Journal of Vascular Surgery
Kasashima S, Kawashima A, Zen Y, Ozaki S, Kasashima F, Endo M, Matsumoto Y, Kawakami K.
PMID: 28434701 | DOI: 10.1016/j.jvs.2016.12.140
Abstract
OBJECTIVE:
Immunoglobulin (Ig) G4-related aortic aneurysms (IgG4-AAs) are a special aortic aneurysm among IgG4-related diseases (IgG4-RDs), which are inflammatory and fibrous conditions characterized by tumorous swelling of affected organs and high serum IgG4 concentrations. Recently, IgG4-RD pathogenesis was shown to be associated with T-helper-2 (Th2) and regulatory T (Treg) dominant cytokine production, such as interleukin (IL)-4, IL-10, and IL-13. IL-6 is a key proinflammatory cytokine contributing to lymphocyte and plasmacyte maturation and to atherosclerosis and aneurysm development. We serologically and histopathologically evaluated the cytokine profile in IgG4-AA patients.
METHODS:
Patients with IgG4-AAs (n = 10), non-IgG4-related inflammatory abdominal aortic aneurysms (non-IgG4-AAAs; n = 5), atherosclerotic AAAs (aAAAs; n = 10), and normal aortas without dilatation (n = 10) were examined for serum IL-10, IL-13, and IL-6 levels. Resected aortic tissues were evaluated for cluster of differentiation (CD) 34 (in the endothelial cells and mesenchymal cells) and CD163 (by macrophages) expression using immunohistochemistry and in situ hybridization.
RESULTS:
Serum IL-10 levels were rather higher in IgG4-AA patients (median, 1.3 pg/mL) than in non-IgG4-AAA and aAAA patients and in patients with normal aortas. Elevated serum IL-13 levels relative to standard values were detected in two IgG4-AA patients but not in the other groups. Cells immunopositive for IL-10 and IL-13 were more frequent in IgG4-AAs and significantly correlated with serum IgG4 levels. Serum IL-6 levels (median, 78.5 pg/mL) were also significantly higher in IgG4-AA patients than in non-IgG4-AAA and aAAA patients and control patients with normal aortas (P = .01, P = .001, and P = .004, respectively). They positively correlated with serum IgG4 levels and adventitial thickness, but other cytokines did not. The number of IL-6-immunopositive cells in the adventitia was significantly higher in IgG4-AA patients (median, 17.8/high-power field) than in aAAA patients or patients with normal aortas (P =.001 and P = .002, respectively). In situ hybridization confirmed frequent IL-6 messenger (m)RNA expression in the endothelium, mesenchymal cells, and histiocytes in IgG4-AA adventitia. In the same cells of IgG4-AAs, coexpression of IL-6 and CD34 mRNA or CD163 mRNA was detected.
CONCLUSIONS:
The cytokine profiles of IgG4-AA patients had two characteristics: local IL-10 and IL-13 upregulation in IgG4-AAs was related to Th2 and Treg-predominant cytokine balance, similar to other IgG4-RDs, and IL-6 upregulation in the adventitia was characterized by activated immune reactions in IgG4-AA patients. IL-6 synthesis, through contributions of mesenchymal cells and macrophages in the adventitia, is strongly involved in IgG4-AA pathogenesis or progression, or both.
Wua HH, Choia S, Levitt P.
PMID: - | DOI: 10.1016/j.placenta.2016.03.013
Abstract
Introduction
Serotonin (5-HT) is an important neuromodulator, but recently has been shown to be involved in neurodevelopment. Although previous studies have demonstrated that the placenta is a major source of forebrain 5-HT during early forebrain development, the processes of how 5-HT production, metabolism, and transport from placenta to fetus are regulated are unknown. As an initial step in determining the mechanisms involved, we investigated the expression patterns of genes critical for 5-HT system function in mouse extraembryonic tissues.
Methods
Mid-through late gestation expression of 5-HT system-related enzymes, Tph1, Ddc,Maoa, and 5-HT transporters, Sert/Slc6a4, Oct3/Slc22a3, Vmat2/Slc18a2, and 5-HT in placenta and yolk sac were examined, with cell type-specific resolution, using multiplex fluorescent in situ hybridization to co-localize transcripts and immunocytochemistry to co-localize the corresponding proteins and neurotransmitter.
Results
Tph1 and Ddc are found in the syncytiotrophoblast I (SynT-I) and sinusoidal trophoblast giant cells (S-TGC), whereas Maoa is expressed in SynT-I, syncytiotrophoblast II (SynT-II) and S-TGC. Oct3 expression is observed in the SynT-II only, while Vmat2 is mainly expressed in S-TGC. Surprisingly, there were comparatively high expression of Tph1,Ddc, and Maoa in the yolk sac visceral endoderm.
