Feline hypertrophic cardiomyopathy: reduced microvascular density and involvement of CD34+ interstitial cells

Veterinary pathology

2021 Dec 27

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.

Varicella-zoster virus proteome-wide T-cell screening demonstrates low prevalence of virus-specific CD8 T-cells in latently infected human trigeminal ganglia

Journal of neuroinflammation

2023 Jun 12

van Gent, M;Ouwendijk, WJD;Campbell, VL;Laing, KJ;Verjans, GMGM;Koelle, DM;
PMID: 37308917 | DOI: 10.1186/s12974-023-02820-y

Trigeminal ganglia (TG) neurons are an important site of lifelong latent varicella-zoster virus (VZV) infection. Although VZV-specific T-cells are considered pivotal to control virus reactivation, their protective role at the site of latency remains uncharacterized.Paired blood and TG specimens were obtained from ten latent VZV-infected adults, of which nine were co-infected with herpes simplex virus type 1 (HSV-1). Short-term TG-derived T-cell lines (TG-TCL), generated by mitogenic stimulation of TG-derived T-cells, were probed for HSV-1- and VZV-specific T-cells using flow cytometry. We also performed VZV proteome-wide screening of TG-TCL to determine the fine antigenic specificity of VZV reactive T-cells. Finally, the relationship between T-cells and latent HSV-1 and VZV infections in TG was analyzed by reverse transcription quantitative PCR (RT-qPCR) and in situ analysis for T-cell proteins and latent viral transcripts.VZV proteome-wide analysis of ten TG-TCL identified two VZV antigens recognized by CD8 T-cells in two separate subjects. The first was an HSV-1/VZV cross-reactive CD8 T-cell epitope, whereas the second TG harbored CD8 T-cells reactive with VZV specifically and not the homologous peptide in HSV-1. In silico analysis showed that HSV-1/VZV cross reactivity of TG-derived CD8 T-cells reactive with ten previously identified HSV-1 epitopes was unlikely, suggesting that HSV-1/VZV cross-reactive T-cells are not a common feature in dually infected TG. Finally, no association was detected between T-cell infiltration and VZV latency transcript abundance in TG by RT-qPCR or in situ analyses.The low presence of VZV- compared to HSV-1-specific CD8 T-cells in human TG suggests that VZV reactive CD8 T-cells play a limited role in maintaining VZV latency.

Therapeutic efficacy of a VSV-GP-based human papilloma virus vaccine in a murine cancer model

Journal of molecular biology

2023 Apr 20

Riepler, L;Frommelt, LS;Wilmschen-Tober, S;Mbuya, W;Held, K;Volland, A;von Laer, D;Geldmacher, C;Kimpel, J;
PMID: 37086948 | DOI: 10.1016/j.jmb.2023.168096

Human papilloma virus (HPV) infections are associated with almost all cervical cancers and to a lower extend also with anogenital or oropharyngeal cancers. HPV proteins expressed in HPV-associated tumors are attractive antigens for cancer vaccination strategies as self-tolerance, which is associated with most endogenous tumor-associated antigens, does not need to be overcome. In this study, we generated a live attenuated cancer vaccine based on the chimeric vesicular stomatitis virus VSV-GP, which has previously proven to be a potent vaccine vector and oncolytic virus. Genes at an earlier position in the genome more to the 3' end are expressed stronger compared to genes located further downstream. By inserting an HPV16-derived antigen cassette consisting of E2, E6 and E7 into VSV-GP either at first (HPVp1) or fifth (HPVp5) position in VSV-GP's genome we aimed to analyze the effect of vaccine antigen position and consequently expression level on viral fitness, immunogenicity, and anti-tumoral efficacy in a syngeneic mouse tumor model. HPVp1 expressed higher amounts of HPV antigens compared to HPVp5 in vitro but had a slightly delayed replication kinetic which overall translated into increased HPV-specific T cell responses upon vaccination of mice. Immunization with both vectors protected mice in prophylactic and in therapeutic TC-1 tumor models with HPVp1 being more effective in the prophylactic setting. Taken together, VSV-GP is a promising candidate as therapeutic HPV vaccine and first position of the vaccine antigen in a VSV-derived vector seems to be superior to fifth position.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development

