Dolled-Filhart M, Locke D, Murphy T, Lynch F, Yearley JH, Frisman D, Pierce R, Weiner R, Wu D, Emancipator K.
PMID: 27788043 | DOI: 10.5858/arpa.2015-0544-OA
Abstract
CONTEXT:
- With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation.
OBJECTIVE:
- To develop a prototype immunohistochemistry assay using the anti-PD-L1 antibody clone 22C3.
DESIGN:
- The assay was developed and optimized using commercially available reagents and archival tumor-bank tissue.
RESULTS:
- The optimized immunohistochemistry method had high precision and reproducibility. Using the prototype assay in 142 non-small cell lung cancer and 79 melanoma archival tumor-bank tissue samples, PD-L1 staining was observed at the plasma membrane of nucleated tumor and nontumor cells and, in some cases, as a distinct lichenoid pattern at the tumor-stroma border. Using a preliminary scoring method, 56% (80 of 142) of non-small cell lung cancer and 53% (42 of 79) of melanoma samples were defined as PD-L1+ based on a modified H-score of 1 or more or the presence of a distinctive staining pattern at the tumor-stroma interface.
CONCLUSIONS:
- The immunohistochemistry assay using the anti-PD-L1 antibody 22C3 merits further investigation in clinical trials and prevalence assessments to further understand the prognostic and predictive value of PD-L1 expression in cancer.
Giardino Torchia, ML;Letizia, M;Gilbreth, R;Merlino, A;Sult, E;Monks, N;Chesebrough, J;Tammali, R;Chu, N;Tong, J;Meekin, J;Schifferli, K;Vashisht, K;DaCosta, K;Clarke, L;Gesse, C;Yao, XT;Bridges, C;Moody, G;
PMID: 35570170 | DOI: 10.1016/j.jcyt.2022.03.008
Chimeric antigen receptor (CAR) T cell therapy has yielded impressive clinical results in hematological malignancies and is a promising approach for solid tumor treatment. However, toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, is a concern hampering its broader use.In selecting a lead CAR-T candidate against the oncofetal antigen glypican 3 (GPC3), we compared CARs bearing a low- and high-affinity single-chain variable fragment (scFv) binding to a similar epitope and cross-reactive with murine GPC3.Where the high-affinity CAR-T cells were toxic in vivo, the low-affinity CAR maintained cytotoxic function against antigen-positive tumor cells but did not show toxicity against normal tissues. High-affinity CAR-induced toxicity was caused by on-target, off-tumor binding, based on the observation that higher doses of the high-affinity CAR-T caused toxicity in non-tumor-bearing mice and accumulated in organs with low expression of GPC3. To explore another layer of controlling CAR-T toxicity, we developed a means to target and eliminate CAR-T cells using anti-TNF-α antibody therapy after CAR-T infusion. The antibody was shown to function by eliminating early antigen-activated, but not all, CAR-T cells, allowing a margin where the toxic response could be effectively decoupled from antitumor efficacy with only a minor loss in tumor control. By exploring additional traits of the CAR-T cells after activation, we identified a mechanism whereby we could use approved therapeutics and apply them as an exogenous kill switch that eliminated early activated CAR-T following antigen engagement in vivo.By combining the reduced-affinity CAR with this exogenous control mechanism, we provide evidence that we can modulate and control CAR-mediated toxicity.
Zhang, Q;Tang, Q;Purohit, NM;Davenport, JB;Brennan, C;Patel, RK;Godschall, E;Zwiefel, LS;Spano, A;Campbell, JN;Güler, AD;
PMID: 36450244 | DOI: 10.1016/j.celrep.2022.111718
Obesity comorbidities such as diabetes and cardiovascular disease are pressing public health concerns. Overconsumption of calories leads to weight gain; however, neural mechanisms underlying excessive food consumption are poorly understood. Here, we demonstrate that dopamine receptor D1 (Drd1) expressed in the agouti-related peptide/neuropeptide Y (AgRP/NPY) neurons of the arcuate hypothalamus is required for appropriate responses to a high-fat diet (HFD). Stimulation of Drd1 and AgRP/NPY co-expressing arcuate neurons is sufficient to induce voracious feeding. Delivery of a HFD after food deprivation acutely induces dopamine (DA) release in the ARC, whereas animals that lack Drd1 expression in ARCAgRP/NPY neurons (Drd1AgRP-KO) exhibit attenuated foraging and refeeding of HFD. These results define a role for the DA input to the ARC that encodes acute responses to food and position Drd1 signaling in the ARCAgRP/NPY neurons as an integrator of the hedonic and homeostatic neuronal feeding circuits.
Lovatt, D;Tamburino, A;Krasowska-Zoladek, A;Sanoja, R;Li, L;Peterson, V;Wang, X;Uslaner, J;
PMID: 36261573 | DOI: 10.1038/s42003-022-03970-0
Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.
Zhang, Y;Roy, DS;Zhu, Y;Chen, Y;Aida, T;Hou, Y;Shen, C;Lea, NE;Schroeder, ME;Skaggs, KM;Sullivan, HA;Fischer, KB;Callaway, EM;Wickersham, IR;Dai, J;Li, XM;Lu, Z;Feng, G;
PMID: 35676479 | DOI: 10.1038/s41586-022-04806-x
Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.
