encounter were most predictive of confidence in performance of the SCE (odds ratios [ORs], 15.35 and 11.48, respectively). Other curricular factors associated with confidence included instruction time of at least 60 minutes on skin cancer (OR, 6.35), lecture on the SCE (OR, 7.54), knowledge of melanoma risk (OR, 3.71), and at least 1 opportunity to observe the SCE (OR, 2.70). Physical diagnosis session and at least 4 opportunities to observe the SCE were most predictive of intent to perform an integrated skin examination (ORs, 4.84 and 4.72, respectively). Other curricular factors associated with intent included knowledge of melanoma risk (OR, 1.83), clinical encounter (OR, 2.39), and at least 1 opportunity to observe the SCE (OR, 1.95). Clinical encounter, physical diagnosis session, and at least 1 opportunity to observe the SCE were most predictive of performance of the SCE (ORs, 21.67, 15.48, and 9.92, respectively). Other curricular factors associated with performance included instruction time of at least 60 minutes on skin cancer (OR, 2.42) and lecture on the SCE (OR, 5.04). CONCLUSIONS AND RELEVANCE: To augment the practice of the SCE among medical students, course directors may design an integrated curriculum that includes at least 60 minutes of instruction related to melanoma and the SCE, a description of the integrated skin examination as part of the physical diagnosis course, and education on high-risk demographic groups and anatomic sites specific to men and women and on the ABCDEs of melanoma, and at least 1 opportunity to observe the SCE.

The Integrated Skin Exam film: an educational intervention to promote early detection of melanoma by medical students.

BACKGROUND: Knowledge of the skin cancer examination (SCE) and its practice remain relevant competency gaps among medical students. OBJECTIVE: We elaborate on a method of SCE known as the Integrated Skin Exam and discuss the development of an instructional film that illustrates its principles. We assess the tool's effect on knowledge, attitudes, and perceptions related to the SCE. METHODS: Second-year students among 8 randomized schools viewed the film and completed pre-post questionnaires. RESULTS: After viewing The Integrated Skin Exam film, students demonstrated improved melanoma knowledge, including identification of high-risk demographic groups (61% vs 42.9%, P < .001), high-risk anatomic sites in women (88.6% vs 46.5%, P < .001) and men (92.1% vs 34.8%, P < .001), and the ABCDEs of melanoma (98.4% vs 91.2%, P < .001). Students demonstrated increased confidence in the SCE (66.93% vs 16.40%, P < .001) and augmented intentions to practice it (99.05% vs 13.9%, P < .001). A greater proportion (70.4% vs 41.9%, P < .001) of students thought less than 3 minutes were required to integrate SCE into the routine examination. LIMITATIONS: Longitudinal impact of the film was not assessed. CONCLUSION: The Integrated Skin Exam film introduces an integrated approach to the SCE that addresses knowledge gaps, mitigates perceived barriers, and augments intention related to practice of the SCE.

Pancreatic laceration in a female collegiate soccer athlete: a case report.

OBJECTIVE: To characterize the diagnosis of pancreatic trauma in an athletic population and to raise awareness among health care providers of the possibility of this life- and organ-threatening injury. BACKGROUND: An 18-year-old, previously healthy female collegiate soccer athlete sustained a direct blow from an opponent's knee between the left and right upper abdominal quadrants while attempting to head the ball. She initially presented with only minimal nausea and discomfort, but this progressed to abdominal pain, tenderness, spasm, and vomiting. She was referred to the emergency department, where she was diagnosed with a pancreatic laceration. DIFFERENTIAL DIAGNOSIS: Duodenal, hepatic, or splenic contusion or laceration; hemorrhagic ovarian cyst. TREATMENT: The patient underwent a distal pancreatectomy and total splenectomy. UNIQUENESS: Pancreatic injuries, particularly those severe enough to warrant surgical intervention, are extremely rare in athletes. CONCLUSIONS: Recognition of a pancreatic injury can be very challenging outside the hospital setting. This is problematic, because a delay in diagnosis is a significant source of preventable morbidity and mortality after this rare injury. Thus, early identification depends on a high index of suspicion, a thorough examination, and close observation. It is imperative that athletic trainers and other health care professionals be able to identify this condition so that referral and management can occur without delay.

GABA supplementation and growth hormone response.

The secretion of growth hormone (GH) is regulated through a complex neuroendocrine control system, especially by the functional interplay of two hypothalamic hormones, GH-releasing hormone and somatostatin. These hormones are subject to modulation by a host of neurotransmitters and are the final mediators of endocrine and neural influences for GH secretion. Interest in the possible role of γ-aminobutyric acid (GABA) in the control of GH secretion began decades ago. However, interest in its role as an ergogenic aid is only recent. It is well accepted that GABAergic neurons are found in the hypothalamus and recent evidence suggests its secretion within the pituitary itself. Inhibition of GABA degradation and blockade of GABA transmission as well as administration of GABA and GABA mimetic drugs have all been shown to affect GH secretion. However, there are many controversial findings. The effects may depend on the site of action within the hypothalamic-pituitary unit and the hormonal milieu. Experimental and clinical evidence support the presence of a dual action of GABA - one mediated centrally, the other exerted directly at the pituitary level. The two sites of action may be responsible for excitatory and inhibitory effects of GABA on GH secretion. This chapter will outline the anatomical basis for possible influences of GABA on GH secretion and present evidence for a role of GABA in the control of GH release by actions at either hypothalamic or pituitary sites. The potential ergogenic benefits of oral GABA supplementation will also be discussed.