The Journal of biological chemistry

2023 Mar 22

Fu, Y;Miyazaki, K;Chiba, Y;Funada, K;Yuta, T;Tian, T;Mizuta, K;Kawahara, J;Zhang, L;Martin, D;Iwamoto, T;Takahashi, I;Fukumoto, S;Yoshizaki, K;
PMID: 36963497 | DOI: 10.1016/j.jbc.2023.104638

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6 (Ly6)/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an ex vivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C-terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; additionally, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Genetic and transcriptomic analyses in a rare case of HPV-related oropharyngeal squamous cell carcinoma combined with small cell carcinoma

Cold Spring Harbor molecular case studies

2021 Aug 30

Sato, K;Nishiyama, K;Taguchi, K;Jiromaru, R;Yamamoto, H;Matsunaga, A;Nagata, R;Rikimaru, F;Toh, S;Higaki, Y;Oda, S;Nakagawa, T;Masuda, M;
PMID: 34462366 | DOI: 10.1101/mcs.a006102

Human papillomavirus (HPV)-related oropharyngeal small cell carcinoma (OPSmCC) is a rare malignancy with aggressive behavior, whereas HPV-related oropharyngeal squamous cell carcinoma (OPSqCC) displays a favorable prognosis. Notably, these two malignancies occasionally arise in an identical tumor. In this case study, we explored the molecular characteristics that distinguishes these two carcinomas employing a rare case of HPV-related oropharyngeal carcinoma (OPC) with the combined histology of SmCC and SqCC. Immunohistochemical analysis and HPV-RNA in situ hybridization (ISH) suggested that both SmCC and SqCC were HPV-related malignancies. Targeted exome sequencing revealed that SmCC and SqCC had no significant difference in mutations of known driver genes. In contrast, RNA sequencing followed by bioinformatic analyses suggested that aberrant transcriptional programs may be responsible for the neuroendocrine differentiation of HPV-related OPC. Compared to SqCC, genes upregulated in SmCC were functionally enriched in inflammatory and immune responses (e.g., arachidonic acid metabolism). We then developed a SmCC-like gene module (top 10 upregulated genes) and found that OPC patients with high module activity showed poor prognosis in The Cancer Genome Atlas (TCGA) and GSE65858 cohort. Gene set enrichment analysis of the SmCC-like gene module suggested its link to MYC proto-oncogene in the TCGA dataset. Taken together, these findings suggest that the SmCC-like gene module may contribute to acquisition of aggressive phenotypes and tumor heterogeneity of HPV-related OPC. The present case study is the first report of genetic and transcriptomic aberrations in HPV-related OPSmCC combined with SqCC.Cold Spring Harbor Laboratory Press.

Detection of HPV infection in urothelial carcinoma using RNAscope: Clinicopathological characterization

Cancer medicine

2021 Jun 23

Musangile, FY;Matsuzaki, I;Okodo, M;Shirasaki, A;Mikasa, Y;Iwamoto, R;Takahashi, Y;Kojima, F;Murata, SI;
PMID: 34164940 | DOI: 10.1002/cam4.4091

Human papillomavirus (HPV) is a well-established mucosotropic carcinogen, but its impact on urothelial neoplasm is unclear. We aimed to clarify the clinical and pathological features of HPV-related urothelial carcinoma (UC).Tissue samples of 228 cases of UC were obtained from the bladder, upper and lower urinary tract, and metastatic sites to construct a tissue microarray. The samples were analyzed for the presence of HPV by a highly sensitive and specific mRNA in situ hybridization (RISH) technique (RNAscope) with a probe that can detect 18 varieties of high-risk HPV. We also conducted immunohistochemistry (IHC) for a major HPV capsid antibody and DNA-PCR.The HPV detection rates varied among the methods; probably due to low HPV copy numbers in UC tissues and the insufficient specificity and sensitivity of the IHC and PCR assays. The RISH method had the highest accuracy and identified HPV infection in 12 (5.2%) of the cases. The histopathological analysis of the HPV-positive UC showed six cases of usual type UC, five cases of UC with squamous differentiation (UC_SqD), and one case of micropapillary UC. The HPV detection rate was six-fold higher in the cases of UC_SqD than in the other variants of UC (odds ratio [OR] =8.9, p = 0.002). In addition, HPV infection showed a significant association with tumor grade (OR =9.8, p = 0.03) and stage (OR =4.7, p = 0.03) of UC. Moreover, the metastatic rate was higher in HPV-positive than in negative UC (OR =3.4).These data indicate that although the incidence of HPV infection in UC is low, it is significantly associated with squamous differentiation and poor prognosis. Furthermore, our observations show that RNAscope is an ideal method for HPV detection in UC compared with the other standard approaches such as IHC and PCR assays.

