Development and characterization of an ETV1 rabbit monoclonal antibody for the immunohistochemical detection of ETV1 expression in cancer tissue specimens

Journal of immunological methods

2023 May 16

Schafer, C;Young, D;Singh, H;Jayakrishnan, R;Banerjee, S;Song, Y;Dobi, A;Petrovics, G;Srivastava, S;Srivastava, S;Sesterhenn, IA;Chesnut, GT;Tan, SH;
PMID: 37196930 | DOI: 10.1016/j.jim.2023.113493

Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function.An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens.Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells.The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.

Racialized Minorities Demonstrate Greater Pain Sensitivity And Sensibility To Heat And Self-Reported Acute Pain Despite Limitations Of Pain Sensitivity Questionnaire In Diverse Populations

The Journal of Pain

2023 Apr 01

Meeker, T;Tulloch, I;Kim, H;Keaser, M;Seminowicz, D;Dorsey, S;
| DOI: 10.1016/j.jpain.2023.02.067

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.

Identification Of Hub Genes Associated With Acute Pain Episodes In Individuals With Sickle Cell Disease

The Journal of Pain

2023 Apr 01

Mucalo, L;Jia, S;Roethle, M;Singh, A;Brousseau, D;Panepinto, J;Hessner, M;Brandow, A;
| DOI: 10.1016/j.jpain.2023.02.062

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.

OP 4.2- 00085 Cytolytic CD8+ T cells infiltrate germinal centers and limit HIV replication in spontaneous controllers

Journal of Virus Eradication

2022 Dec 01

Collins, D;Hitschfel, J;Walker, B;
| DOI: 10.1016/j.jve.2022.100202

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

Defining Inner Ear Cell Type Specification at Single-Cell Resolution in a Model of Human Cranial Development

SSRN Electronic Journal

2021 Dec 09

Steinhart, M;Serdy, S;van der Valk, W;Zhang, J;Kim, J;Lee, J;Koehler, K;
| DOI: 10.2139/ssrn.3974124

Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of _in vitro_ human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of _in vitro_ development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathway-related gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue.

Direct healthcare costs of lip, oral cavity and oropharyngeal cancer in Brazil

PloS one

2021 Feb 17

Milani, V;Zara, ALSA;da Silva, EN;Cardoso, LB;Curado, MP;Ribeiro-Rotta, RF;
PMID: 33596233 | DOI: 10.1371/journal.pone.0246475

The efficiency of public policies includes the measurement of the health resources used and their associated costs. There is a lack of studies evaluating the economic impact of oral cancer (OC). This study aims to estimate the healthcare costs of OC in Brazil from 2008 to 2016. This is a partial economic evaluation using the gross costing top-down method, considering the direct healthcare costs related to outpatients, inpatients, intensive care units, and the number of procedures, from the perspective of the public health sector. The data were extracted from the Outpatient and Inpatient Information System of the National Health System, by diagnosis according to the 10th Revision of the International Classification of Diseases, according to sites of interest: C00 to C06, C09 and C10. The values were adjusted for annual accumulated inflation and expressed in 2018 I$ (1 I$ = R$2,044). Expenditure on OC healthcare in Brazil was I$495.6 million, which was composed of 50.8% (I$251.6 million) outpatient and 49.2% (I$244.0 million) inpatient healthcare. About 177,317 admissions and 6,224,236 outpatient procedures were registered. Chemotherapy and radiotherapy comprised the largest number of procedures (88.8%) and costs (94.9%). Most of the costs were spent on people over 50 years old (72.9%) and on males (75.6%). Direct healthcare costs in Brazil for OC are substantial. Outpatient procedures were responsible for the highest total cost; however, inpatient procedures had a higher cost per procedure. Men over 50 years old consumed most of the cost and procedures for OC. The oropharynx and tongue were the sites with the highest expenditure. Further studies are needed to investigate the cost per individual, as well as direct non-medical and indirect costs of OC.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo

Gut

2021 Jan 28

Allweiss, L;Giersch, K;Pirosu, A;Volz, T;Muench, RC;Beran, RK;Urban, S;Javanbakht, H;Fletcher, SP;Lütgehetmann, M;Dandri, M;
PMID: 33509930 | DOI: 10.1136/gutjnl-2020-322571

Silencing of the therapeutic strategies and reducing the HBV reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys

Science translational medicine

2021 Jan 27

Speranza, E;Williamson, BN;Feldmann, F;Sturdevant, GL;Pérez-Pérez, L;Meade-White, K;Smith, BJ;Lovaglio, J;Martens, C;Munster, VJ;Okumura, A;Shaia, C;Feldmann, H;Best, SM;de Wit, E;
PMID: 33431511 | DOI: 10.1126/scitranslmed.abe8146

Detailed knowledge about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is important for uncovering the viral and host factors that contribute to coronavirus disease 2019 (COVID-19) pathogenesis. Old-World nonhuman primates recapitulate mild to moderate cases of COVID-19, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with infectious SARS-CoV-2 or irradiated, inactivated virus to study the dynamics of virus replication throughout the respiratory tract. Genomic RNA from the animals inoculated with the irradiated virus was found to be highly stable, whereas subgenomic RNA, an indicator of viral replication, was found to degrade quickly. We combined this information with single-cell RNA sequencing of cells isolated from the lung and lung-draining mediastinal lymph nodes and developed new analysis methods for unbiased targeting of important cells in the host response to SARS-CoV-2 infection. Through detection of reads to the viral genome, we were able to determine that replication of the virus in the lungs appeared to occur mainly in pneumocytes, whereas macrophages drove the inflammatory response. Monocyte-derived macrophages recruited to the lungs, rather than tissue-resident alveolar macrophages, were most likely to be responsible for phagocytosis of infected cells and cellular debris early in infection, with their roles switching during clearance of infection. Together, our dataset provides a detailed view of the dynamics of virus replication and host responses over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.

