Correlation between ellipsoid zone thickness and the presence of subretinal drusenoid deposits in age-related macular degeneration.

PURPOSE: Subretinal drusenoid deposits (SDDs) in age-related macular degeneration (AMD) are associated with systemic vascular diseases that compromise ocular perfusion. We demonstrate that SDDs are associated with decreased ellipsoid zone (EZ) thickness, further evidence of hypoxic damage. METHODS: Post hoc analysis of a cross-sectional study. 165 AMD subjects (aged 51-100; 61% women). Spectral-domain optical coherence tomography was obtained in both eyes. Masked readers assigned subjects to three groups: drusen only, SDD+drusen (SDD+D) and SDD only. EZ thickness was measured subfoveally and 2000 µm nasally, temporally, superiorly and inferiorly from the fovea. Univariate testing was performed using two-tailed t-tests with Bonferroni correction. RESULTS: The mean EZ thickness differences between the SDD+D and drusen-only groups were (in µm) 1.10, 0.67, 1.21, 1.10 and 0.50 at the foveal, nasal, temporal, superior and inferior locations, respectively (p=0.08 inferiorly, otherwise p≤0.01); between the SDD-only and drusen-only groups, the differences were 3.48, 2.48, 2.42, 2.08 and 1.42 (p≤0.0002). Differences in EZ thicknesses across all subjects and between groups were not significantly different based on gender, race or age. CONCLUSION: Subjects with SDDs (±drusen) had thinner EZs than those with drusen only, and the inferior EZ was least affected. EZs were thinnest in SDD-only subjects. This thinning gradation is consistent with progressive destruction of highly oxygen-sensitive mitochondria in the EZ from hypoxia. These findings support the reduced ophthalmic perfusion hypothesis for the formation of SDDs secondary to high-risk systemic vasculopathy.

Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

SOX17 integrates HOXA and arterial programs in hemogenic endothelium to drive definitive lympho-myeloid hematopoiesis.

SOX17 has been implicated in arterial specification and the maintenance of hematopoietic stem cells (HSCs) in the murine embryo. However, knowledge about molecular pathways and stage-specific effects of SOX17 in humans remains limited. Here, using SOX17-knockout and SOX17-inducible human pluripotent stem cells (hPSCs), paired with molecular profiling studies, we reveal that SOX17 is a master regulator of HOXA and arterial programs in hemogenic endothelium (HE) and is required for the specification of HE with robust lympho-myeloid potential and DLL4+CXCR4+ phenotype resembling arterial HE at the sites of HSC emergence. Along with the activation of NOTCH signaling, SOX17 directly activates CDX2 expression, leading to the upregulation of the HOXA cluster genes. Since deficiencies in HOXA and NOTCH signaling contribute to the impaired in vivo engraftment of hPSC-derived hematopoietic cells, the identification of SOX17 as a key regulator linking arterial and HOXA programs in HE may help to program HSC fate from hPSCs.

Utility of intrathecal baclofen pump in primary lateral sclerosis.

Primary lateral sclerosis is a disorder categorized by insidious onset of progressive upper motor neuron dysfunction without lower motor neuron involvement. PLS often presents with gradual-onset, progressive lower extremity stiffness and pain due to muscle spasticity. Intrathecal Baclofen pumps (ITB) have been used to effectively treat spasticity in several neurologic conditions including MS and spinal cord injury. This study aimed at reviewing a cohort of PLS patients with spasticity requiring ITB to assess the clinical course, benefits, and complications in these patients. A series of 5 patients were identified who were diagnosed with PLS and received ITB as treatment for spasticity. The average age of the patients at the time of ITB insertion was 56.4 years. The average length of treatment was 10.4 years with a range of 4-15 years. All patients reported improvement in spasticity as measured by clinical examinations and Ashworth scores; 1/5 had complications with the pump related to migration of catheter. No patients required permanent removal of the ITB. ITB is a safe and effective treatment for spasticity in PLS and should be considered in other patients.

