Aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix-Per-Arnt-Sim transcription factor family, plays a critical role in the developing nervous system of invertebrates and vertebrates. Dioxin, a ubiquitous environmental pollutant, avidly binds to this receptor, and maternal exposure to dioxin has been shown to impair higher brain functions and dendritic morphogenesis, possibly via an AhR-dependent mechanism. However, there is little information on AhR expression in the developing mammalian brain. To address this issue, the present study analyzed AhR mRNA expression in the brains of embryonic, juvenile, and adult mice by reverse transcription (RT)-PCR and in situhybridization. In early brain development (embryonic day 12.5), AhR transcript was detected in the innermost cortical layer. The mRNA was also expressed in the hippocampus, cerebral cortex, cerebellum, olfactory bulb, and rostral migratory stream on embryonic day 18.5, postnatal days 3, 7, and 14, and in 12-week-old (adult) mice. Hippocampal expression was abundant in the CA1 and CA3 pyramidal and dentate gyrus granule cell layers, where expression level of AhR mRNA in 12-week old is higher than that in 7-day old. These results reveal temporal and spatial patterns of AhR mRNA expression in the mouse brain, providing the information that may contribute to the elucidation of the physiologic and toxicologic significance of AhR in the developing brain.

Abstract

BACKGROUND:

Investigating disease mechanisms and treatment responses in obstructive airway diseases with invasive sampling are hampered by the small size and mechanical artefacts that conventional forceps biopsies suffer from. Endoscopic cryobiopsies are larger and more intact and are being increasingly used. However, the technique has not yet been explored for obtaining mucosa biopsies.

OBJECTIVE:

To investigate differences in size and quality of endobronchial mucosal biopsies obtained with cryotechnique and forceps. Further, to check for eligibility of cryobiopsies to be evaluated with immunohistochemistry and in situ hybridization and to investigate tolerability and safety of the technique.

METHODS:

Endobronchial mucosal biopsies were obtained with cryotechnique and forceps from patients with haemoptysis undergoing bronchoscopy and evaluated by quantitative morphometry, automated immunohistochemistry and in situ hybridization.

RESULTS:

A total of 40 biopsies were obtained from 10 patients. Cross-sectional areas were threefold larger in cryobiopsies (median: 3.08 mm2 (IQR: 1.79) vs 1.03 mm2 (IQR: 1.10), P < 0.001). Stretches of intact epithelium were 8-fold longer (median: 4.61 mm (IQR: 4.50) vs 0.55 mm (IQR: 1.23), P = 0.001). Content of glands (median: 0.095 mm2 (IQR: 0.30) vs 0.00 mm2 (IQR: 0.01), P = 0.002) and airway smooth muscle (median: 0.25 mm2 (IQR: 0.30) vs 0.060 mm2 (IQR: 0.11), P = 0.02) was higher in the cryobiopsies compared with forceps biopsies. Further, the cryobiopsies had well-preserved protein antigens and mRNA. Mild to moderate bleeding was the only complication observed.

CONCLUSION AND CLINICAL RELEVANCE:

By yielding significantly larger and more intact biopsies, the cryotechnique represents a valuable new research tool to explore the bronchi in airway disease. Ultimately with the potential to create better understanding of underlying disease mechanisms and improvement of treatments.

Abstract

BACKGROUND:

In 2014, a notification of porcine transmissible gastroenteritis virus (TGEV) was made by the National Services of Animal Health of Argentina (SENASA) to the World Organization of Animal Health (OIE). The notification was based on a serological diagnosis in a small farm with a morbidity rate of 2.3% without enteric clinical signs. In order to determine if TGEV was circulating before the official report, a retrospective study on cases of neonatal diarrhea was performed. The selection criteria was a sudden increase in mortality in 1- to 21-day-old piglets with watery diarrhea that did not respond to antibiotics. Based on these criteria, three clinical cases were identified during 2010-2015.

RESULTS:

All animals that were evaluated presented histological lesions consistent with enteric viral infection. The feces and ultrathin sections of intestine that were evaluated by electron microscopy confirmed the presence of round particles of approximately 80 nm in size and characterized by finely granular electrodense nucleoids consistent with complete particles of coronavirus. The presence of the TGEV antigen was confirmed by monoclonal specific immunohistochemistry, and final confirmation of a metabolically-active virus was performed by in situ hybridization to detect a TGE mRNA encoding spike protein. All sections evaluated in this case were negative for PEDV and rotavirus A.

CONCLUSIONS:

This is the first case series describing neonatal mortality with etiological confirmation of TGEV in Argentina. The clinical diagnosis of TGEV infections in endemic regions is challenging due to the epidemiological distribution and coinfection with other enteric pathogens that mask the clinical presentation.

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air-filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain-of-function disorders are clinically characterized, those in the FGFR2 loss-of-function disorder lacrimo-auriculo-dento-digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra-articular ligaments of the incudomalleal joint is derived from Scx-expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.

Abstract

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re‐induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situhybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell‐specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5‐diethoxycarbony1‐1,4‐dihydrocollidine (DDC), and multidrug‐resistant gene 2 knockout (Mdr2–/–) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double‐positive H19RNA+/sex‐determining region Y (SRY)‐box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple‐positive H19RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

KCNQ2/3 channels, ubiquitously expressed neuronal potassium channels, have emerged as indispensable regulators of brain network activity. Despite their critical role in brain homeostasis, the mechanisms by which KCNQ2/3 dysfunction lead to hypersychrony are not fully known. Here, we show that deletion of KCNQ2/3 channels changed PV+ interneurons', but not SST+ interneurons', firing properties. We also find that deletion of either KCNQ2/3 or KCNQ2 channels from PV+ interneurons led to elevated homeostatic potentiation of fast excitatory transmission in pyramidal neurons. Pvalb-Kcnq2 null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.

