Abstract BACKGROUND: Corneal transplant failure with neovascularisation is a leading indication for full-thickness grafts in patients. Lymphangiogenesis is implicated in the pathology of graft failure, and here we systematically evaluate failed human corneal transplants with neovascularisation for the presence of lymphatic vessels. METHODS: Nine failed grafts with neovascularisation, based on H&E staining with subsequent immunoperoxidase staining for CD31, a blood vessel marker, were selected. Lymphatics were investigated by immunohistochemical and immunofluorescence approaches using podoplanin as a lymphatic marker. In two of nine cases, fluorescence in situ hybridisation (FISH) was used for detection of lymphatic mRNAs including podoplanin, VEGFR-3 and LYVE-1. All immunofluorescence and FISH samples were compared with positive and negative controls and visualised by confocal microscopy. RESULTS: Corneal neovascularisation was established in all cases by H&E and further confirmed by CD31 immunoreactive profiles. Immunohistochemistry for the podoplanin antibody was positive in all cases and showed morphologies ranging from distinct luminal structures to elongated profiles. Simultaneous immunofluorescence using CD31 and podoplanin showed lymphatic vessels distinct from blood vessels. Podoplanin immunofluorescence was noted in seven of nine cases and revealed clear lumina of varying sizes, in addition to lumen-like and elongated profiles. The presence of lymphatic mRNA was confirmed by FISH studies using a combination of at least two of podoplanin, VEGFR-3 and LYVE-1 mRNAs. CONCLUSIONS: The consistent finding of lymphatic vessels in failed grafts with neovascularisation implicates them in the pathogenesis of corneal transplant failure, and points to the lymphatics as a potential new therapeutic target.

The melanocortin-1 receptor (MC1R) in podocytes has been suggested as the mediator of the ACTH renoprotective effect in patients with nephrotic syndrome with the mechanism of action beeing stabilization of the podocyte actin cytoskeleton. To understand how melanocortin receptors are regulated in nephrotic syndrome and how they are involved in restoration of filtration barrier function, melanocortin receptor expression was evaluated in patients and a rat model of nephrotic syndrome in combination with cell culture analysis. Phosphoproteomics was applied and identified MC1R pathways confirmed using biochemical analysis. We found that glomerular MC1R expression was increased in nephrotic syndrome, both in humans and in a rat model. A MC1R agonist protected podocytes from protamine sulfate induced stress fiber loss with the top ranked phoshoproteomic MC1R activated pathway beeing actin cytoskeleton signaling. Actin stabilization through the MC1R consisted of ERK1/2 dependent phosphorylation and inactivation of EGFR signaling with stabilization of synaptopodin and stressfibers in podocytes. These results further explain how patients with nephrotic syndrome show responsiveness to MC1R receptor activation by decreasing EGFR signaling and as a consequence restore filtration barrier function by stabilizing the podocyte actin cytoskeleton

Abstract RATIONALE: MUC5AC and MUC5B are the predominant gel-forming mucins in the mucus layer of human airways. Each mucin has distinct functions and site-specific expression. However, the regional distribution of expression and cell types that secrete each mucin in normal/healthy human airways are not fully understood. OBJECTIVES: To characterize the regional distribution of MUC5B and MUC5AC in normal/healthy human airways and assess which cell types produce these mucins, referenced to the club cell secretory protein (CCSP). METHODS: Multiple airway regions from 16 non-smoker lungs without a history of lung disease were studied. MUC5AC, MUC5B, and CCSP expression/co-localization were assessed by RNA in situ hybridization (ISH) and immunohistochemistry in 5 lungs with histologically healthy airways. Droplet digital PCR and cell culture were performed for absolute quantification of MUC5AC/5B ratios and protein secretion, respectively. RESULTS: Submucosal glands expressed MUC5B, but not MUC5AC. However, MUC5B was also extensively expressed in superficial epithelia throughout the airways except for the terminal bronchioles. Morphometric calculations revealed that the distal airway superficial epithelium was the predominant site for MUC5B expression, whereas MUC5AC expression was concentrated in proximal, cartilaginous airways. RNA ISH revealed both MUC5AC and MUC5B were co-localized with CCSP-positive secretory cells in proximal superficial epithelia, whereas MUC5B and CCSP-co-positive cells dominated distal regions. CONCLUSIONS: In normal/healthy human airways, MUC5B is the dominant secretory mucin in the superficial epithelium as well as glands, with distal airways being a major site of expression. MUC5B and MUC5AC expression is a property of CCSP positive secretory cells in superficial airway epithelia.

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes in vivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an in vivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.

Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.

Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting β-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic β-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.

Abstract OBJECTIVES: To evaluate the clinical utility of immune receptor translocation-associated protein 1 (IRTA1) and myeloid nuclear differentiation antigen (MNDA) expression in the diagnosis and classification of marginal zone lymphomas (MZLs). METHODS: IRTA1 was examined using a novel RNA in situ hybridization assay and MNDA expression determined by immunohistochemistry in 127 small B-cell neoplasms, including 80 cases of MZL. RESULTS: IRTA1 expression was detected in 31 (42%) of 74 MZLs vs one (2%) of 43 other small B-cell neoplasms (P < .001). MNDA staining was positive in 51 (64%) of 79 MZLs vs 21 (45%) of 46 non-MZLs (P = .06). MNDA expression was particularly uncommon in follicular lymphoma (3/14, 21%; P = .003 vs MZL). There was no association between MNDA and IRTA1 expression and the presence of monocytoid cytology. IRTA1 expression was less frequent in cases with a diffuse growth pattern. CONCLUSIONS: IRTA1 and MNDA are useful markers in the differential diagnosis of MZLs.