Design and implementation of the infection prevention program into risk management: Managing high level disinfection and sterilization in the outpatient setting.

Reusable, invasive medical devices within the outpatient setting pose a risk for patient harm. Ineffective disinfection of medical devices can potentially lead to transmission of pathogens between patients; and improper handling can lead to patient injury. A risk assessment was conducted, and the results strongly supported the necessity to develop a robust infection prevention program within the risk management department. This exclusive program was a proactive approach to preventing patient exposure within our healthcare system. Designing and integrating an Infection Prevention program into the Risk Management Department presented challenges, especially with the magnitude of devices and lack of standardization throughout our 33 clinics. Key components of the program included: capturing an accurate inventory of devices throughout the system, hiring a sterile processing expert, engaging support from senior leadership, adhering to rigorous auditing processes, and establishing a staff competency training structure. Since the program was launched 2 years ago, outcomes include: identification of high-risk practices with immediate resolution, increase in average clinic compliance to device reprocessing standards from 88% to 99%, elimination of 71% of scope reprocessing and 39% of instrument sterilization by clinic staff with allocation to central sterile processing departments, and development of a staff competency training structure.

NOTCH signaling specifies arterial-type definitive hemogenic endothelium from human pluripotent stem cells.

NOTCH signaling is required for the arterial specification and formation of hematopoietic stem cells (HSCs) and lympho-myeloid progenitors in the embryonic aorta-gonad-mesonephros region and extraembryonic vasculature from a distinct lineage of vascular endothelial cells with hemogenic potential. However, the role of NOTCH signaling in hemogenic endothelium (HE) specification from human pluripotent stem cell (hPSC) has not been studied. Here, using a chemically defined hPSC differentiation system combined with the use of DLL1-Fc and DAPT to manipulate NOTCH, we discover that NOTCH activation in hPSC-derived immature HE progenitors leads to formation of CD144+CD43-CD73-DLL4+Runx1 + 23-GFP+ arterial-type HE, which requires NOTCH signaling to undergo endothelial-to-hematopoietic transition and produce definitive lympho-myeloid and erythroid cells. These findings demonstrate that NOTCH-mediated arterialization of HE is an essential prerequisite for establishing definitive lympho-myeloid program and suggest that exploring molecular pathways that lead to arterial specification may aid in vitro approaches to enhance definitive hematopoiesis from hPSCs.

West Nile Virus Infection in Human and Mouse Cornea Tissue.

The purpose of this study was to determine the in vitro and ex vivo susceptibility of human corneal cells to West Nile virus (WNV) infection and evaluate the ability of the virus to disseminate to the corneas of infected mice. Human corneal epithelial cells were challenged with WNV, incubated for 1-6 days, and tested for evidence of WNV infection. Viral RNA and antigen were detected at every time point, and the virus reached a peak titer of 2.5 × 107 plaque-forming units (pfu)/mL at 3 days postinoculation (PI). Corneas procured from donors were incubated in culture dishes containing WNV for 1-5 days and tested for evidence of WNV. Viral RNA and antigen were detected, and the virus reached a mean peak titer of 4.9 × 104 pfu/mL at 5 days PI. Mice were inoculated intraperitoneally with WNV, and their eyes were harvested at 2, 5, and 8 days PI and tested for evidence of WNV. Viral RNA was detected in corneas of four of nine systemically infected mice as early as 2 days PI. We conclude that human corneal cells support WNV replication in vitro and ex vivo, and WNV may disseminate into the corneas of experimentally infected mice. These findings indicate that corneal transmission cannot be ruled out as a novel mode of human-to-human WNV transmission and additional experiments should be conducted to assess this risk further.

Two clinical isolates of Mycoplasma hyosynoviae showed differing pattern of lameness and pathogen detection in experimentally challenged pigs.

