Fibroblast growth factor 23 (FGF23) secreted by osteocytes is a circulating factor essential for phosphate homeostasis. High plasma FGF23 levels are associated with cardiovascular complications and mortality. Increases of plasma FGF23 in uremia antedate high levels of phosphate, suggesting a disrupted feedback regulatory loop or an extra-skeletal source of this phosphatonin. Since induction of FGF23 expression in injured organs has been reported we decided to examine the regulation of FGF23 gene and protein expressions in the kidney and whether kidney-derived FGF23 contributes to the high plasma levels of FGF23 in uremia. FGF23 mRNA was not detected in normal kidneys, but was clearly demonstrated in injured kidneys, already after four hours in obstructive nephropathy and at 8 weeks in the remnant kidney of 5/6 nephrectomized rats. No renal extraction was found in uremic rats in contrast to normal rats. Removal of the remnant kidney had no effect on plasma FGF23 levels. Well-known regulators of FGF23 expression in bone, such as parathyroid hormone, calcitriol, and inhibition of the FGF receptor by PD173074, had no impact on kidney expression of FGF23. Thus, the only direct contribution of the injured kidney to circulating FGF23 levels in uremia appears to be reduced renal extraction of bone-derived FGF23. Kidney-derived FGF23 does not generate high plasma FGF23 levels in uremia and is regulated differently than the corresponding regulation of FGF23 gene expression in bone.

Basolateral amygdala (BLA) principal cells are capable of driving and antagonizing behaviors of opposing valence. BLA neurons project to the central amygdala (CeA), which also participates in negative and positive behaviors. However, the CeA has primarily been studied as the site for negative behaviors, and the causal role for CeA circuits underlying appetitive behaviors is poorly understood. Here, we identify several genetically distinct populations of CeA neurons that mediate appetitive behaviors and dissect the BLA-to-CeA circuit for appetitive behaviors. Protein phosphatase 1 regulatory subunit 1B+ BLA pyramidal neurons to dopamine receptor 1+ CeA neurons define a pathway for promoting appetitive behaviors, while R-spondin 2+ BLA pyramidal neurons to dopamine receptor 2+ CeA neurons define a pathway for suppressing appetitive behaviors. These data reveal genetically defined neural circuits in the amygdala that promote and suppress appetitive behaviors analogous to the direct and indirect pathways of the basal ganglia.

Interleukin-1β (IL-1β) is known to trigger beta cell dysfunction in vitro and could potentially play a role during the pathogenesis of type 1 diabetes and type 2 diabetes. However, several clinical trials attempting to block IL-1β function have had minimal success. We therefore re-investigated local expression of IL-1β in human diabetic and non-diabetic pancreata. We obtained pancreatic tissue sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) including non-diabetic (n = 9), non-diabetic auto-antibody positive (AAb+, n = 5), type 1 diabetes (n = 6), and type 2 diabetes (n = 6) donors. Islets were systematically investigated for the presence of IL-1β mRNA by in situ hybridization and IL-1β protein by indirect immunofluorescence. We found that intra-islet IL-1β was produced at comparable level in both non-diabetic and diabetic donors. Interestingly, the main source for IL-1β was alpha cells but not beta cells. Our findings call into question the role of IL-1β in the diabetic pancreas as it has been proposed in previous literature. Additionally, our results regarding the localization of IL-1β should lead to further investigation into the role of IL-1β in the physiology of pancreatic alpha cells.

αKlotho (Klotho), a type I transmembrane protein and a coreceptor for Fibroblast growth factor-23, was initially thought to be expressed only in a limited number of tissues, most importantly the kidney, parathyroid gland and choroid plexus. Emerging data may suggest a more ubiquitous Klotho expression pattern which has prompted reevaluation of the restricted Klotho paradigm. Herein we systematically review the evidence for Klotho expression in various tissues and cell types in humans and other mammals, and discuss potential reasons behind existing conflicting data. Based on current literature and tissue expression atlases, we propose a classification of tissues into high, intermediate and low/absent Klotho expression. The functional relevance of Klotho in organs with low expression levels remain uncertain and there is currently limited data on a role for membrane-bound Klotho outside the kidney. Finally, we review the evidence for the tissue source of soluble Klotho, and conclude that the kidney is likely to be the principal source of circulating Klotho in physiology.

The intestine is maintained by stem cells, marked by LGR5 expression, located at the base of crypts. Genetically engineered mouse models have provided information about marker genes and stem cell pathways. Less is known about human intestinal stem cells due to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas, and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC) associated genes. Normal and neoplastic colon tissue organoids were analyzed for LGR5 expression by immunohistochemistry. LGR5-positive cells were isolated from 4 adenoma organoid lines and analyzed by RNA-sequencing. LGR5 expression in epithelium and stroma was associated with tumor stage. Integrating functional experiments with RNA-seq data from LGR5-positive adenoma organoid cells and normal colon, we associated expression of CRC-specific genes, including DKK4, with LGR5 expression. This system can be used to study LGR5-expressing cells in human tissue homeostasis and carcinogenesis.

