Chimeric antigen receptor (CAR) T cells are a new and powerful class of cancer immunotherapeutics that have shown potential for the treatment of hematopoietic malignancies. The tremendous promise of this approach is tempered by safety concerns, including potentially fatal neurotoxicity, sometimes but not universally associated with cytokine release syndrome. We describe the postmortem examination of a brain from a 21-year-old patient with relapsed pre-B cell acute lymphoblastic leukemia (ALL) who died from fulminant cerebral edema following CAR T-cell infusion. We found a range of changes that included activation of microglia, expansion of perivascular spaces by proteinaceous exudate, and clasmatodendrosis-a beading of glial fibrillary acidic protein consistent with astrocyte injury. Notably, within the brain parenchyma, we identified only infrequent T cells and did not identify ALL cells or CAR T cells. The overall findings are nonspecific but raise the possibility of astrocyte and blood-brain barrier dysfunction as a potential etiology of fatal CAR T-cell neurotoxicity in this patient.

Epstein-Barr virus (EBV) is a herpesvirus associated with approximately 1% of tumors worldwide. Although EBV is consistently detected in nasopharyngeal carcinoma(NPC) biopsy, it is hardly detected in normal nasopharyngeal epithelium. The mechanism how virus establishes latent infection in tumor epithelial cells, including the source of virus and the route of entry, has not been fully elucidated largely due to the lack of appropriate in vivo models. We herein aim to investigate the potential route that epithelial cells are infected with EBV. To this end, we established in vivo model system by injection of cell-free EBV or EBV producer line Akata cells together with EBV negative NPC line HONE-1 cells. Akin to in vitro infections, we presented the first in vivo evidence that cell-mediated transfer infection via Akata cells was much more efficient than cell-free virus. These cells then expressed the EBV latency-associated small RNA EBERs, but not lytic antigens, such as BZLF1. However, when cells were inoculated at separate sites, EBV producer line Akata cell failed to demonstrate the ability of migrating from distant location to interact with HONE-1 cell to establish latent infection. In conclusion, cell-cell contact is critical for in vivo EBV infection of nasopharyngeal epithelial cells.

Microdeletions involving TBX1 result in variable congenital malformations known collectively as 22q11.2 deletion syndrome (22q11.2DS). Tbx1-deficient mice and zebrafish recapitulate several disease phenotypes, including pharyngeal arch artery (PAA), head muscle (HM), and cardiac outflow tract (OFT) deficiencies. In zebrafish, these structures arise from nkx2.5+ progenitors in pharyngeal arches 2–6. Because pharyngeal arch morphogenesis is compromised in Tbx1-deficient animals, the malformations were considered secondary. Here, we report that the PAA, HM, and OFT phenotypes in tbx1 mutant zebrafish are primary and arise prior to pharyngeal arch morphogenesis from failed specification of the nkx2.5+pharyngeal lineage. Through in situ analysis and lineage tracing, we reveal that nkx2.5 and tbx1 are co-expressed in this progenitor population. Furthermore, we present evidence suggesting that gdf3-ALK4 signaling is a downstream mediator of nkx2.5+ pharyngeal lineage specification. Collectively, these studies support a cellular mechanism potentially underlying the cardiovascular and craniofacial defects observed in the 22q11.2DS population.

Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating a broad spectrum of hematological disorders including cancer. However, the limited number of HSCs in a single hUCB unit restricts its widespread use. Although extensive efforts have led to multiple methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it remains unknown whether it is possible to simultaneously manipulate the large number of targets essential for stem cell self-renewal. Recent studies indicate that N6-methyladenosine (m6A) modulates the expression of a group of mRNAs critical for stem cell-fate determination by influencing their stability. Among several m6A readers, YTHDF2 is recognized as promoting targeted mRNA decay. However, the physiological functions of YTHDF2 in adult stem cells are unknown. Here we show that following the conditional knockout (KO) of mouse Ythdf2 the numbers of functional HSC were increased without skewing lineage differentiation or leading to hematopoietic malignancies. Furthermore, knockdown (KD) of human YTHDF2 led to more than a 10-fold increase in the ex vivo expansion of hUCB HSCs, a fivefold increase in colony-forming units (CFUs), and more than an eightfold increase in functional hUCB HSCs in the secondary serial of a limiting dilution transplantation assay. Mapping of m6A in RNAs from mouse hematopoietic stem and progenitor cells (HSPCs) as well as from hUCB HSCs revealed its enrichment in mRNAs encoding transcription factors critical for stem cell self-renewal. These m6A-marked mRNAs were recognized by Ythdf2 and underwent decay. In Ythdf2 KO HSPCs and YTHDF2 KD hUCB HSCs, these mRNAs were stabilized, facilitating HSC expansion. Knocking down one of YTHDF2′s key targets, Tal1 mRNA, partially rescued the phenotype. Our study provides the first demonstration of the function of YTHDF2 in adult stem cell maintenance and identifies its important role in regulating HSC ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal, thus identifying potential for future clinical applications.

Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.