Altered vascular expression of EphrinB2 and EphB4 in a model of oxygen-induced retinopathy.

EphrinB2 ligands and EphB4 receptors are expressed on endothelial cells (EC) of arteries and veins, respectively, and are essential for vascular development. To understand how these molecules regulate retinal neovascularization (NV), we evaluated their expression in a model of oxygen-induced retinopathy (OIR). EphrinB2 and EphB4 were expressed on arterial and venous trunks, respectively, and on a subset of deep capillary vessels. EphB4 expression was reduced following hyperoxia, while ephrinB2 expression remained unaltered. In addition, a subset of EphB4-positive veins regressed in a caspase-3-dependent manner during hyperoxia. Arteriovenous malformations were also observed with loss of arterial-venous boundaries. Finally, both ephrinB2 and EphB4 were expressed on a subset of neovascular tufts following hyperoxia. These data confirm the contribution of ECs from both venous and arterial origins to the development of retinal NV.

TRAIL-deficient mice exhibit delayed regression of retinal neovascularization.

While it is well established that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cell types, the role of TRAIL in regulation of retinal neovascularization (NV) has not been described. Here we determined the role of TRAIL in retinal NV during oxygen-induced retinopathy using TRAIL deficient ((-/-)) mice. TRAIL and its receptor, DR5, were expressed in wild-type retinas at all time points evaluated (postnatal days 12, 17, 21, 24) during oxygen-induced retinopathy and in age-matched room air control animals. Localization of TRAIL(+) cells within the neovascular tufts of hyperoxia- exposed wild-type mice suggested TRAIL plays a role in oxygen-induced retinopathy. Retinal vascular development appeared normal in the TRAIL(-/-) mice, except for a small but significant difference in the capillary-free zone surrounding major arteries. A minimal difference in avascularity was observed at postnatal day 12 in the retinas of TRAIL(-/-) mice after hyperoxia-exposure compared with wild-type mice, suggesting that TRAIL does not play a major role in the vaso-obliterative phase of oxygen-induced retinopathy. However, at the peak of NV, TRAIL(-/-) mice had a significant increase in retinal neovascularization. In addition, when NV naturally regresses in wild-type mice, TRAIL(-/-) mice continued to display significantly high levels of NV. This was attributed to a significant decrease in neovascular tuft cells undergoing apoptosis in TRAIL(-/-) mice. Together, these data strongly suggest that TRAIL plays a role in the control of retinal NV.

Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells.

PURPOSE: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. METHODS: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 microL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). RESULTS: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2+/-VEGF compared to IgG control+/-VEGF (P<0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. CONCLUSIONS: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

Rhesus macaque rhadinovirus-associated non-Hodgkin lymphoma: animal model for KSHV-associated malignancies.

Rhesus macaque rhadinovirus (RRV) is closely related to Kaposi sarcoma-associated herpesvirus (KSHV) and is associated with the development of B-cell hyperplasia and persistent lymphadenopathy resembling multicentric Castleman disease in rhesus macaques (RMs) coinfected with simian immunodeficiency virus (SIV). Here we investigated whether RMs experimentally infected with SIV and RRV can develop other disease manifestations observed in HIV- and KSHV-infected patients. As reported earlier, inoculation of SIV-infected RMs with RRV results in persistent RRV infection, whereas immunocompetent animals infected with RRV exhibit viremia 2 weeks after infection, followed by a period of no virus detection until they are subsequently made immunodeficient by SIV infection. A subset of animals developed abnormal cellular proliferations characterized as extranodal lymphoma and a proliferative mesenchymal lesion. In situ hybridization and immunohistochemistry analysis indicate RRV is present in both malignancies, and DNA microarray analysis detected viral interleukin-6 (vIL-6) and viral FLICE-like inhibitory protein (vFLIP) transcripts. Reverse-transcriptase polymerase chain reaction analysis confirmed vIL-6 and vFLIP expression, and that of RRV open reading frames 72 and 73, homologs of KSHV open reading frames shown to be expressed in primary effusion lymphoma. These data support the utility of the RRV-/SIV-infected RM as an excellent animal model to investigate KSHV-like pathogenesis.

MCP-1 deficiency delays regression of pathologic retinal neovascularization in a model of ischemic retinopathy.

