Squamous and Neuroendocrine Specific Immunohistochemical Markers in Head and Neck Squamous Cell Carcinoma: A Tissue Microarray Study.

Head Neck Pathol.

2017 May 20

Lewis JS Jr, Chernock RD, Bishop JA.
PMID: 28528398 | DOI: 10.1007/s12105-017-0825-y

The performance characteristics of neuroendocrine-specific and squamous-specific immunohistochemical markers in head and neck squamous cell carcinomas (SCC), in particular in oropharyngeal tumors in this era of human papillomavirus (HPV)-induced cases, are not well-established. The differential diagnosis for poorly differentiated SCCs, for nonkeratinizing oropharyngeal SCCs, and for other specific SCC variants such as basaloid SCC and undifferentiated (or lymphoepithelial-like) carcinomas includes neuroendocrine carcinomas. Given that neuroendocrine carcinomas of the head and neck are aggressive regardless of HPV status, separating them from SCC is critically important. In this study, we examined the neuroendocrine markers CD56, synaptophysin, and chromogranin-A along with the squamous markers p40 and cytokeratin 5/6 in a large tissue microarray cohort of oral, oropharyngeal, laryngeal, and hypopharyngeal SCCs with known HPV results by RNA in situ hybridization for the oropharyngeal tumors. Results were stratified by site and specific SCC variant. The neuroendocrine stains were rarely expressed in SCC (<1% overall) with CD56 the least, and chromogranin-A the most, specific markers. Further, p40 and cytokeratin 5/6 were very consistently expressed in all head and neck SCC (>98% overall), including very strong, consistent staining in oropharyngeal HPV-related nonkeratinizing SCC. Undifferentiated (or lymphoepithelial-like) carcinomas of the oropharynx are more frequently p40 or cytokeratin 5/6 negative or show only weak or focal expression. In summary, markers of neuroendocrine and squamous differentiation show very high specificity and sensitivity, respectively, across the different types of head and neck SCC.

Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics

Acta Pharmacol Sin.

2018 Sep 10

Liu QR, Huang NS, Qu H, O’Connell JF, Gonzalez-Mariscal I, Santa-Cruz-Calvo S, Doyle ME, Xi ZX, Wang Y, Onaivi ES, Egan J.
PMID: 30202012 | DOI: 10.1038/s41401-018-0152-1

Targeting peripheral CB1R is desirable for the treatment of metabolic syndromes without adverse neuropsychiatric effects. We previously reported a human hCB1b isoform that is selectively enriched in pancreatic beta-cells and hepatocytes, providing a potential peripheral therapeutic hCB1R target. It is unknown whether there are peripherally enriched mouse and rat CB1R (mCB1 and rCB1, respectively) isoforms. In this study, we found no evidence of peripherally enriched rodent CB1 isoforms; however, some mCB1R isoforms are absent in peripheral tissues. We show that the mouse Cnr1 gene contains six exons that are transcribed from a single promoter. We found that mCB1A is a spliced variant of extended exon 1 and protein-coding exon 6; mCB1B is a novel spliced variant containing unspliced exon 1, intron 1, and exon 2, which is then spliced to exon 6; and mCB1C is a spliced variant including all 6 exons. Using RNAscope in situ hybridization, we show that the isoforms mCB1A and mCB1B are expressed at a cellular level and colocalized in GABAergic neurons in the hippocampus and cortex. RT-qPCR reveals that mCB1A and mCB1B are enriched in the brain, while mCB1B is not expressed in the pancreas or the liver. Rat rCB1R isoforms are differentially expressed in primary cultured neurons, astrocytes, and microglia. We also investigated modulation of Cnr1 expression by insulin in vivo and carried out in silico modeling of CB1R with JD5037, a peripherally restricted CB1R inverse agonist, using the published crystal structure of hCB1R. The results provide models for future CB1R peripheral targeting.