Conforti, P;Bocchi, VD;Campus, I;Scaramuzza, L;Galimberti, M;Lischetti, T;Talpo, F;Pedrazzoli, M;Murgia, A;Ferrari, I;Cordiglieri, C;Fasciani, A;Arenas, E;Felsenfeld, D;Biella, G;Besusso, D;Cattaneo, E;
PMID: 36590694 | DOI: 10.1016/j.crmeth.2022.100367
Stem cell engineering of striatal medium spiny neurons (MSNs) is a promising strategy to understand diseases affecting the striatum and for cell-replacement therapies in different neurological diseases. Protocols to generate cells from human pluripotent stem cells (PSCs) are scarce and how well they recapitulate the endogenous fetal cells remains poorly understood. We have developed a protocol that modulates cell seeding density and exposure to specific morphogens that generates authentic and functional D1- and D2-MSNs with a high degree of reproducibility in 25 days of differentiation. Single-cell RNA sequencing (scRNA-seq) shows that our cells can mimic the cell-fate acquisition steps observed in vivo in terms of cell type composition, gene expression, and signaling pathways. Finally, by modulating the midkine pathway we show that we can increase the yield of MSNs. We expect that this protocol will help decode pathogenesis factors in striatal diseases and eventually facilitate cell-replacement therapies for Huntington's disease (HD).
Development (Cambridge, England)
Negretti, NM;Plosa, EJ;Benjamin, JT;Schuler, BA;Habermann, AC;Jetter, CS;Gulleman, P;Bunn, C;Hackett, AN;Ransom, M;Taylor, CJ;Nichols, D;Matlock, BK;Guttentag, SH;Blackwell, TS;Banovich, NE;Kropski, JA;Sucre, JMS;
PMID: 34927678 | DOI: 10.1242/dev.199512
Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages - wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.
bioRxiv : the preprint server for biology
Sharafeddin, F;Ghaly, M;Simon, TB;Ontiveros-Ángel, P;Figueroa, JD;
PMID: 36747666 | DOI: 10.1101/2023.01.23.525269
Childhood traumatic stress profoundly affects prefrontal cortical networks regulating top-down control of eating and body weight. However, the neurobiological mechanisms contributing to trauma-induced aberrant eating behaviors remain largely unknown. Traumatic stress influences brain immune responses, which may, in turn, disrupt prefrontal cortical networks and behaviors. The tumor necrosis factor alpha-converting enzyme / a disintegrin and metalloproteinase 17 (TACE/ADAM17) is a sheddase with essential functions in brain maturation, behavior, and neuroinflammation. This study aimed to determine the role of TACE/ADAM17 on traumatic stress-induced disruption of eating patterns. We demonstrate a novel mechanistic connection between prefrontal cortical TACE/ADAM17 and trauma-induced eating behaviors. Fifty-two (52) adolescent Lewis rats (postnatal day, PND, 15) were injected intracerebrally either with a novel Accell SMARTpool ADAM17 siRNA or a corresponding siRNA vehicle. The RNAscope Multiplex Fluorescent v2 Assay was used to visualize mRNA expression. Observation cages were used to monitor ethological behaviors in a more naturalistic environment over long periods. We found that traumatic stress blunts startle reactivity and alter eating behaviors (increased intake and disrupted eating patterns). We also found that the rats that received prefrontal cortical TACE/ADAM17 siRNA administration exhibited decreased eating and increased grooming behaviors compared to controls. These changes were associated with decreased AIF-1 expression (a typical marker of microglia and neuroinflammation). This study demonstrates that prefrontal cortical TACE/ADAM17 is involved in neuroinflammation and may play essential roles in regulating feeding patterns under stress conditions. TACE/ADAM17 represents a promising target to ameliorate inflammation-induced brain and behavior alterations.
Appl Immunohistochem Mol Morphol.
Gafeer MM, Hosny Mohammed K, Ormenisan-Gherasim C, Choudhary F, Siddiqui MT, Cohen C.
PMID: 28968265 | DOI: 10.1097/PAI.0000000000000595
Abstract
BACKGROUND:
Programmed death receptor and programmed death ligand (PD-L1) are immunoregulatory proteins. Nonsmall cell lung cancer bypasses the immune system through the induction of protumorigenic immunosuppressive changes. The better understanding of immunology and antitumor immune responses has brought the promising development of novel immunotherapy agents like programmed death receptor checkpoint inhibitors. The aim of this study was to investigate the expression of PD-L1 in lung adenocarcinoma (ADC), comparing 2 different technologies: immunohistochemistry (IHC) by 2 methods versus RNA in situ hybridization (RISH).
METHODOLOGY:
In total, 20 cases of ADC of the lung and 4 samples of metastatic colon ADC were selected. Evaluation of PD-L1 expression was performed by IHC and RISH. RISH was performed using RNAscope. Both methods were scored in tumor cells and quantified using combined intensity and proportion scores.