Discussion
In addition to trophoblast cells, visceral endoderm cells in the yolk sac may contribute to fetal 5-HT production. The findings raise the possibility of a more complex regulation of 5-HT access to the fetus through the differential roles of trophoblasts that surround maternal and fetal blood space and of yolk sac endoderm prior to normal degeneration.
Improving function of cytotoxic T-lymphocytes by transforming growth factor-β inhibitor in oral squamous cell carcinoma
Kondo, Y;Suzuki, S;Takahara, T;Ono, S;Goto, M;Miyabe, S;Sugita, Y;Ogawa, T;Ito, H;Satou, A;Tsuzuki, T;Yoshikawa, K;Ueda, R;Nagao, T;
PMID: 34309966 | DOI: 10.1111/cas.15081
Immunotherapy with immune-checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor-β (TGF-β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T-cells (Tregs) and cancer-associated fibroblasts and inhibiting the function of cytotoxic T-lymphocytes (CTLs) and natural killer cells. TGF-β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF-β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T-cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF-β suppressed the function of antigen-specific CTLs in the priming and effector phases in vitro. Additionally, TGF-β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T-cell/Treg ratio and between TGFB1 mRNA expression and the Ki-67 expression in CD8+ T-cells, indicating that TGF-β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF-β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune-checkpoint inhibitors and TGF-β inhibitors, for OSCCs.
Rodríguez, JMM;Fonfara, S;Hetzel, U;Kipar, A;
PMID: 34955067 | DOI: 10.1177/03009858211062631
The sequence of pathological events in feline hypertrophic cardiomyopathy (fHCM) is still largely unknown, although we know that fHCM is characterized by interstitial remodeling in a macrophage-driven pro-inflammatory environment and that myocardial ischemia might contribute to its progression. This study aimed to gain further insights into the structural changes associated with interstitial remodeling in fHCM with special focus on the myocardial microvasculature and the phenotype of the interstitial cells. Twenty-eight hearts (16 hearts with fHCM and 12 without cardiac disease) were evaluated in the current study, with immunohistochemistry, RNA-in situ hybridization, and transmission electron microscopy. Morphometrical evaluations revealed a statistically significant lower microvascular density in fHCM. This was associated with structural alterations in capillaries that go along with a widening of the interstitium due to the accumulation of edema fluid, collagen fibers, and mononuclear cells that also proliferated locally. The interstitial cells were mainly of fibroblastic or vascular phenotype, with a substantial contribution of predominantly resident macrophages. A large proportion expressed CD34 mRNA, which suggests a progenitor cell potential. Our results indicate that microvascular alterations are key events in the pathogenesis of fHCM and that myocardial interstitial cell populations with CD34+ phenotype play a role in the pathogenesis of the disease.
Characterization of SARS-CoV-2 and host entry factors distribution in a COVID-19 autopsy series
Wang, X;Mannan, R;Xiao, L;Abdulfatah, E;Qiao, Y;Farver, C;Myers, J;Zelenka-Wang, S;McMurry, L;Su, F;Wang, R;Pantanowitz, L;Jentzen, J;Wilson, A;Zhang, Y;Cao, X;Chinnaiyan, A;Mehra, R;
| DOI: 10.1038/s43856-021-00025-z
Background SARS-CoV-2 is a highly contagious virus that causes the disease COVID-19. We have recently reported that androgens regulate the expression of SARS-CoV-2 host entry factors ACE2 and TMPRSS2, and androgen receptor (AR) in lung epithelial cells. We also demonstrated that the transcriptional repression of the AR enhanceosome inhibited SARS-CoV-2 infection in vitro. Methods To better understand the various sites of SARS-CoV-2 infection, and presence of host entry factors, we extensively characterized the tissue distribution and localization of SARS-CoV-2 virus, viral replication, and host entry factors in various anatomical sites sampled via autopsy. We applied RNA in-situ-hybridization (RNA-ISH), immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) approaches. We also assessed histopathological changes in SARS-CoV-2 infected tissues. Results We detect SARS-CoV-2 virus and viral replication in pulmonary tissues by RNA-ISH and IHC and a variety of non-pulmonary tissues including kidney, heart, liver, spleen, thyroid, lymph node, prostate, uterus, and colon by qRT-PCR. We observe heterogeneity in viral load and viral cytopathic effects among various organ systems, between individuals and within the same patient. In a patient with a history of kidney transplant and under immunosuppressant therapy, we observe an unusually high viral load in lung tissue by RNA-ISH, IHC and qRT-PCR. SARS-CoV-2 virus is also detected in this patent’s kidney, liver and uterus. We find ACE2, TMPRSS2 and AR expression to overlap with the infection sites. Conclusions This study portrays the impact of dispersed SARS-CoV-2 infection in diverse organ systems, thereby facilitating avenues for systematic therapeutic approaches.
Veerman K, Tardiveau C, Martins F, Coudert J, Girard JP.