Mycoplasma (M.) hyosynoviae is known to colonize and cause disease in growing-finishing pigs. In this study, two clinical isolates of M. hyosynoviae were compared by inoculating cesarean-derived colostrum-deprived and specific-pathogen-free growing pigs. After intranasal or intravenous inoculation, the proportion and distribution pattern of clinical cases was compared in addition to the severity of lameness. Tonsils were found to be the primary site of colonization, while bacteremia was rarely detected prior to the observation of clinical signs. Regardless of the clinical isolate, route of inoculation, or volume of inocula, histopathological alterations and tissue invasion were detected in multiple joints, indicating an apparent lack of specific joint tropism. Acute disease was primarily observed 7 to 10 days post-inoculation. The variability in the severity of synovial microscopic lesions and pathogen detection in joint cavities suggests that the duration of joint infection may influence the diagnostic accuracy. In summary, these findings demonstrate that diagnosis of M. hyosynoviae-associated arthritis can be influenced by the clinical isolate, and provides a study platform to investigate the colonization and virulence potential of field isolates. This approach can be particularly relevant to auxiliate in surveillance and testing of therapeutic and/or vaccine candidates.

Endothelin-B Receptor Activation in Astrocytes Regulates the Rate of Oligodendrocyte Regeneration during Remyelination.

Reactive astrogliosis is an essential and ubiquitous response to CNS injury, but in some cases, aberrant activation of astrocytes and their release of inhibitory signaling molecules can impair endogenous neural repair processes. Our lab previously identified a secreted intercellular signaling molecule, called endothelin-1 (ET-1), which is expressed at high levels by reactive astrocytes in multiple sclerosis (MS) lesions and limits repair by delaying oligodendrocyte progenitor cell (OPC) maturation. However, as ET receptors are widely expressed on neural cells, the cell- and receptor-specific mechanisms of OPC inhibition by ET-1 action remain undefined. Using pharmacological approaches and cell-specific endothelin receptor (EDNR) ablation, we show that ET-1 acts selectively through EDNRB on astrocytes--and not OPCs--to indirectly inhibit remyelination. These results demonstrate that targeting specific pathways in reactive astrocytes represents a promising therapeutic target in diseases with extensive reactive astrogliosis, including MS.

Quantitative real-time polymerase chain reaction for detecting Mycoplasma hyosynoviae and Mycoplasma hyorhinis in pen-based oral, tonsillar, and nasal fluids.

Mycoplasma (M.) hyorhinis and M. hyosynoviae are pathogens known to cause disease in pigs post-weaning. Due to their fastidious nature, there is increased need for culture-independent diagnostic platforms to detect these microorganisms. Therefore, this study was performed to develop and optimize quantitative real-time PCR (qPCR) assays to rapidly detect M. hyorhinis and M. hyosynoviae in pen-based oral fluids as well as nasal and tonsillar fluids as proxies for samples used in swine herd surveillance. Two methods of genomic DNA extraction, automated versus manual, were used to compare diagnostic test performance. A wean-to-finish longitudinal study was also carried out to demonstrate the reproducibility of using pen-based oral fluids. Overall, pen-based oral and tonsillar fluids were more likely to be positive for both types of bacteria whereas only M. hyorhinis was detected in nasal fluids. DNA extraction protocols were shown to significantly influence test result. Although the initial detection time somewhat differed, both organisms were repeatedly detected in the longitudinal study. Overall, this study evaluated two qPCR methods for rapid and specific detection of either mycoplasma. Results from the present investigation can serve as a foundation for future studies to determine the prevalence of the two microorganisms, environmental load, and effectiveness of veterinary interventions for infection control.

Antibody responses of swine following infection with Mycoplasma hyopneumoniae, M. hyorhinis, M. hyosynoviae and M. flocculare.