Preclinical development of an AAV8-hUGT1A1 vector for the treatment of Crigler-Najjar syndrome.

Molecular Therapy - Methods & Clinical Development (2018)

2018 Dec 26

Collaud F, Bortolussi G, Guianvarc’h L, Aronson SJ, Bordet T, Veron P, Charles S, Vidal P, Sola MS, Rundwasser S, Dufour DG, Lacoste F, Luc C, Wittenberghe Lv, Martin S, Le Bec C, Bosma PJ, Muro AF, Ronzitti G, Hebben M, Mingozzi F.
| DOI: 10.1016/j.omtm.2018.12.011

Adeno-associated viruses (AAV) are among the most efficient vectors for liver gene therapy. results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed a (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase- family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo. Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their sc counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple-transfection of human embryonic kidney (HEK) 293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology/biodistribution study were also conducted using large scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.

Growth plate borderline chondrocytes behave as transient mesenchymal precursor cells

J Bone Miner Res

2019 Mar 19

Mizuhashi K, Nagata M, Matsushita Y, Ono W and Ono N
PMID: 30888720 | DOI: 10.1002/jbmr.3719

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly, at the periphery of the growth plate immediately adjacent to the perichondrium are 'borderline' chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell-type specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow and became Col(2.3kb)-GFP(+) osteoblasts and Cxcl12-GFP(high) reticular stromal 'CAR' cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. This article is protected by copyright. All rights reserved.

The Amphiregulin/EGFR axis protects from lupus nephritis via downregulation of pathogenic CD4+ T helper cell responses

Journal of autoimmunity

2022 Apr 22

Melderis, S;Warkotsch, MT;Dang, J;Hagenstein, J;Ehnold, LI;Herrnstadt, GR;Niehus, CB;Feindt, FC;Kylies, D;Puelles, VG;Berasain, C;Avila, MA;Neumann, K;Tiegs, G;Huber, TB;Tharaux, PL;Steinmetz, OM;
PMID: 35468361 | DOI: 10.1016/j.jaut.2022.102829

Systemic lupus erythematosus (SLE) is a common autoimmune disorder with a complex and poorly understood immuno-pathogenesis. Lupus nephritis (LN) is a frequent and difficult to treat complication, which causes high morbidity and mortality. The multifunctional cytokine amphiregulin (AREG) has been implicated in SLE pathogenesis, but its function in LN currently remains unknown. We thus studied the model of pristane-induced LN and found increasing renal and systemic AREG expression during the course of disease. Importantly, renal injury was significantly aggravated in the absence of AREG, revealing a net anti-inflammatory role. Analyses of immune responses showed dual effects. On the one hand, AREG enhanced activation of pro-inflammatory myeloid cells, which however did not play a major role for the course of LN. More importantly, on the other hand, AREG strongly suppressed pathogenic cytokine production by T helper effector cells. This effect was more general in nature and could be reproduced in response to antigen immunization. Since AREG has been postulated to downregulate T cell responses via enhancing Treg suppressive capacity, we followed up on this aspect. Interestingly, however, in vitro studies revealed potential direct and Treg independent effects of AREG on T helper effector cells. In favor of this notion, we found significantly enhanced T cell responses and consecutive aggravation of LN, only if epidermal growth factor receptor (EGFR) signaling was abrogated in total T cells, but not if the EGFR was absent on Tregs alone. Finally, we also found enhanced AREG expression in plasma and renal biopsies of patients with LN, supporting the relevance of our findings for human disease. In summary, our data identify AREG as an anti-inflammatory mediator of LN via broad downregulation of pathogenic T cell immunity. These findings further highlight the AREG/EGFR axis as a potential therapeutic target.