RESULTS:
Eight of 20 (40%) lung ADC and 2 of 4 (50%) colon ADC were positive for PD-L1 with Cell Signaling IHC, and 65% lung ADC were positive by Dako IHC (13/20). All 4 cases of colon ADC were negative. When evaluated by RISH, 12 lung ADC (60%) and 1 colon ADC (25%) were PD-L1 positive.
CONCLUSIONS:
RNAscope probes provide sensitive and specific detection of PD-L1 in lung ADC. Both IHC methods (Cell Signaling and Dako) show PD-L1 expression, with the Dako method more sensitive (40% vs. 65%). This study illustrates the utility of RISH and Cell Signaling IHC as complementary diagnostic tests, and Food and Drug Administration approved Dako IHC as a companion diagnostic test.
Tejeda HA, Wu J, Kornspun AR, Pignatelli M, Kashtelyan V, Krashes MJ, Lowell BB, Carlezon WA Jr, Bonci A.
PMID: 28056342 | DOI: 10.1016/j.neuron.2016.12.005
Endogenous dynorphin signaling via the kappa-opioid receptor (KOR) in the nucleus accumbens (NAcc) powerfully mediates negative affective states and stress reactivity. Excitatory inputs from the hippocampus and amygdala play a fundamental role in shaping the activity of both NAcc D1 and D2 MSNs, which encode positive and negative motivational valences, respectively. However, a circuit-based mechanism by which KOR modulation of excitation-inhibition balance modifies D1 and D2 MSN activity is lacking. Here, we provide a comprehensive synaptic framework wherein presynaptic KOR inhibition decreases the excitatory drive of D1 MSN activity by the amygdala, but not the hippocampus. Conversely, presynaptic inhibition by KORs of inhibitory synapses on D2 MSNs enhances integration of excitatory drive by the amygdala and hippocampus. In conclusion, we describe a circuit-based mechanism showing differential gating of afferent control of D1 and D2 MSN activity by KORs in a pathway-specific manner.
Bakheet AMH, Zhao C, Chen JN, Zhang JY, Huang JT, Du Y, Gong LP, Bi YH, Shao CK
PMID: 31832976 | DOI: 10.1007/s12072-019-10006-z
BACKGROUND:
The diagnostic and prognostic values of glypican3 (GPC3) and glutamine synthetase (GS) proteins in hepatocellular carcinoma (HCC) have been reported, but their specificity and sensitivity remain low. Here, we applied RNAscope to improve HCC early pathological and differential diagnosis by estimating GPC3 and GS mRNAs.
METHODS:
We performed RNAscope and immunohistochemistry (IHC) to detect GPC3 and GS biomarkers on the tissue sections of 194 cases, including high- and low-grade liver dysplastic nodules; highly, moderately, and poorly differentiated HCCs; intrahepatic cholangiocarcinomas (ICCs); metastatic HCC; and carcinomas from other organs.
RESULTS:
The results showed that all the cases that were negative for GPC3 by RNAscope were also negative for this protein by IHC. The use of RNAscope assay improved the GPC3 and GS specificity and sensitivity by 20-30%. Hence, HCC shows early recognition and upgrades the metastatic HCC differentiation by 23% compared with IHC (p?=?0.0001, 0.0064). Meanwhile, all liver cirrhosis, cholangiocytes and non-HCC samples were negative for GPC3 and GS except lymphocytes in lymphomas, and 2 (8.3%) out of the 24 ICC samples but not in the cancer cells.
CONCLUSION:
RNAscope for GPC3 and GS panel was highly specific and sensitive for the pathological identification of dysplastic nodules, early stages of HCCs, and would differentiate them from HCCs and metastatic tumors compared with IHC.
Liu, Z;Le, Q;Lv, Y;Chen, X;Cui, J;Zhou, Y;Cheng, D;Ma, C;Su, X;Xiao, L;Yang, R;Zhang, J;Ma, L;Liu, X;
PMID: 34848869 | DOI: 10.1038/s41422-021-00588-5
Dopamine (DA) level in the nucleus accumbens (NAc) is critical for reward and aversion encoding. DA released from the ventral mesencephalon (VM) DAergic neurons increases the excitability of VM-projecting D1-dopamine receptor-expressing medium spiny neurons (D1-MSNs) in the NAc to enhance DA release and augment rewards. However, how such a DA positive feedback loop is regulated to maintain DA homeostasis and reward-aversion balance remains elusive. Here we report that the ventral pallidum (VP) projection of NAc D1-MSNs (D1NAc-VP) is inhibited by rewarding stimuli and activated by aversive stimuli. In contrast to the VM projection of D1-MSN (D1NAc-VM), activation of D1NAc-VP projection induces aversion, but not reward. D1NAc-VP MSNs are distinct from the D1NAc-VM MSNs, which exhibit conventional functions of D1-MSNs. Activation of D1NAc-VP projection stimulates VM GABAergic transmission, inhibits VM DAergic neurons, and reduces DA release into the NAc. Thus, D1NAc-VP and D1NAc-VM MSNs cooperatively control NAc dopamine balance and reward-aversion states.