Abstract BACKGROUND: Mitochondrial dysfunction has been implicated in the pathologies of a number of retinal degenerative diseases in both the outer and inner retina. In the outer retina, photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and sensitivity to oxidative stress. However, it is unclear how defective mitochondrial biogenesis affects neural development and contributes to neural degeneration. In this report, we investigated the in vivo function of nuclear respiratory factor 1 (Nrf1), a major transcriptional regulator of mitochondrial biogenesis in both proliferating retinal progenitor cells (RPCs) and postmitotic rod photoreceptor cells (PRs). METHODS: We used mouse genetic techniques to generate RPC-specific and rod PR-specific Nrf1 conditional knockout mouse models. We then applied a comprehensive set of tools, including histopathological and molecular analyses, RNA-seq, and electroretinography on these mouse lines to study Nrf1-regulated genes and Nrf1's roles in both developing retinas and differentiated rod PRs. For all comparisons between genotypes, a two-tailed two-sample student's t-test was used. Results were considered significant when P < 0.05. RESULTS: We uncovered essential roles of Nrf1 in cell proliferation in RPCs, cell migration and survival of newly specified retinal ganglion cells (RGCs), neurite outgrowth in retinal explants, reconfiguration of metabolic pathways in RPCs, and mitochondrial morphology, position, and function in rod PRs. CONCLUSIONS: Our findings provide in vivo evidence that Nrf1 and Nrf1-mediated pathways have context-dependent and cell-state-specific functions during neural development, and disruption of Nrf1-mediated mitochondrial biogenesis in rod PRs results in impaired mitochondria and a slow, progressive degeneration of rod PRs. These results offer new insights into the roles of Nrf1 in retinal development and neuronal homeostasis and the differential sensitivities of diverse neuronal tissues and cell types of dysfunctional mitochondria. Moreover, the conditional Nrf1 allele we have generated provides the opportunity to develop novel mouse models to understand how defective mitochondrial biogenesis contributes to the pathologies and disease progression of several neurodegenerative diseases, including glaucoma, age-related macular degeneration, Parkinson's diseases, and Huntington's disease.

Zika virus (ZIKV) has elicited global concern due to its unique biological features, unusual transmission routes, and unexpected clinical outcomes. Although ZIKV transmission through anal intercourse has been reported in humans, it remains unclear if ZIKV is detectable in the stool, if it can infect the host through the anal canal mucosa, and what the pathogenesis of such a route of infection might be in the mouse model. Herein, we demonstrate that ZIKV RNA can be recovered from stools in multiple mouse models, as well as from the stool of a ZIKV patient. Remarkably, intra-anal (i.a.) inoculation with ZIKV leads to efficient infection in both Ifnar1-/- and immunocompetent mice, characterized by extensive viral replication in the blood and multiple organs, including the brain, small intestine, testes, and rectum, as well as robust humoral and innate immune responses. Moreover, i.a. inoculation of ZIKV in pregnant mice resulted in transplacental infection and delayed fetal development. Overall, our results identify the anorectal mucosa as a potential site of ZIKV infection in mice, reveal the associated pathogenesis of i.a. infection, and highlight the complexity of ZIKV transmission through anal intercourse.

Epithelial tissues, such as the skin, rely on cellular plasticity of stem cells (SCs) from different niches to restore tissue function after injury. How these molecularly and functionally diverse SC populations respond to injury remains elusive. Here, we genetically labeled Lgr5- or Lgr6-expressing cells from the hair follicle bulge and interfollicular epidermis (IFE), respectively, and monitored their individual transcriptional adaptations during wound healing using single-cell transcriptomics. Both Lgr5 and Lgr6 progeny rapidly induced a genetic wound signature that, for Lgr5 progeny, included the remodeling of receptors to permit interactions with the wound environment, a property that Lgr6 progeny possessed even before wounding. When contributing to re-epithelialization, Lgr5 progeny gradually replaced their bulge identity with an IFE identity, and this process started already before Lgr5 progeny left the bulge. Altogether, this study reveals how different SCs respond and adapt to a new environment, potentially explaining cellular plasticity of many epithelial tissues.

Progress in understanding the molecular bases of human health and disease in recent decades has flourished making it possible for the field of gene therapy (GT) to offer new possibilities for treating, and even curing, a plethora of medical conditions such as monogenic disorders and metabolic diseases. GT is a therapeutic intervention to genetically alter or modify living cells by means of gene delivery achieved using either viral vectors or nonviral vectors, with adeno-associated virus (AAV) vectors constituting market-share majority. Although GT is conceptually attractive, adverse and even fatal iatrogenic complications have marred the initial enthusiasm of clinical successes. The properties of investigational AAV-based GT may pose safety concerns unique from those of small molecule drugs and other macromolecular biologics, such as ectopic or unregulated expression of the transgene, long-term persistence, and off-target distribution. Herein, we discuss considerations in the design of a comprehensive preclinical safety program for AAV-based GT prior to administration in humans.