Development And Validation Of Painface, A Software Platform That Simplifies And Standardizes Mouse Grimace Analyses

The Journal of Pain

2023 Apr 01

Zylka, M;McCoy, E;Park, S;Patel, R;Ryan, D;Mullen, Z;Nesbitt, J;Lopez, J;Taylor-Blake, B;Krantz, J;Hu, W;Garris, R;Lima, L;Sotocinal, S;Austin, J;Kashlan, A;Jimenez, J;Shah, S;Trocinski, A;Vanden, K;Major, R;Bazick, H;Klein, M;Mogil, J;Wu, G;
| DOI: 10.1016/j.jpain.2023.02.113

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.

Exploring Corticospinal Functional Connectome Using Resting-State Functional Magnetic Resonance Imaging

The Journal of Pain

2023 Apr 01

Kaptan, M;Law, C;Weber, K;Pfyffer, D;Zhang, X;Maronesy, T;Glover, G;Mackey, S;
| DOI: 10.1016/j.jpain.2023.02.065

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.

The Protective Effect of Social Reward on Opioid and Psychostimulant Reward and Relapse: Behavior, Pharmacology, and Brain Regions

The Journal of neuroscience : the official journal of the Society for Neuroscience

2022 Dec 14

Venniro, M;Marino, RAM;Chow, JJ;Caprioli, D;Epstein, DH;Ramsey, LA;Shaham, Y;
PMID: 36517252 | DOI: 10.1523/JNEUROSCI.0931-22.2022

Until recently, most modern neuroscience research on addiction using animal models did not incorporate manipulations of social factors. Social factors play a critical role in human addiction: social isolation and exclusion can promote drug use and relapse, while social connections and inclusion tend to be protective. Here, we discuss the state of the literature on social factors in animal models of opioid and psychostimulant preference, self-administration, and relapse. We first summarize results from rodent studies on behavioral, pharmacological, and circuit mechanisms of the protective effect of traditional experimenter-controlled social interaction procedures on opioid and psychostimulant conditioned place preference, self-administration, and relapse. Next, we summarize behavioral and brain-mechanism results from studies using newer operant social-interaction procedures that inhibit opioid and psychostimulant self-administration and relapse. We conclude by discussing how the reviewed studies point to future directions for the addiction field and other neuroscience and psychiatric fields, and their implications for mechanistic understanding of addiction and development of new treatments.SIGNIFICANCE STATEMENT In this review, we propose that incorporating social factors into modern neuroscience research on addiction could improve mechanistic accounts of addiction and help close gaps in translating discovery to treatment. We first summarize rodent studies on behavioral, pharmacological, and circuit mechanisms of the protective effect of both traditional experimenter-controlled and newer operant social-interaction procedures. We then discuss potential future directions and clinical implications.

Female Urethral Carcinoma: A contemporary review of the clinicopathologic features, with emphasis on the histo-anatomic landmarks and potential staging issues

Human pathology

2022 Aug 26

Lagarde-Lenon, MS;Aron, M;
PMID: 36037997 | DOI: 10.1016/j.humpath.2022.08.003

Primary female urethral carcinoma (PUC-F) accounts for less than 1% of all genitourinary malignancies and comprises a histologically diverse group of tumors that are usually associated with poor prognosis. The carcinomas documented at this site include adenocarcinoma (clear cell adenocarcinoma, columnar cell carcinoma and Skene gland adenocarcinoma), urothelial carcinoma (UCa) and squamous cell carcinoma (SCC). Recent studies have shown adenocarcinomas to be the most common type of primary urethral carcinoma in females. Since most of the urethral carcinomas morphologically resemble carcinomas arising from surrounding pelvic organs or metastases, these should be ruled out before making the diagnosis of PUC-F. These tumors are currently staged according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging system. However, the AJCC system has limitations, including the staging of tumors involving the anterior wall of the urethra. Staging systems like the recently proposed histology based female urethral carcinoma staging system (UCS) takes into account the unique histological landmarks of the female urethra to better stratify pT2 and pT3 tumors into prognostic groups, that correlate with clinical outcomes including recurrence rates, disease-specific and overall survival. Further larger multi-institutional cohorts are however required to validate the results of this staging system. There is very limited information regarding the molecular profiling of PUC-F. 31% of clear cell adenocarcinomas have been reported to show PIK3CA alterations, while 15% of adenocarcinomas show PTEN mutations. Higher tumor mutational burden (TMB) and PD-L1 staining have been reported in UCa and SCC. Although multimodality treatment is usually recommended in locally advanced and metastatic disease, the role of immunotherapy and targeted therapy is promising in select PUC-F cases.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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