Probes for INS

Actinic keratosis (AK) is considered as precursor lesion of invasive squamous cell carcinoma. Molecular studies on AK are limited because of too small size of the biopsy specimen to obtain enough DNA or RNA.Twenty biopsy cases of AK, followed by second same-sited biopsies, were included. Ten cases were diagnosed with total regression (regression group), while the other 10 were diagnosed with invasive carcinoma (progression group) in the follow-up biopsies. Using digital spatial profiling (DSP) technology, whole-gene expression analysis defined by specific regions of interest was performed for all 20 cases. After the clinicopathological features were assessed, separate and integrated analyses of these features and gene expression patterns were performed using machine-learning technology. All analyses were performed on both lesion keratinocytes (KT) and infiltrated stromal lymphocytes (LC).Among the 18,667 genes assessed, 33 and 72 differentially expressed genes (DEGs) between the regression and progression groups were found in KT and LC respectively. The primary genes distinguishing the two groups were KRT10 for KT and CARD18 for LC. Clinicopathological features were weaker in risk stratification of AK progression than the gene expression patterns. Pathways associated with various cancers were upregulated in the progression group of KT, whereas the nucleotide-binding oligomerization domain (NOD)-like receptor signalling pathway was upregulated in the progression of LC.Gene expression patterns were effective for risk stratification of AK progression, and their distinguishing power was higher than that of clinicopathological features.

The epidermis is a barrier that prevents water loss while keeping harmful substances from penetrating the host. The impermeable cornified layer of the stratum corneum is maintained by balancing continuous turnover driven by epidermal basal cell proliferation, suprabasal cell differentiation, and corneal shedding. The epidermal desquamation process is tightly regulated by balance of the activities of serine proteases of the Kallikrein-related peptidases (KLK) family and their cognate inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI), which is encoded by the serine peptidase inhibitor Kazal type 5 gene. Imbalance of proteolytic activity caused by a deficiency of LEKTI leads to excessive desquamation due to increased activities of KLK5, KLK7, and KLK14 and results in Netherton syndrome (NS), a debilitating condition with an unmet clinical need. Increased activity of KLKs may also be pathological in other dermatoses such as atopic dermatitis (AD). Here, we describe the discovery of inhibitory antibodies against murine KLK5 and KLK7 that could compensate for the deficiency of LEKTI in NS. These antibodies are protective in mouse models of NS and AD and, when combined, promote improved skin barrier integrity and reduced inflammation. To translate these findings, we engineered a humanized bispecific antibody capable of potent inhibition of human KLK5 and KLK7. A crystal structure of KLK5 bound to the inhibitory Fab revealed that the antibody binds distal to its active site and uses a relatively unappreciated allosteric inhibition mechanism. Treatment with the bispecific anti-KLK5/7 antibody represents a promising therapy for clinical development in NS and other inflammatory dermatoses.