NMDA receptors are important for cognition and are implicated in neuropsychiatric disorders. GluN1 knockdown (GluN1KD) mice have reduced NMDA receptor levels, striatal spine density deficits, and cognitive impairments. However, how NMDA depletion leads to these effects is unclear. Since Rho GTPases are known to regulate spine density and cognition, we examined the levels of RhoA, Rac1, and Cdc42 signaling proteins. Striatal Rac1-pathway components are reduced in GluN1KD mice, with Rac1 and WAVE-1 deficits at 6 and 12 weeks of age. Concurrently, medium spiny neuron (MSN) spine density deficits are present in mice at these ages. To determine whether WAVE-1 deficits were causal or compensatory in relation to these phenotypes, we intercrossed GluN1KD mice with WAVE-1 overexpressing (WAVE-Tg) mice to restore WAVE-1 levels. GluN1KD-WAVE-Tg hybrids showed rescue of striatal WAVE-1 protein levels and MSN spine density, as well as selective behavioral rescue in the Y-maze and 8-arm radial maze tests. GluN1KD-WAVE-Tg mice expressed normalized WAVE-1 protein levels in the hippocampus, yet spine density of hippocampal CA1 pyramidal neurons was not significantly altered. Our data suggest a nuanced role for WAVE-1 effects on cognition and a delineation of specific cognitive domains served by the striatum. Rescue of striatal WAVE-1 and MSN spine density may be significant for goal-directed exploration and associated long-term memory in mice.

New myelin sheaths can be restored to demyelinated axons in a spontaneous regenerative process called remyelination. In general, new myelin sheaths are made by oligodendrocytes newly generated from a widespread population of adult CNS progenitors called oligodendrocyte progenitor cells (OPCs). New myelin in CNS remyelination in both experimental models and clinical diseases can also be generated by Schwann cells (SCs), the myelin-forming cells of the PNS. Fate-mapping studies have shown that SCs contributing to remyelination in the CNS are often derived from OPCs and appear not to be derived from myelinating SCs from the PNS. In this study, we address whether CNS remyelinating SCs can also be generated from PNS-derived cells other than myelinating SCs. Using a genetic fate-mapping approach, we have found that a subpopulation of nonmyelinating SCs identified by the expression of the transcription factor Foxj1 also contribute to CNS SC remyelination, as well as to remyelination in the PNS. We also find that the ependymal cells lining the central canal of the spinal cord, which also express Foxj1, do not generate cells that contribute to CNS remyelination. These findings therefore identify a previously unrecognized population of PNS glia that can participate in the regeneration of new myelin sheaths following CNS demyelination.SIGNIFICANCE STATEMENT Remyelination failure in chronic demyelinating diseases such as multiple sclerosis drives the current quest for developing means by which remyelination in CNS can be enhanced therapeutically. Critical to this endeavor is the need to understand the mechanisms of remyelination, including the nature and identity of the cells capable of generating new myelin sheath-forming cells. Here, we report a previously unrecognized subpopulation of nonmyelinating Schwann cells (SCs) in the PNS, identified by the expression of the transcription factor Foxj1, which can give rise to SCs that are capable of remyelinating both PNS and CNS axons. These cells therefore represent a new cellular target for myelin regenerative strategies for the treatment of CNS disorders characterized by persistent demyelination.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the Abca4 gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in Abca4 -/- mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness. No effective treatment currently exists for STGD1. Here we show by several approaches that ABCA4 is additionally expressed in RPE cells. (i) By in situ hybridization analysis and by RNA-sequencing analysis, we show the Abca4 mRNA is expressed in human and mouse RPE cells. (ii) By quantitative immunoblotting, we show that the level of ABCA4 protein in homogenates of wild-type mouse RPE is about 1% of the level in neural retina homogenates. (iii) ABCA4 immunofluorescence is present in RPE cells of wild-type and Mertk -/- but not Abca4 -/- mouse retina sections, where it colocalizes with endolysosomal proteins. To elucidate the role of ABCA4 in RPE cells, we generated a line of genetically modified mice that express ABCA4 in RPE cells but not in photoreceptors. Mice from this line on the Abca4 -/- background showed partial rescue of photoreceptor degeneration and decreased lipofuscin accumulation compared with nontransgenic Abca4 -/- mice. We propose that ABCA4 functions to recycle retinaldehyde released during proteolysis of rhodopsin in RPE endolysosomes following daily phagocytosis of distal photoreceptor OS. ABCA4 deficiency in the RPE may play a role in the pathogenesis of STGD1.

Aim: The presence of cells within meningioma (MG) that express embryonic stem cell (ESC) markers has been previously reported. However, the precise location of these cells has yet to be determined.

Methods: 3,3-Diaminobenzidine (DAB) immunohistochemical (IHC) staining was performed on 11 WHO grade I MG tissue samples for the expression of the ESC markers OCT4, NANOG, SOX2, KLF4 and c-MYC. Immunofluorescence (IF) IHC staining was performed to investigate the localization of each of these ESC markers. NanoString and colorimetric in situ hybridization (CISH) mRNA expression analyses were performed on six snap-frozen MG tissue samples to confirm transcriptional activation of these proteins, respectively.

Results: DAB IHC staining demonstrated expression of OCT4, NANOG, SOX2, KLF4, and c-MYC within all 11 MG tissue samples. IF IHC staining demonstrated the expression of the ESC markers OCT4, NANOG, SOX2, KLF4, and c-MYC on both the endothelial and pericyte layers of the microvessels. NanoString and CISH mRNA analyses confirmed transcription activation of these ESC markers.

Conclusion: This novel finding of the expression of all aforementioned ESC markers in WHO grade I MG infers the presence of a putative stem cells population which may give rise to MG.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.