The Role of TRPA1 Channels in the Central Processing of Odours Contributing to the Behavioural Responses of Mice

Pharmaceuticals (Basel, Switzerland)

2021 Dec 20

Konkoly, J;Kormos, V;Gaszner, B;Sándor, Z;Kecskés, A;Alomari, A;Szilágyi, A;Szilágyi, B;Zelena, D;Pintér, E;
PMID: 34959735 | DOI: 10.3390/ph14121336

Transient receptor potential ankyrin 1 (TRPA1), a nonselective cation channel, contributes to several (patho)physiological processes. Smell loss is an early sign in several neurodegenerative disorders, such as multiple sclerosis, Parkinson's and Alzheimer's diseases; therefore, we focused on its role in olfaction and social behaviour with the aim to reveal its potential therapeutic use. The presence of Trpa1 mRNA was studied along the olfactory tract of mice by combined RNAscope in situ hybridisation and immunohistochemistry. The aversive effects of fox and cat odour were examined in parallel with stress hormone levels. In vitro calcium imaging was applied to test if these substances can directly activate TRPA1 receptors. The role of TRPA1 in social behaviour was investigated by comparing Trpa1 wild-type and knockout mice (KO). Trpa1 mRNA was detected in the olfactory bulb and piriform cortex, while its expression was weak in the olfactory epithelium. Fox, but not cat odour directly activated TRPA1 channels in TRPA1-overexpressing Chinese Hamster Ovary cell lines. Accordingly, KO animals showed less aversion against fox, but not cat odour. The social interest of KO mice was reduced during social habituation-dishabituation and social interaction, but not during resident-intruder tests. TRPA1 may contribute to odour processing at several points of the olfactory tract and may play an important role in shaping the social behaviour of mice. Thus, TRPA1 may influence the development of certain social disorders, serving as a potential drug target in the future.

Human papillomavirus (HPV) status of non-tobacco related squamous cell carcinomas of the lateral tongue.

Oral Oncol. Apr; 50(4):306–310.

Poling JS, Ma XJ, Bui S, Luo Y, Li R, Koch WM, Westra WH (2014).
PMID: 24485566 | DOI: 10.1016/j.oraloncology.2014.01.006.

OBJECTIVES: The human papillomavirus (HPV) is an important cause of some head and neck squamous cell carcinomas (HNSCCs), but its role in cancer of the lateral tongue is debatable. Suspicion of HPV causation is heightened when these lateral tongue carcinomas arise in patients that are young and/or have never smoked. The purpose of this study was to determine the incidence of transcriptionally active high risk HPV in these tumors, with a particular emphasis on non-smoking patients who are often presumed to have HPV-positive tumors. METHODS: We evaluated 78 HNSCCs of the lateral tongue for the presence of HPV using p16 immunohistochemistry and an RNA in situ hybridization assay targeting HPV E6/E7 mRNA. The study population was enriched for patients without traditional risk factors such as smoking and drinking. RESULTS: P16 overexpression was detected in 9 (11.5%) of 78 cases, but HPV E6/E7 mRNA transcripts were detected in only 1 (1.3%) case (positive predictive value of p16 staining for the presence of transcriptionally active HPV=0.12). HPV mRNA transcripts were not detected in any patient under 40 (n=11), or in patients who had never smoked (n=44), had quit smoking (n=15), and/or were only light consumers of alcohol (n=57). CONCLUSIONS: HPV is not detected in the vast majority of lateral tongue carcinomas. In light of the observation that HPV plays little if any role in the development of these cancers, routine HPV testing is unwarranted , even for patients without traditional risk factors. P16 staining is not a reliable marker for the presence of transcriptionally active HPV at this particular anatomic site.

Correlation of Circulating CD64+/CD163+ Monocyte Ratio and stroma/peri-tumoral CD163+ Monocyte Density with Human Papillomavirus Infected Cervical Lesion Severity

Cancer Microenviron.