Targeted inhibition of programmed cell death-1 (PD-1) and its ligand (PD-L1) has emerged as first-line therapy for advanced non-small cell lung cancer. While patients with high PD-L1 expression have improved outcomes with anti-PD-1/PD-L1 directed therapies, use as a predictive biomarker is complicated by robust responses in some patients with low-level expression. Furthermore, reported PD-L1 levels in lung cancers vary widely and discrepancies exist with different antibodies. PD-L1 expression was thus compared by immunohistochemistry (IHC) versus RNA in situ hybridization (ISH) in 112 lung cancers by tissue microarray: 51 adenocarcinoma, 42 squamous cell carcinoma, 9 adenosquamous carcinoma, 5 carcinoid, 3 undifferentiated large-cell carcinoma, 1 large-cell neuroendocrine carcinoma, and 1 small cell carcinoma. At least 1% tumor cell staining was considered positive in each modality. A positive concordance of only 60% (67/112) was found between IHC and ISH. 50% (56/112) were positive by IHC and 50% (56/112) by ISH, however 20% (22/112) were ISH positive but IHC negative. Conversely, 21% (23/112) were IHC positive but ISH negative. There was no significant stratification of PD-L1 positivity by histologic subtype. A trend of more PD-L1 positive stage I cancers identified by ISH versus IHC was observed, however was not statistically significant [50% (27/54) by IHC and 64% (35/55) by ISH, P=.18]. No significant difference in survival was identified, with an average of 5.3months in IHC versus 5.2months in ISH positive cases. The results demonstrate discordance between PD-L1 RNA levels and protein expression in non-small cell lung cancers, warranting comparison as predictive biomarkers.

We and others have reported that taste cells in taste buds express many peptides in common with cells in the gut and islets of Langerhans in the pancreas. Islets and taste bud cells express the hormones glucagon and ghrelin, the same ATP-sensitive potassium channel (KATP) responsible for depolarizing the insulin secreting beta (β) cell during glucose-induced insulin secretion, as well as the propeptide processing enzymes PC1/3 and PC2. Given the common expression of functionally specific proteins in taste buds and islets, it is surprising that no one has investigated whether insulin is synthesized in taste bud cells until now. Using immunofluorescence, we demonstrate the presence of insulin in mouse, rat and human taste bud cells. We further prove that insulin is synthesized in individual taste buds and not taken up from the parenchyma by: detection of the post-processing insulin molecule C-peptide and green fluorescence protein (GFP) in taste cells of both insulin 1- and insulin 2-GFP mice, and the presence of the mouse insulin transcript by in situ hybridization (ISH). In addition to our cytology data we measured the level of insulin transcript by qRT-PCR in the anterior and posterior lingual epithelium. These analyses show insulin is translated in the circumvallate and foliate papillae in the posterior but only insulin transcript was detected in the anterior fungiform papillae of rodent tongue. Thus, some taste cells are insulin synthesizing cells generated from a continually replenished source of precursor cells in adult mammalian lingual epithelium.

Subtype C HIV-1 is responsible for the largest proportion of people living with HIV-1 infection. However, there is limited information about the roles of the brain and its cell types as a potential sanctuary for this subtype and how the sanctuary may be affected by the administration of anti-retroviral therapy (ART). To address this issue, we collected postmortem brain tissues from ART treated HIV-1 infected Zambian individuals who experienced complete viral suppression and those who did not. Tissues from various brain compartments were collected from each individual as frozen and formalin-fixed paraffin embedded brain specimens, for detection and quantification of HIV-1 genomes and identification of the infected cell type. Genomic DNA and RNA were extracted from frozen brain tissues. The extracted DNA and RNA were then subjected to droplet digital PCR for HIV-1 quantification. RNA/DNAscope in situ hybridization (ISH) for HIV-1 was performed on formalin-fixed paraffin embedded brain tissues in conjugation with immunohistochemistry to identify the infected cell types. Droplet digital PCR revealed that HIV-1 gag DNA and RNA were detectable in half of the cases studied regardless of ART success or failure. The presence of HIV-1 lacked specific tissue compartmentalization since detection was random among various brain tissues. When combined with immunohistochemistry, RNA/DNAscope ISH demonstrated co-localization of HIV-1 DNA with CD68 expressing cells indicative of microglia or peripheral macrophage. Our study showed that brain is a potential sanctuary for subtype C HIV-1, as HIV-1 can be detected in the brain of infected individuals irrespective of ART treatment outcome and no compartmentalization of HIV-1 to specific brain compartments was evident.

