DNA-based detection for onychomycosis correlates better to histopathology than does fungal culture.

Onychomycosis is a prevalent disease of the nail. Traditional methods for diagnosis include direct microscopy with potassium hydroxide (KOH microscopy) and fungal culture. Other techniques using histochemical staining have higher sensitivity, but cannot identify genus or species of the infecting agent. PCR assays are sensitive, specific, and capable of genus and species level identification. We describe a real-time PCR assay for 15 different fungi that are associated with onychomycosis. Of 425 clinical samples suspected of onychomycosis analyzed by fungal culture and PCR, 219 samples were positive for both (52% agreement). Of the 206 discordant samples, 95% were resolved in favor of PCR by DNA sequencing. On a larger data set of 2,452 samples, positivity rates for histopathology, PCR, and culture were 85%, 73%, and 54% respectively. Further, 48% of PCR positive and 51% of histopathology positive samples were negative by culture. PCR outperformed culture compared to histopathology for sensitivity (80% versus 49%), specificity (92% versus 79%), positive predictive value (94% versus 77%), and negative predictive value (76% versus 52%). These results indicate the culture method lacks the sensitivity to be a reliable assay for onychomycosis, that PCR and histopathology are highly concordant, and that PCR provides the highest degree of diagnostic accuracy available.

An Electronic Dashboard to Improve Dosing of Hydroxychloroquine Within the Veterans Health Care System: Time Series Analysis.

BACKGROUND: Hydroxychloroquine (HCQ) is commonly used for patients with autoimmune conditions. Long-term use of HCQ can cause retinal toxicity, but this risk can be reduced if high doses are avoided. OBJECTIVE: We developed and piloted an electronic health record-based dashboard to improve the safe prescribing of HCQ within the Veterans Health Administration (VHA). We observed pilot facilities over a 1-year period to determine whether they were able to improve the proportion of patients receiving inappropriate doses of HCQ. METHODS: Patients receiving HCQ were identified from the VHA corporate data warehouse. Using PowerBI (Microsoft Corp), we constructed a dashboard to display patient identifiers and the most recent HCQ dose and weight (flagged if ≥5.2 mg/kg/day). Six VHA pilot facilities were enlisted to test the dashboard and invited to participate in monthly webinars. We performed an interrupted time series analysis using synthetic controls to assess changes in the proportion of patients receiving HCQ ≥5.2 mg/kg/day between October 2020 and November 2021. RESULTS: At the start of the study period, we identified 18,525 total users of HCQ nationwide at 128 facilities in the VHA, including 1365 patients at the 6 pilot facilities. Nationwide, at baseline, 19.8% (3671/18,525) of patients were receiving high doses of HCQ. We observed significant improvements in the proportion of HCQ prescribed at doses ≥5.2 mg/kg/day among pilot facilities after the dashboard was deployed (-0.06; 95% CI -0.08 to -0.04). The difference in the postintervention linear trend for pilot versus synthetic controls was also significant (-0.06; 95% CI -0.08 to -0.05). CONCLUSIONS: The use of an electronic health record-based dashboard reduced the proportion of patients receiving higher than recommended doses of HCQ and significantly improved performance at 6 VHA facilities. National roll-out of the dashboard will enable further improvements in the safe prescribing of HCQ.

Circumventricular organs: a novel site of neural stem cells in the adult brain.

Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin+, GFAP+, vimentin+ cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin+, beta-tubulin III+) and glial (S100beta+, GFAP+, RIP+) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.

B-cell deficiency and reduced B-cell reconstitution in hemoglobin-deficit mice.