PRION DISEASES are a group of incurable neurodegenerative diseases caused by the prions, affecting both humans and animals. The atypical folding and aggregation of the soluble cellular prion proteins (PrPC) into scrapie isoform (PrPSc) in the CNS, results in brain damage and other symptoms associated with it. Different therapeutic approaches ranging from organic compounds to antibodies have been proposed, including stalling PrPCto PrPScconversion, increasing PrPScremoval, and/or PrPCstabilization using different research methodologies like cell-therapy, immunotherapy, pharmacotherapy and compounds ranging from chemicals to proteins have been studied to target the disease with special attention to PrPScaggregation inhibition. Compounds destabilizing PrPScand reducing infection have also been identified. A few important CHEMICAL COMPOUNDS (Sulfated polyanions, diazo dyes, Phenothiazine derivative, Cyclic Tetrapyrroles, Diphenylmethane derivatives, Diphenylpyrazole derivatives, Indole-3-Glyoxylamides, 2-Aminothiazoles, Carbazole derivatives, Benzoxazole derivative, Ethanolamine, Dimethyl sulfoxide); REPURPOSED DRUGS (Quinacrine, _Chlorpromazine_, Celecoxib, Flupirtine, Imatinib, Efavirenz, Simvastatin, Glimepiride, Doxycycline); NATURAL PRODUCTS (Polydatin, Curcumin, Resveratrol, Epigallocatechin gallate, Cannabidiol, Baicalein, Hinokitiol, Ginsenoside, Bile acids) and ANTI-PRION ANTIBODIES (6H4, D13, D18, 8B4, 8H4, ICSM18, ICSM35, POM1-2, 4H11, 44B1). All these compounds have displayed anti-prion activity _in vitro_, but only a few were effective _in vivo_. Recently, encouraging results of a PRION PROTEIN MONOCLONAL ANTIBODY (an IgG4κ isotype; PNR100) in a clinical trial study on CJD patients have been announced which stabilized PrPCand increased the survival in infected mice. After such promising results, PRN100 will be evaluated for Phase-II trials. The success story doesn’t end here as another promising molecule MC and GN8 are ready for human clinical trials on prion-diseases while Anle138b is in Phase 1b for PD patients. The ineffectiveness of most of the molecules tested was either due to inability to cross BBB, toxicity or transitory accumulation of drug resistant prions. Hence, it is incredibly important to learn from the backstory. By understanding what all has already been done, what is the mechanism of drug action, why the clinical trials failed, we can comprehend prion diseases better and chalk out the right direction leading to more effective treatment. Also, targeting more than one pathway involved in prion diseases may provide synergistic benefits.

Previous studies have demonstrated effects of racialized minority status on thermal pain sensitivity, sensibility, and tolerance. However, there is limited evidence demonstrating effects of minority status on painful punctate mechanical stimuli and self-report pain. We analyzed the effects of racialized minority status on heat pain sensitivity, sensibility to painful heat and punctate mechanical stimuli, and Pain Sensitivity Questionnaire (PSQ) scores. Our secondary purpose was to test face validity of the PSQ in a US population. Using quantitative sensory testing for painful heat and punctate mechanical stimuli (forces: 64, 128, 256 and 512 mN), and self-report PSQ, we evaluated pain sensitivity and sensibility in 134 healthy participants (34 Asian, 25 Black, and 75 White). We used linear mixed models to analyze outcomes allowing maximal inclusion of incomplete data sets. Racialized minority status was associated with greater heat pain sensitivity (F=7.63; p=0.00074) and PSQ scores (F=15.45; p=9.84 × 10-7) but had no effect on painful suprathreshold heat (model improvement by addition of race: Χ2=2.199; p=0.333) or punctate mechanical stimuli (F=1.50; p=0.229). Face validity of the PSQ in racialized minorities was limited by differential experience of individual items (F=19.87; p=3.28 × 10-8). Ratings of painful suprathreshold heat (R=0.204; p=0.00020) and punctate mechanical stimuli (R=0.333; p=0.00062) positively correlated with PSQ scores. Consistent with previous research, sensitivity to painful heat was affected by racialized minority status. In contrast, there was no significant effect of racialized minority status on suprathreshold painful heat or punctate mechanical stimuli. Certain items of the PSQ are inapplicable to healthy participants from racialized minority groups. NIH National Institute of Nursing Research P30NR014129.

Sudden, unpredictable, severe acute pain episodes are the most common sickle cell disease (SCD) complication. Some SCD patients experience frequent pain episodes while others experience rare episodes. Knowledge of the biology driving this variability is limited. Using gene transcription analyses, we previously showed an elevated inflammatory response is associated with increased SCD pain episode frequency. We sought to replicate these findings in a larger SCD cohort and identify hub genes closely associated with increased pain frequency. We conducted plasma-induced transcription analyses in 132 SCD patients (baseline health) and 60 Black controls (4-21 years, both groups). 3028 differentially expressed genes between SCD patients and controls were retained for subsequent analyses with Weighted Gene Co-Expression Network Analysis (WGCNA). WGCNA was used to define modules (functionally grouped genes) and we correlated these modules with number of pain episodes requiring health care utilization in prior three years. Of 11 identified modules, four showed significant correlation with number of pain episodes. Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for ontological analysis of the four significant modules and key biological processes identified were inflammatory response and cellular response to lipopolysaccharide. Cytoscape was used to construct a protein-protein interaction network and the 10 top hub genes identified in hierarchical order were: TNF, CCR5, CCR1, CCL2, CXCL2, ITGAM, CCL7, CXCL3, TLR2 and MMP9. These genes, as part of the inflammatory response, support the immune system contributes to increased pain episode frequency. Identified hub genes may be leveraged as therapeutic targets for reducing SCD pain episodes. 1R61NS114954-01.