2017 Oct 24

Swangphon P, Pientong C, Sunthamala N, Bumrungthai S, Azuma M, Kleebkaow P, Tangsiriwatthana T, Sangkomkamhang U, Kongyingyoes B, Ekalaksananan T.
PMID: 29064053 | DOI: 10.1007/s12307-017-0200-2

HPV infected cervical cells secrete mediators that are gradually changed and have influence on infiltrating M2 phenotypic monocytes in cervical lesions. However, profiles of circulating immune cells in women with cervical lesions and M2 phenotypic monocyte activity in HPV infected cervical lesions are limited. This study aimed to investigate circulating monocyte populations correlated with M2 phenotype density and its activity in HPV infected cervical lesions. HPV DNA was investigated in cervical tissues using PCR. High risk HPV E6/E7 mRNA was detected using in situ hybridization. CD163 immunohistochemical staining was performed for M2 macrophage. CD163 and Arg1 mRNA expression were detected using real-time PCR. Circulating monocyte subpopulations were analyzed using flow cytometry. CD163 and Arg1 mRNA expression were increased according to cervical lesion severity and corresponding with density of M2 macrophage in HSIL and SCC in stroma and peri-tumoral areas. Additionally, the relationship between M2 macrophage infiltration and high risk HPV E6/E7 mRNA expression was found and corresponded with cervical lesion severity. Circulating CD14+CD16+ and CD14+CD163+ monocytes were elevated in No-SIL and cervical lesions. Interestingly, CD14+CD64+ monocyte was greatly elevated in HSIL and SCC, whereas intracellular IL-10+monocytes were not significantly different between cervical lesions. The correlation between increasing ratio of circulating CD64+/CD163+monocyte and density of infiltrating CD163+ monocytes was associated with severity of HPV infected cervical lesions. The elevated circulating CD64+/CD163+ monocyte ratio correlates to severity of HPV infected cervical lesions and might be a prognostic marker in cervical cancer progression.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification

Cell discovery

2022 Nov 29

Zhao, M;Su, HZ;Zeng, YH;Sun, Y;Guo, XX;Li, YL;Wang, C;Zhao, ZY;Huang, XJ;Lin, KJ;Ye, ZL;Lin, BW;Hong, S;Zheng, J;Liu, YB;Yao, XP;Yang, D;Lu, YQ;Chen, HZ;Zuo, E;Yang, G;Wang, HT;Huang, CW;Lin, XH;Cen, Z;Lai, LL;Zhang, YK;Li, X;Lai, T;Lin, J;Zuo, DD;Lin, MT;Liou, CW;Kong, QX;Yan, CZ;Xiong, ZQ;Wang, N;Luo, W;Zhao, CP;Cheng, X;Chen, WJ;
PMID: 36443312 | DOI: 10.1038/s41421-022-00475-2

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.

Somatostatin Interneurons of the Insula Mediate QR2-Dependent Novel Taste Memory Enhancement

eNeuro

2021 Sep 29

Gould, NL;Kolatt Chandran, S;Kayyal, H;Edry, E;Rosenblum, K;
PMID: 34518366 | DOI: 10.1523/ENEURO.0152-21.2021

Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information.

Mucoepidermoid Carcinoma Does Not Harbor Transcriptionally Active High Risk Human Papillomavirus Even in the Absence of the MAML2 Translocation

Head Neck Pathol. 2014 Apr 5

Bishop JA, Yonescu R, Batista D, Yemelyanova A, Ha PK, Westra WH
PMID: 24706055 | DOI: 10.1007/s12105-014-0541-9

High risk human papillomavirus (HPV) is firmly established as an important cause of oropharyngeal carcinoma. Recent studies have also implicated HPV as a cause of mucoepidermoid carcinoma (MEC)—a tumor of salivary gland origin that frequently harbors MAML2 translocations. The purpose of this study was to determine the prevalence of transcriptionally active HPV in a large group of MECs and to determine whether HPV infection and the MAML2 translocation are mutually exclusive events. Break-apart fluorescence in situ hybridization for MAML2 was performed on a tissue microarray containing 92 MECs. HPV testing was performed using RNA in situ hybridization targeting high risk HPV mRNA E6/E7 transcripts. Of the 71 MECs that could be evaluated by FISH, 57 (80 %) harbored the MAML2 rearrangement. HPV was not detected in any of the 57 MECs that contained a MAML2 rearrangement, in any of the 14 MECs that did not contain the rearrangement, or in any of the 21 MECs where MAML2 status was unknown. High risk HPV does not appear to play any significant role in the development of MEC. It neither complements nor replaces MAML2 translocation in the tumorigenesis of MEC

Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia

Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology

2016 Jun 22

Bhosale PG, Pandey M, Desai RS, Patil A, Kane S, Prabhash K, Mahimkar MB.
PMID: - | DOI: 10.1016/j.oooo.2016.06.006

Abstract

Objectives

In the present study, we comprehensively analyzed the prevalence of transcriptionally active HPV in tissue samples of Indian patients with leukoplakia - predominantly hyperplastic lesions and HNSCC. In addition, saliva samples from patients with HNSCC were screened for HPV detection.

Study Design

p16 overexpression was analyzed by immunohistochemistry. Leukoplakia (n = 121) and HNSCC (n = 427) tissue samples and the saliva of patients with HNSCC (n = 215) were tested for HPV using nested PCR. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in-situ hybridization.

Results

p16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, either in saliva and/or tissue. E6/E7 mRNA positivity was observed in eight HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx with very little concordance between p16 overexpression and HPV positivity. All patients with HPV positive saliva samples had transcriptionally active HPV present in their tumors.

Conclusion

Presence of HPV-DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV associated tumors.

GABA Neuronal Deletion of Shank3 Exons 14-16 in Mice Suppresses Striatal Excitatory Synaptic Input and Induces Social and Locomotor Abnormalities.

Front Cell Neurosci. 2018 Oct 9;12:341.

2018 Oct 09

Yoo T, Cho H, Lee J, Park H, Yoo YE, Yang E, Kim JY, Kim H, Kim E.
PMID: 30356810 | DOI: 10.3389/fncel.2018.00341

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.

Synaptic Targets of Glycinergic Neurons in Laminae I-III of the Spinal Dorsal Horn

International journal of molecular sciences

2023 Apr 08

Miranda, CO;Hegedüs, K;Kis, G;Antal, M;
PMID: 37108107 | DOI: 10.3390/ijms24086943

A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I-III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I-III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I-III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I-III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs.

CDX2 Expression in Primary Skin Tumors- Case Series and Review of the Literature

Human pathology

2022 Aug 01

Tekin, B;Kundert, P;Yang, HH;Guo, R;
PMID: 35926811 | DOI: 10.1016/j.humpath.2022.07.013

CDX2 expression characterizes tumors of gastrointestinal origin, including those of intestinal-type differentiation. In dermatopathology, CDX2 expression is reported in four settings: cutaneous metastases from carcinomas of intestinal origin or differentiation, extramammary Paget's disease associated with an underlying colorectal or urothelial tumor, pilomatricomas and pilomatrical carcinomas, and rare primary cutaneous (adeno)squamous carcinomas with intestinal immunophenotype. Over 4 years (10/2017-10/2021), 252 dermatopathology cases with CDX2 immunostain were reviewed, revealing 46 cases with confirmed positive staining. Among them, 11 cases confirmed as primary non-intestinal type cutaneous carcinoma with definitively positive CDX2 nuclear staining were further studied. All cases demonstrated basaloid morphology with atypia, variable necrosis, and brisk mitotic activity. Cases 1-5 had heterogeneous features that cannot be further classified, including two cases with neuroendocrine or pseudoglandular/pseudopapillary features, and one case with HPV high risk E6/E7 ISH positivity. In cases 6 through 11, the diagnosis of pilomatrical carcinoma was supported morphologically. This study substantiates the association of CDX2 with pilomatrical carcinoma. In addition, CDX2 positivity was observed in a subset of basaloid cutaneous carcinomas of ambiguous classification. However, this finding also raises a diagnostic pitfall in clinical diagnostic specificity of the CDX2 immunostain in skin cancers, which can be observed in rare while heterogenous subsets of primary cutaneous carcinomas with primitive cytomorphology.

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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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Abstract

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Results

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