Several mycoplasma species possessing a range of virulence have been described in swine. The most commonly described are Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, and Mycoplasma flocculare. They are ubiquitious in many pig producing areas of the world, and except for M. hyopneumoniae, commercial antibody-based assays are lacking for most of these. Antibody cross-reactivity among these four mycoplasma species is not well characterized. Recently, the use of pen-based oral fluids for herd surveillance is of increasing interest. Thus, this study sought to measure pig antibody responses and the level of cross-reactivity in serum and pen-based oral fluids after challenge with four species of swine mycoplasmas. Four groups of four mycoplasma-free growing pigs were separately inoculated with the different mycoplasma species. Pen-based oral fluids and serum samples were collected weekly until necropsy. Species-specific Tween 20 ELISAs were used to measure antibody responses along with four other commercial M. hyopneumoniae ELISAs. Animals from all groups seroconverted to the challenge species of mycoplasma and no evidence of cross-contamination was observed. A delayed antibody response was seen with all but M. hyorhinis-infected pigs. Cross-reactive IgG responses were detected in M. hyopneumoniae- and M. flocculare-infected animals by the M. hyorhinis Tween 20 ELISA, while sera from M. hyosynoviae and M. flocculare-infected pigs were positive in one commercial assay. In pen-based oral fluids, specific anti-M. hyopneumoniae IgA responses were detected earlier after infection than serum IgG responses. In summary, while some antibody-based assays may have the potential for false positives, evidence of this was observed in the current study.

Chronic perinatal hypoxia reduces glutamate-aspartate transporter function in astrocytes through the Janus kinase/signal transducer and activator of transcription pathway.

The cellular and molecular mechanisms that govern the response of the perinatal brain to injury remain largely unexplored. We investigated the role of white matter astrocytes in a rodent model of diffuse white matter injury produced by exposing neonatal mice to chronic hypoxia-a paradigm that mimics brain injury in premature infants. We demonstrate the absence of reactive gliosis in the immature white matter following chronic hypoxia, as determined by astrocyte proliferation index and glial fibrillary acidic protein levels. Instead, Nestin expression in astrocytes is transiently increased, and the glial-specific glutamate transporters glutamate-aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) are reduced. Finally, we demonstrate that Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling-which is important in both astrocyte development and response to injury-is reduced in the white matter following hypoxia, as well as in primary astrocytes exposed to hypoxia in vitro. Hypoxia and JAK/STAT inhibition reduce glutamate transporter expression in astrocytes, but unlike hypoxia JAK/STAT inhibition downregulates GLAST expression without affecting GLT-1, as demonstrated in vitro by treatment with JAK inhibitor I and in vivo by treatment with the JAK/STAT inhibitor AG490 [(E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide]. Our findings (1) demonstrate specific changes in astrocyte function after perinatal hypoxia, which might contribute to the particular pathogenesis of perinatal white matter injury, (2) provide evidence that at least part of these changes result from a disturbance of the JAK/STAT pathway by hypoxia, and (3) identify JAK/STAT signaling as a potential therapeutic target to restore normal GLAST expression and uptake of glutamate after perinatal brain injury.

Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination.

The mechanisms that regulate the developmental potential of adult neural progenitor populations under physiological and pathological conditions remain poorly defined. Glutamic acid decarboxylase 65 (GAD65)- and Doublecortin (Dcx)-expressing cells constitute major progenitor populations in the adult mouse subventricular zone (SVZ). Under normal physiological conditions, SVZ-derived GAD65-positive and Dcx-positive cells expressed the transcription factor Pax6 and migrated along the rostral migratory stream to the olfactory bulb to generate interneurons. After lysolecithin-induced demyelination of corpus callosum, however, these cells altered their molecular and cellular properties and migratory path. Demyelination upregulated chordin in the SVZ, which redirected GAD65-positive and Dcx-positive progenitors from neuronal to glial fates, generating new oligodendrocytes in the corpus callosum. Our findings suggest that the lineage plasticity of SVZ progenitor cells could be a potential therapeutic strategy for diseased or injured brain.