The sympathetic nervous system (SNS) serves to maintain homeostasis of vital organ systems throughout the body, and its dysfunction plays a major role in human disease. The SNS also links the central nervous system to the immune system during different types of stress via innervation of the lymph nodes, spleen, thymus, and bone marrow. Previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP, gene name adcyap1) exhibits anti-inflammatory properties in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Because PACAP is known to regulate SNS function, we hypothesized that part of the immunoprotective action of PACAP is due to its neuromodulatory effects on sympathetic neurons. To examine this, we used an inducible, targeted approach to conditionally disrupt not only the PACAP-preferring PAC1 receptor gene (adcyap1r1) in dopamine β-hydroxylase-expressing cells, which includes postganglionic sympathetic neurons, but also catecholaminergic neurons in the brain and adrenomedullary chromaffin cells. In contrast to our previous EAE studies using PACAP global knockout mice which developed severe and prolonged EAE, we found that mice with conditional loss of PAC1 receptors in catecholaminergic cells developed a delayed time course of EAE with reduced helper T cell type 1 (Th1) and Th17 and enhanced Th2 cell polarization. At later time points, similar to mice with global PACAP loss, mice with conditional loss of PAC1 exhibited more severe clinical disease than controls. The latter was associated with a reduction in the abundance of thymic regulatory T cells (Tregs). These studies indicate that PAC1 receptor signaling acts in catecholaminergic cells in a time-dependent manner. At early stages of disease development, it enhances the ability of the SNS to polarize the Th response towards a more inflammatory state. Then, after disease is established, it enhances the ability of the SNS to dampen the inflammatory response via Tregs. The lack of concordance in results between global PACAP KO mice and mice with the PAC1 deletion targeted to catecholaminergic cells during early EAE may be explained by the fact that PACAP acts to regulate inflammation via multiple receptor subtypes and multiple targets, including inflammatory cells.

The striatum integrates motor behavior using a well-defined microcircuit whose individual components are independently affected in several neurological diseases. The glial cell line-derived neurotrophic factor (GDNF), synthesized by striatal interneurons, and Sonic hedgehog (Shh), produced by the dopaminergic neurons of the substantia nigra (DA SNpc), are both involved in the nigrostriatal maintenance but the reciprocal neurotrophic relationships among these neurons are only partially understood. To define the postnatal neurotrophic connections among fast-spiking GABAergic interneurons (FS), cholinergic interneurons (ACh), and DA SNpc, we used a genetically induced mouse model of postnatal DA SNpc neurodegeneration and separately eliminated Smoothened (Smo), the obligatory transducer of Shh signaling, in striatal interneurons. We show that FS postnatal survival relies on DA SNpc and is independent of Shh signaling. On the contrary, Shh signaling but not dopaminergic striatal innervation is required to maintain ACh in the postnatal striatum. ACh are required for DA SNpc survival in a GDNF-independent manner. These data demonstrate the existence of three parallel but interdependent neurotrophic relationships between SN and striatal interneurons, partially defined by Shh and GDNF. The definition of these new neurotrophic interactions opens the search for new molecules involved in the striatal modulatory circuit maintenance with potential therapeutic value.

HPV-related multiphenotypic sinonasal carcinoma (HMSC) is associated with high-risk human papillomavirus (HR-HPV) infection. Using HR-HPV mRNA in situ hybridization (ISH), we reported six new HMSC cases and compared their histopathology with that of sinonasal adenoid cystic carcinoma (ACC). Using p16 immunohistochemistry (IHC) and HR-HPV ISH, we retrospectively identified six HMSC cases. All HMSC cases were positive for HR-HPV mRNA ISH and p16 IHC. Two HMSC cases had overlying atypical squamous epithelium and one also had invasive squamous cell carcinoma (SCC). All HMSC were SOX10-positive whereas the overlying atypical squamous epithelium and the SCC were SOX10-negative. One atypical HMSC-like case was also identified which was positive for HR-HPV mRNA ISH, HR-HPV DNA ISH, SOX10 IHC, but negative for p16 IHC. This study showed that HR-HPV mRNA ISH was a useful tool to diagnose HMSC and had stronger signals than HR-HPV DNA ISH. HR-HPV E6/E7 mRNA could be identified in the overlying atypical squamous epithelium as well as the invasive SCC. A combination of p16 and SOX10 IHC will be a useful screening panel for HMSC followed by confirmatory HR-HPV mRNA ISH test.

Long noncoding RNAs (LncRNAs) have been reported to play vital roles in non-small cell lung cancer (NSCLC). Recently, LncRNA/VPS9D1-AS1 has been reported to be overexpressed in various cancers. In this study, we aimed to investigate its expression pattern and clinical significance and further evaluate its prognostic value for NSCLC. VPS9D1-AS1 expression was examined in 184 NSCLC patients using a highly sensitive in situ hybridization protocol (RNAscope), and the expression values were correlated with the clinicopathological features. Another cohort including 12 NSCLC patients was used to validate the differential expression of VPS9D1-AS1 by qRT-PCR. TCGA datasets were further used to validate the main findings. We found that the levels of VPS9D1-AS1 were significantly higher in cancer tissues than in paired normal tissues from both lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC) (P < 0.001). Importantly, the levels of VPS9D1-AS1 in patients with lung SCC were significantly higher than those in patients with lung ADC. The high levels of VPS9D1-AS1 were found to be associated with cancer lymph node metastasis (P = 0.020). Prognostic analysis revealed that the survival time for SCC patients with high levels of VPS9D1-AS1 was significantly shorter than that of patients with low levels of VPS9D1-AS1 (P = 0.007). Therefore, our findings suggest that the overexpression of VPS9D1-AS1 serves as a promising biomarker to predict the prognosis of NSCLC.