PURPOSE: The present study investigates whether retinal neovascularization (NV) and apoptosis are altered in MCP-1-deficient ((-/-)) mice in the OIR model. METHODS: Postnatal day (P) 7 MCP-1(-/-) and C57BL/6 (B6) mice were exposed to 75% oxygen for 5 days and then recovered in room air. Immunostaining was performed to localize macrophages/microglia within retinal whole mounts and cross-sections. Retinopathy was qualitatively assessed in FITC-dextran-perfused retinas, and preretinal NV was quantified on P17, P21, and P24. TUNEL analysis was used to compare apoptosis between B6 and MCP-1(-/-) mice. RESULTS: MCP-1(-/-) and B6 mice revealed normal vascular development in room air controls and similar vaso-obliteration in oxygen-exposed mice on P12. MCP-1(-/-) mice exhibited significantly reduced vascular tuft-associated F4/80(+) cells compared with B6 mice. FITC-dextran-perfused retinas exhibited prominent neovascular tufts on P17, and quantification of preretinal nuclei revealed no significant differences between MCP-1(-/-) and B6 mice. In contrast, on P21 and P24, MCP-1(-/-) mice exhibited significant increases in preretinal neovascular nuclei compared with B6 controls. These increases in NV in the MCP-1(-/-) mice were associated with a significant reduction in vascular tuft apoptosis. CONCLUSIONS: The results demonstrate that the absence of MCP-1 does not alter normal retinal vascular development. Furthermore, MCP-1(-/-) mice exhibit a similar neovascular response on P17. However, the reduction in tuft-associated macrophages/microglia in the MCP-1(-/-) mice correlates with reduced vascular tuft apoptosis and delayed regression of retinal NV. These findings suggest that macrophages/microglia may contribute to tuft regression through their proapoptotic properties.

Identification of novel alternatively spliced isoforms of RTEF-1 within human ocular vascular endothelial cells and murine retina.

PURPOSE: Identification of transcription factors that regulate the transcription of the vascular endothelial growth factor (VEGF) gene may facilitate understanding of the etiology and progression of ocular neovascular diseases. The purpose of this study was to determine whether transcriptional enhancer factor 1-related (RTEF-1) was present within ocular vascular endothelial cells and whether it played a role in the control of the transcription of the VEGF gene. METHODS: Primary cultures of human retinal vascular endothelial cells (RVECs) were maintained under normoxic or hypoxic conditions before isolation of mRNA. RT-PCR was performed to detect RTEF-1 transcripts. Amplified products were cloned into an expression plasmid. Human VEGF promoter and deletion constructs were cloned into a pSEAP reporter vector. Various RTEF-1 isoforms and VEGF promoter constructs were coelectroporated into human cells, and reporter expression levels were determined. Retinal tissue from a mouse model of retinopathy of prematurity (ROP) was analyzed by RT-PCR for the presence of RTEF-1 transcripts. RESULTS: Full-length 1305-bp and novel 936-bp RTEF-1 transcripts were identified in cultured human RVECs under normoxic conditions. A novel 447-bp isoform was present in cells maintained in a hypoxic environment. Four of the 11 translated exons predicted to code for the 1305-bp product were spliced out of the 936-bp transcript. The 1305-bp product enhanced expression from the VEGF promoter 4-fold greater than background, whereas the 936-bp and the 447-bp isoforms enhanced expression 3x and 12x, respectively. Analysis with deletion promoter constructs determined that all isoforms required the presence of Sp1 elements for efficient activation and that the hypoxia response element (HRE) was not essential for enhancement. Transcripts for novel RTEF-1 isoforms were also identified in neural retinal tissue of mice. Different murine-specific isoforms were present at different stages of postnatal development. CONCLUSIONS: Novel RTEF-1 transcripts are present within human ocular vascular endothelial cells and mouse neural retina during normal and ROP development, and alternatively spliced products are produced under hyperoxic and hypoxic conditions. Alternative spliced variants of human RTEF-1 transcripts are able to potentiate expression from the VEGF 5' proximal promoter region.

Deployable, field-sustainable, reverse transcription-polymerase chain reaction assays for rapid screening and serotype identification of dengue virus in mosquitoes.

Dengue virus universal and serotype 1 to 4 fluorogenic probe hydrolysis, reverse transcription (RT)-polymerase chain reaction (PCR) assays and positive-control RNA template were freeze-dried in a thermally stable, hydrolytic enzyme-resistant format and deployed for testing in a dengue fever-endemic region of Thailand. The study site presented austere testing conditions. Field-collected Aedes aegypti mosquitoes spiked with inoculated A. aegypti mosquitoes and individual and pooled, field-collected, A. aegypti, A. albopictus, and Culex tritaeniorhynchus mosquitoes were used for RT-PCR assay evaluations. For dengue virus-inoculated A. aegypti mosquitoes and spiked samples, in vitro sensitivity and specificity results for all five assays were concordant with indirect fluorescent antibody assay results. A single pool of field-collected, female, A. aegypti mosquitoes was identified as dengue virus positive. Cross-reactivity was not observed across heterologous serotypes, mosquito vectors, or human DNA. The limit of detection was >7 to < or =70 genomic equivalents. Sample processing and analysis required <2 hours. These results show promise of field-formatted RT-PCR reagents for rapid, sensitive, specific dengue virus screening and serotype identification in mosquitoes under field-deployed conditions.