OBJECTIVE: To study the effect of persistent hemoglobin-deficit mutation (hbd/hbd) on hematopoiesis and the function of hematopoietic stem cells (HSCs). METHODS: Young and old mice homozygous for the spontaneous hbd/hbd mutation were compared to young and old wild-type control mice, all on the C57BL/6 background, over cellular composition in blood and bone marrow (BM) using cell counting, complete blood counts, and flow cytometry. BM cells from hbd/hbd mutants and normal controls were also tested for HSC engraftment in vivo using the competitive repopulation assay. RESULTS: Both young and old hbd/hbd mutants exhibited a microcytic anemia with significantly (p<0.01) lower levels of hemoglobin and mean corpuscular volume. There were significant declines in CD45R+ B cells in both blood (p<0.01) and BM (p<0.05) in old hbd/hbd mice, suggesting that B-cell homeostasis was compromised. Total BM cells per mouse was significantly increased (p<0.05) in old hbd/hbd mice. In the competitive repopulation assay in vivo, BM cells from old hbd/hbd donors showed slightly decreased contribution to T cells and myeloid cells but a significantly (p<0.01) decreased engraftment in B lymphocytes, indicating that B-cell hematopoiesis was compromised in old hbd/hbd mice. These data differ from results previously obtained from normal C57BL/6 mice in which BM cell engraftment ability does not decrease with donor age. CONCLUSION: Persistent hbd/hbd mutation causes hematopoiesis defects in the B cell lineage.

A dorsal root ganglia cell line derived from trisomy 16 fetal mice, a model for Down syndrome.

We have established two immortalized cell lines from dorsal root ganglia of normal (G4b) and trisomy 16 mice (GT1), a model for Down syndrome. By immunohistochemistry, both cell lines exhibit neuronal traits and lack glial markers. GTl cells exhibited greater [3H]choline uptake than G4b cells. K+ and nicotine-mediated acetylcholine release was greater in GT1 cells. Basal intracellular Ca2+ concentration ([Ca2+]i) was significantly lower in GTl cells. More GTl cells responded to neurotransmitters with a transient [Ca2+]i increase compared to G4b cells, but both cell types showed similar amplitudes of [Ca2+]i responses. The results show that both cell lines retain neuronal characteristics and respond to specific neurotransmitter stimuli. Altered GT1 cell responses could be related to neuronal pathophysiology in Down's syndrome.

Heterotopically transplanted CVO neural stem cells generate neurons and migrate with SVZ cells in the adult mouse brain.

Production of new neurons throughout adulthood has been well characterized in two brain regions, the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ (Bennett et al.). The present study demonstrates that NSCs derived from two astrogliogenic CVOs, the median eminence and organum vasculosum of the lamina terminalis of the nestin-GFP mouse, possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs, following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice, migrate caudally to the SVZ and express early neuronal markers (TUC-4, PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU(+) cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively, these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ, they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain.

Presenilin-dependent gamma-secretase-like intramembrane cleavage of ErbB4.

An unusual protease gamma-secretase requires functional presenilins and cleaves substrates (e.g. amyloid beta-protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent gamma-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the approximately 80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a gamma-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro gamma-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.

Establishment and characterization of immortalized neuronal cell lines derived from the spinal cord of normal and trisomy 16 fetal mice, an animal model of Down syndrome.

We report the establishment of continuously growing cell lines from spinal cords of normal and trisomy 16 fetal mice. We show that both cell lines, named M4b (derived from a normal animal) and MTh (trisomic) possess neurological markers by immunohistochemistry (neuron specific enolase, synaptophysin, microtubule associated protein-2 [MAP-2], and choline acetyltransferase) and lack glial traits (glial fibrillary acidic protein and S100). MTh cells were shown to overexpress mRNA of Cu/Zn superoxide dismutase, whose gene is present in autosome 16. We also studied intracellular Ca2+ signals ([Ca2+]i) induced by different agonists in Indo-1 loaded cells. Basal [Ca2+]i was significantly higher in MTh cells compared to M4b cells. Glutamate (200 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACDP) (100 microM) induced rapid, transient increases in [Ca2+]i in M4b and MTh cells, indicating the presence of glutamatergic metabotropic receptors. N-methyl-D-aspartate (NMDA) and kainate, but not alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), produced [Ca2+)]i rises in both cell types. MTh cells exhibited faster time-dependent decay phase kinetics in glutamate-induced responses compared to M4b cells. Nicotine induced a transient increase in [Ca2+]i in M4b and MTh cells, with significantly greater amplitudes in the latter compared to the former. Further, both cell types responded to noradrenaline. Finally, we examined cholinergic function in both cell lines and found no significant differences in the [3H]-choline uptake, but fractional acetylcholine release induced by either K+, glutamate or nicotine was significantly higher in MTh cells. These results show that M4b and MTh cells have neuronal characteristics and the MTh line shows differences which could be related to neuronal pathophysiology in Down's syndrome.