Kidney-Specific KO of the Circadian Clock Protein PER1 Alters Renal Sodium Handling, Aldosterone Levels, and Kidney/Adrenal Gene Expression

American journal of physiology. Renal physiology

2022 Feb 07

Douma, LG;Costello, HM;Crislip, GR;Cheng, KY;Lynch, IJ;Juffre, A;Barral, D;Masten, SH;Roig, E;Beguiristain, K;Li, W;Bratanatawira, P;Wingo, CS;Gumz, ML;
PMID: 35129370 | DOI: 10.1152/ajprenal.00385.2021

PER1 is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and sodium retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in sodium excretion in response to a high salt diet plus desoxycorticosterone pivalate treatment (HS+DOCP), a model of salt-sensitive hypertension. Additionally, global Per1 KO mice exhibit higher aldosterone levels on a normal salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased sodium retention, but have normal diurnal differences in sodium excretion in response to HS+DOCP. The increased sodium retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared to control (CNTL) mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in the KS-Per1 KO mice compared to CNTL mice. These results emphasize the importance of the circadian clock, not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS+ DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.

Follicle-stimulating hormone promotes growth of human prostate cancer cell line-derived tumor xenografts

FASEB journal : official publication of the Federation of American Societies for Experimental Biology

2021 Apr 01

Oduwole, OO;Poliandri, A;Okolo, A;Rawson, P;Doroszko, M;Chrusciel, M;Rahman, NA;Serrano de Almeida, G;Bevan, CL;Koechling, W;Huhtaniemi, IT;
PMID: 33724574 | DOI: 10.1096/fj.202002168RR

Chemical castration in prostate cancer can be achieved with gonadotropin-releasing hormone (GnRH) agonists or antagonists. Their effects differ by the initial flare of gonadotropin and testosterone secretion with agonists and the immediate pituitary-testicular suppression by antagonists. While both suppress luteinizing hormone (LH) and follicle-stimulating hormone (FSH) initially, a rebound in FSH levels occurs during agonist treatment. This rebound is potentially harmful, taken the expression of FSH receptors (R) in prostate cancer tissue. We herein assessed the role of FSH in promoting the growth of androgen-independent (PC-3, DU145) and androgen-dependent (VCaP) human prostate cancer cell line xenografts in nude mice. Gonadotropins were suppressed with the GnRH antagonist degarelix, and effects of add-back human recombinant FSH were assessed on tumor growth. All tumors expressed GnRHR and FSHR, and degarelix treatment suppressed their growth. FSH supplementation reversed the degarelix-evoked suppression of PC-3 tumors, both in preventive (degarelix and FSH treatment started upon cell inoculation) and therapeutic (treatments initiated 3 weeks after cell inoculation) setting. A less marked, though significant FSH effect occurred in DU145, but not in VCaP xenografts. FSHR expression in the xenografts supports direct FSH stimulation of tumor growth. Testosterone supplementation, to maintain the VCaP xenografts, apparently masked the FSH effect on their growth. Treatment with the LH analogue hCG did not affect PC-3 tumor growth despite their expression of luteinizing hormone/choriongonadotropin receptor. In conclusion, FSH, but not LH, may directly stimulate the growth of androgen-independent prostate cancer, suggesting that persistent FSH suppression upon GnRH antagonist treatment offers a therapeutic advantage over agonist.

Spatial Sequencing in a Model of Early Onset Retinal Degeneration

Investigative Ophthalmology & Visual Science

2022 Jan 01

Huffman, K;Sasik, R;Borooah, S;

RESULTS : Uniform Manifold Approximation and Projection clustering identified distinct expression signatures from the ganglion cell layer(GCL), inner nuclear layer(INL), retinal pigment epithelium (RPE)/choroid/sclera, optic nerve, and ciliary body (Fig, 1) but not the outer nuclear layer(ONL) which was contaminated with expression from other layers. Our findings highlight Clu, C4b, Apoe, and C1qa genes (z-score 3.0, 2.4, 2.3, and 2.2) as potential markers of disease in the RPE. Gene Set Enrichment analysis between rd6 and WT eyes showed upregulation of glycolysis and carbon metabolism pathways in the GCL and Rap1, Hippo and lysosome pathways in the RPE/Choroid/sclera. The ribosomal pathway was downregulated in these layers. No significant pathways were found in the INL, ciliary body or optic nerve.

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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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