The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1-/- mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1-/- mice or wild-type mice treated with anti-type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Canine parvovirus-2 (CPV-2) is nearly indistinguishable from feline panleukopenia virus (FPV) and is a well-known cause of viral myocarditis in young puppies; however, it is not known whether either FPV or CPV-2 naturally infects feline cardiomyocytes and causes myocarditis. Endomyocarditis (EMC) and left ventricular endomyocardial fibrosis (LVEF), clinically known as “endomyocardial restrictive cardiomyopathy,” are important feline heart diseases suspected to have an infectious etiology. A continuum is suggested with EMC representing the acute reaction to an unknown infectious agent and LVEF the chronic manifestation of repair. The purpose of this study was to determine (1) whether there is natural parvovirus infection of the feline myocardium and (2) whether parvoviral infection is associated with feline EMC and/or LVEF. In a retrospective study, polymerase chain reaction and sequencing for the parvovirus VP1/2 gene was performed on archived heart tissue from cats with endomyocardial disease and controls. Similar methods were used prospectively on myocardial tissues from shelter-source kittens. Although 8 of 36 (22%; 95% confidence interval [CI], 11%–40%) shelter kittens had parvoviral DNA in myocardial tissue, VP1/2 DNA was not detected in 33 adult cases or 34 controls (95% CI, 0% to ∼11%). These findings were confirmed by in situ hybridization: adult cats did not have detectable parvovirus DNA, although rare intranuclear signal was confirmed in 7 of 8 shelter-source kittens. In kittens, parvovirus was not significantly associated with myocarditis, and in situ hybridization signal did not colocalize with inflammation. Although infection of cardiomyocytes was demonstrated in kittens, these data do not support a role for parvovirus in EMC-LVEF.

Abstract

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression through their complex interactions with cancer cells. The secreted bone morphogenetic protein antagonist, gremlin1 (GREM1) is expressed by the CAFs of basal cell carcinomas (BCCs), and promotes the growth of cancer cells. In this study, we investigated the expression of GREM1 mRNAs in various benign and malignant skin tumors, including various BCC subtypes. Analysis by RNA in situ hybridization (ISH) revealed that fibroblasts in the scar tissue expressed GREM1 and α-smooth muscle actin (α-SMA), whereas resident fibroblasts in the dermis of the normal skin did not express GREM1. Real-time polymerase chain reaction analysis showed significantly higher GREM1 expression in skin cancers and pilomatricomas (PMCs) than in other benign skin tumors. Tissue microarrays analyzed by RNA ISH for GREM1 expression also demonstrated that 23% of BCCs, 42% of squamous cell carcinomas, 20% of melanomas, and 90% of PMCs were positive for GREM1 expression, whereas trichoepitheliomas, eccrine poromas, hidradenomas, and spiradenomas were negative for GREM1 expression. Most BCCs that were GREM1 expression positive were of desmoplastic or mixed subtypes, and GREM1 expression was localized to activated myofibroblasts at the tumoral-stromal interface. Interestingly, most PMCs harbored GREM1-expressing fibroblasts, probably because of the inflammatory responses caused by foreign body reactions to keratin. Additionally, in BCCs, stromal GREM1 expression had a strong correlation with CD10 expression. In conclusion, GREM1 is frequently expressed by myofibroblasts in scars or in the stroma of basal cell carcinomas, suggesting that GREM1 expression can be a marker for activated myofibroblasts in the cancer stroma or in scar tissue.

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.

Abstract: Objective: To investigate the potential involvement of secreted protein acidic and rich in cysteine (SPARC) in the progression of the breast tumor and to determine its association with outcome variables and matrix metalloproteinases (MMPs) expression in patients with breast carcinoma (BC). Methods: SPARC expression was examined in 8 pairs of BC tissues and surrounding normal tissues at mRNA and protein levels by qRT-PCR, RNAscope in situ hybridization (ISH), Western blotting, and immunohistochemistry techniques. Immunohistochemical staining of SPARC was done in 26 normal breasts, 76 ductal carcinoma in situ (DCIS), and 198 BC samples. In addition, immunohistochemical staining was performed for MMP-2 and MMP-9 in BC. Results: SPARC expression at mRNA and protein levels was significantly increased in BC tissues compared to the surrounding normal tissues (P < 0.05 and P < 0.01, respectively). RNAscope ISH and immunohistochemistry of SPARC confirmed an increase in SPARC expression in BC tissues compared with the normal tissues. Epithelial SPARC expression increased continuously from normal breast through DCIS to BC (P < 0.001). In patients with BC, high epithelial SPARC expression was associated with worse disease-free survival and overall survival (P = 0.002 and P = 0.048, respectively) and independently predicted worse disease-free survival (P = 0.002). Epithelial SPARC expression was significantly correlated with MMP-2 expression (P < 0.05). Conclusion: Up-regulation of SPARC contributes to breast tumor progression. SPARC expression may be a useful biomarker for the prognostic prediction in patients with BC. SPARC can control extracellular matrix degradation through up-regulation of MMP-2.