PMID: 30865898 | DOI: 10.1016/j.celrep.2019.02.042
High-endothelial venules (HEVs) are specialized blood vessels allowing recirculation of naive lymphocytes through lymphoid organs. Here, using full-length, single-cell RNA sequencing, RNA fluorescence in situ hybridization (FISH), flow cytometry, and immunohistofluorescence, we reveal the heterogeneity of HEVs in adult mouse peripheral lymph nodes (PLNs) under conditions of homeostasis, antigenic stimulation, and after inhibition of lymphotoxin-β receptor (LTβR) signaling. We demonstrate that HEV endothelial cells are in an activated state during homeostasis, and we identify the genes characteristic of the differentiated HEV phenotype. We show that LTβR signaling regulates many HEV genes and pathways in resting PLNs and that immune stimulation induces a global and temporary inflammatory phenotype in HEVs without compromising their ability to recruit naive lymphocytes. Most importantly, we uncover differences in the regulation of genes controlling lymphocyte trafficking, Glycam1, Fut7, Gcnt1, Chst4, B3gnt3, and Ccl21a, that have implications for HEV function and regulation in health and disease.
Mavropoulos A, Allo B, He M, Park E, Majonis D, Ornatsky O.
PMID: 29194963 | DOI: 10.1002/cyto.a.23281
Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope™ platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells.
Gucy2d selectively marks inhibitory dynorphin neurons in the spinal dorsal horn but is dispensable for pain and itch sensitivity
Serafin, EK;Burns, R;Yoo, J;Baccei, ML;
PMID: 34296052 | DOI: 10.1097/PR9.0000000000000947
Inhibitory neurons in the spinal dorsal horn can be classified based on expression of neurochemical marker genes. However, these marker genes are often expressed throughout the central nervous system, which poses challenges for manipulating genetically identified spinal neurons without undesired off-target effects.We investigated whether Gucy2d, previously identified as a highly selective marker of dynorphin-lineage neurons in the dorsal horn, is expressed in other locations within the adult mouse spinal cord, dorsal root ganglia (DRG), or brain. In addition, we sought to molecularly characterize Gucy2d-expressing dorsal horn neurons and investigate whether the disruption of Gucy2d gene expression affects sensitivity to itch or pain.In situ hybridization experiments assessed Gucy2d mRNA expression in the adult mouse spinal cord, DRG, and brain, and its colocalization with Pax2, Bhlhb5, and Pde2a in dorsal horn neurons. Knockout mice lacking Gucy2d expression were compared with littermate controls to assess sensitivity to chloroquine-induced itch and dry skin-mediated chronic itch, as well as heat, cold, or mechanical stimuli.Gucy2d is selectively expressed in dynorphin-lineage neurons in lamina I-III of the adult mouse spinal cord but not in the brain or DRG. Spinal Gucy2d-expressing neurons are inhibitory neurons that also express the transcription factor Bhlhb5 and the cGMP-dependent phosphodiesterase Pde2a. Gucy2d knockout mice did not exhibit altered responses to itch or pain.The selective expression of Gucy2d within a subpopulation of inhibitory dorsal horn neurons may yield a means to selectively manipulate inhibitory signaling at the level of the spinal cord without effects on the brain.
Δ133p53β isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells
Arsic, N;Slatter, T;Gadea, G;Villain, E;Fournet, A;Kazantseva, M;Allemand, F;Sibille, N;Seveno, M;de Rossi, S;Mehta, S;Urbach, S;Bourdon, JC;Bernado, P;Kajava, AV;Braithwaite, A;Roux, P;
PMID: 34526502 | DOI: 10.1038/s41467-021-25550-2
The p53 isoform, Δ133p53β, is critical in promoting cancer. Here we report that Δ133p53β activity is regulated through an aggregation-dependent mechanism. Δ133p53β aggregates were observed in cancer cells and tumour biopsies. The Δ133p53β aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53β aggregates and loss of Δ133p53β dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53β from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion.
Anderson CM, Zhang B, Miller M, Butko E, Wu X, Laver T, Kernag C, Kim J, Luo Y, Lamparski H, Park E, Su N, Ma XJ.
PMID: 27191821 | DOI: 10.1002/jcb.25606.
Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development.
Ruggiero, AD;Davis, MA;Davis, AT;DeStephanis, D;Williams, AG;Vemuri, R;Fanning, KM;Sherrill, C;Cline, JM;Caudell, DL;Kavanagh, K;
PMID: 36136223 | DOI: 10.1007/s11357-022-00660-x
The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAβ-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-βgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.
Expression of immunoglobulin constant domain genes in neurons of the mouse central nervous system
Scheurer, L;Das Gupta, RR;Saebisch, A;Grampp, T;Benke, D;Zeilhofer, HU;Wildner, H;
PMID: 34433614 | DOI: 10.26508/lsa.202101154
General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.