Background: HIV infection persists predominantly within follicular helper CD4+ T cell-rich B cell follicles of lymphoid tissues. Cytotoxic CD8+ T cells, which are associated with natural control of HIV infection in peripheral blood, are relatively excluded from this niche, representing a potential barrier to cellular immunity and HIV cure. To better understand the mechanisms of HIV control within lymph nodes (LN), we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics and transcriptional profiles of LN-resident virus-specific and CXCR5-expressing follicular CD8+ T cells (fCD8) in persons who control HIV without medications. Methods: We obtained paired excisional inguinal LN biopsies and peripheral blood (PB) from 19 spontaneous HIV controllers and 17 HIV+ individuals on long-term ART. HIV-specific CD8+ T cell responses were identified by IFN-γ ELISpot and functional response to antigenic stimulation was measured by flow cytometry and CFSE-based proliferation assay. Clonotypic compartmentalization and transcriptional signatures associated with localization of HIV-specific CD8+ T cells were assessed via TCR and RNA-sequencing. Spatial relationships between ongoing viral replication and fCD8 cytotoxic effector potential in GCs were measured by HIV gagpol RNAscope and immunofluorescence on fixed LN sections. Results: Antigen-induced HIV-specific CD8+ T cell proliferation and cytolytic effector upregulation consistently distinguished spontaneous controllers from noncontrollers in PB (p=0.03) and LN (p=0.04). HIV-specific CD8+ T cells from both compartments shared TCR clonotypic composition (Morisita-Horn Similarity Index 0.8-1.0), consistent with ongoing infiltration from circulation. Migration into LNs was associated with gene signatures of inflammatory chemotaxis and antigen-induced effector function. The cytolytic effectors perforin and granzyme B were elevated among virus-specific CXCR5 + fCD8 s (p

Facial grimaces are now commonly used to quantify spontaneous pain in mice and other mammalian species, but scoring remains subjective and relies on humans with very different levels of proficiency. Here, we developed a Mouse Grimace Scale (MGS) for black-coated (C57BL/6) mice consisting of four facial action units (orbitals, nose, ears, whiskers). We used this scale to generate ground truth data from over 70,000 images of black mice in different settings. With this large data set, we developed a deep neural network and cloud-based software platform called PainFace (http://painface.net) that accurately scores facial grimaces of black mice on a 0-8 scale. PainFace generates over two orders of magnitude more MGS data than humans can realistically achieve, and at superhuman speed. By analyzing the frequency distribution of grimace scores, we found that mice spent >7x more time in a high grimace state following laparotomy surgery relative to sham surgery controls. The analgesic carprofen reduced the amount of time animals spent in this high grimace state after surgery. Specific facial action unit score combinations were overrepresented following laparotomy surgery, suggesting that characteristic facial expressions are associated with a high grimace state. While this study is focused on mice, PainFace was designed to simplify, standardize, and scale up grimace analyses with many other mammalian species, including humans. This work was supported by a grant from the NINDS (R01NS114259) to M.J.Z. NSF GRFP awarded to R.P.P.

Investigation of spontaneous- so-called‘resting-state'-activity of the central nervous system with functional magnetic resonance imaging (fMRI) holds great clinical potential to identify possible prognostic and diagnostic biomarkers for pain disorders and provides novel insights into the functional architecture of the central nervous system. Although previous resting-state studies in humans characterized functional networks of the brain and recently of the spinal cord, the resting-state networks of the entire central nervous system-delineating the interaction between the cord and the brain-have not been well characterized, possibly due to technical difficulties of corticospinal fMRI. Given the important role of ascending and descending pathways to understand disorders chronic pain disorders, here we characterize the resting-state functional connectivity networks along the whole neuroaxis in 29 healthy humans as a step prior to clinical studies. 31 brain slices and 12 cervical spinal cord slices from were acquired with a tailored fMRI sequence on a 3T system. Time courses of dorsal and ventral horns were used to map spinal cord's connection to the brain via a seed-based approach. Functional connectivity maps revealed that dorsal and ventral horn are significantly correlated with sensory and motor areas in the brain such as primary and somatosensory and motor cortices as well as with the thalamus. At the same time, we have observed that they somewhat distinct functional connectivity profiles in line with their functional segregation; frontal, occipital and insular cortices were more synchronized with ventral horn whereas caudate and thalamus appeared to be more synchronized with dorsal horn reflecting their functional division. NIH NINDS R01 NS109450.

METHODS : Gene expression of ABCA1 and ApoA1 on human donor tissue and iPSC-RPE were examined by qPCR (n=3). Bulk RNAseq examined transcript changes in key RCT genes on donor retinas across different stages of disease progression. RNAscope probes (ACDBio) were designed against abca1 transcripts with appropriate mismatch controls. Neutral lipid stain with oil-red O on 10um cryo-sections of abca1 KO and wild type (WT) eyes (N= 5). Two siRNAs knocked down abca1 in iPSC-RPE cells to assess abca1 contribution to cholesterol efflux (n=3). Samples were analyzed with the cholesterol efflux kit (ab196985) and compared to non-targeting control siRNAs. Histological analysis of ABCA1 protein using anti-ABCA1 (Invitrogen-MA516026) on human donor retinas (AMD1 vs AMD3).

Background: Adrenocortical carcinoma (ACC) is a very rare and aggressive, endocrine malignancy with still limited treatment options. Approximately 60% of patients with ACC show endogenous glucocorticoid excess which could be one potential cause, why first clinical trials with immunotherapies, like immune checkpoint inhibitors, showed only modest results. Due to the lack of an ACC-specific antigen structure, other immunotherapeutic approaches, like specialized cancer treatments using chimeric antigen receptor (CAR) therapy in ACC, have not been tested so far. In this study, we evaluated the expression of a new enticing tumor antigen (TA*) structure and investigated the effect of TA-specific CAR-T cells _in vitro_.

The most abundant circulating CD3- population in patients with cHL was a newly identified monocyte subset with increased expression of multiple immunosuppressive and tumorigenic cytokines and chemokines, PD-L1 and SIRPa. This newly identified monocytic population was virtually absent from the blood of healthy donors. RNAscope analysis of the intact tumor microenvironment localized these tumor-infiltrating monocytes/macrophages to the immediate proximity of HRS cells. Monocytes from patients whose disease progressed following PD-1 blockade expressed significantly higher levels of immunosuppressive cytokine/chemokine signature which led to the development of a predictive transcriptional assay. We identified a comparable circulating monocyte population and transcriptional signature associated with unresponsiveness to PD-1 blockade in an additional solid tumor underscoring the broad-based significance of these findings.

Vascular co-option is a non-angiogenic mechanism whereby tumor growth and progression move on by hijacking the pre-existing and nonmalignant blood vessels and is employed by various tumors to grow and metastasize.The histopathological identification of co-opted blood vessels is complex, and no specific markers were defined, but it is critical to develop new and possibly more effective therapeutic strategies. Here, in glioblastoma, we show that the co-opted blood vessels can be identified, by double immunohistochemical staining, as weak CD31+ vessels with reduced P-gp expression and proliferation and surrounded by highly proliferating and P-gp- or S100A10-expressing tumor cells. Results can be quantified by the Aperio Colocalization algorithm, which is a valid and robust method to handle and investigate large data sets.