NR4A2 Expression Patterns in Mouse Models of Rheumatoid Arthritis

Arthritis is a group of over 100 musculoskeletal disorders affecting approximately 50 million adults in the US. In an effort to develop new drugs to treat arthritis, we are exploring the function of the orphan nuclear receptor 4A2 (NR4A2), a transcription factor over-expressed in inflamed joints. The transcriptional targets of NR4A2 include angiogenesis factors and matrix metalloproteinases (MMPs). NR4A2 appears to have a deleterious effect in synoviocytes by promoting tissue degradation, while in chondrocytes it seems to have a protective function. Previous work on human synoviocytes has shown NR4A2 to rise early in response to inflammation, leading us to hypothesize that NR4A2 may be a preliminary mediator of arthritis. To test this hypothesis in vivo, we studied NR4A2 expression patterns in two mouse models of RA: Antigen Induced Arthritis (AIA) and Serum Transfer Arthritis (STA). Tissue sections were obtained from healthy and arthritic mice at early, mid, and late time-points following induction. Joint cross-sections were examined via immunohistochemical staining, and NR4A2 positive cells were quantified in synovial and cartilage tissues. In the AIA model, NR4A2 protein levels peaked in synovium at day 10 of disease (mid stage, 50% positive) and declined later in disease. In cartilage, protein levels reached a maximum at day 8 (early stage, 70%) and subsequently declined as well. In contrast, NR4A2 was not expressed in the STA model, despite apparent joint degradation. NR4A2 has been shown to be expressed in STA arthritis by other researchers, so it is unknown why STA samples did not show NR4A2 expression in our experiment.

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Upregulations of Clcn3 and P-Gp Provoked by Lens Osmotic Expansion in Rat Galactosemic Cataract

Journal of Diabetes Research ◽

10.1155/2017/3472735 ◽

2017 ◽

Vol 2017 ◽

pp. 1-8

Author(s):

Lixia Ji ◽

Lixia Cheng ◽

Zhihong Yang

Keyword(s):

Osmotic Stress ◽

Early Stage ◽

Lens Epithelial Cells ◽

Anterior Capsule ◽

Glycoprotein P ◽

Protein Levels ◽

P Glycoprotein ◽

Sugar Cataract

Objective.Lens osmotic expansion, provoked by overactivated aldose reductase (AR), is the most essential event of sugar cataract. Chloride channel 3 (Clcn3) is a volume-sensitive channel, mainly participating in the regulation of cell fundamental volume, and P-glycoprotein (P-gp) acts as its modulator. We aim to study whether P-gp and Clcn3 are involved in lens osmotic expansion of galactosemic cataract.Methods and Results.In vitro, lens epithelial cells (LECs) were primarily cultured in gradient galactose medium (10–60 mM), more and more vacuoles appeared in LEC cytoplasm, and mRNA and protein levels of AR, P-gp, and Clcn3 were synchronously upregulated along with the increase of galactose concentration. In vivo, we focused on the early stage of rat galactosemic cataract, amount of vacuoles arose from equatorial area and scattered to the whole anterior capsule of lenses from the 3rd day to the 9th day, and mRNA and protein levels of P-gp and Clcn3 reached the peak around the 9th or 12th day.Conclusion. Galactosemia caused the osmotic stress in lenses; it also markedly leads to the upregulations of AR, P-gp, and Clcn3 in LECs, together resulting in obvious osmotic expansion in vitro and in vivo.

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A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-17 ◽

2018 ◽

Vol 62 (4) ◽

pp. e01505-17 ◽

Cited By ~ 17

Author(s):

R. S. Jumani ◽

K. Bessoff ◽

M. S. Love ◽

P. Miller ◽

E. E. Stebbins ◽

...

Keyword(s):

Mouse Model ◽

Drug Development ◽

Early Stage ◽

New Drugs ◽

Cryptosporidium Hominis ◽

Content Type ◽

Knockout Mouse Model ◽

Malaria Box

ABSTRACTCryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drivein vivoefficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound forCryptosporidiumdrug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies,in vitrotoxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound againstCryptosporidium parvumIowa and field isolates was comparable to that againstCryptosporidium hominis. Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic forC. parvum, we developed a novelin vitroparasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stageCryptosporidiumdrug leads and may aid in planningin vivoefficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.

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Hepatic gene expression patterns in thyroid hormone-treated hypothyroid rats

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310291 ◽

2003 ◽

Vol 31 (2) ◽

pp. 291-303 ◽

Cited By ~ 42

Author(s):

JM Weitzel ◽

S Hamann ◽

M Jauk ◽

M Lacey ◽

A Filbry ◽

...

Keyword(s):

Gene Expression ◽

Thyroid Hormone ◽

Expression Patterns ◽

Regulatory Elements ◽

Activation Mechanism ◽

Gene Expression Patterns ◽

Peroxisome Proliferator ◽

Gene Promoters ◽

Protein Levels

Thyroid hormone (T3) is essential for normal development, differentiation and metabolic balance. We have performed DNA microarray experiments using hepatic RNA from hypothyroid and T3-treated hypothyroid rats in order to characterize T3-induced gene expression patterns after various time points (6, 24 and 48 h after the administration of the hormone). Sixty-two of 4608 different genes displayed a reproducible T3-response, and cluster analysis divided these differentially regulated genes into six expression patterns. Thirty-six genes were not significantly regulated within the first 24 h. Transient transfection experiments of eight late-induced gene promoters failed to detect a thyroid hormone response element within their regulatory elements, suggesting an indirect activation mechanism(s). In search for an intermediate factor of T3 action, we examined whether various rather ubiquitous transcription factors, peroxisome proliferator-activated receptors (PPARs) and coactivators of the PPARgamma coactivator 1 family (PGC-1) are regulated by T3. Only PPARgamma and PERC/PGC-1beta exhibit a significant T3-response within the first 6 h after treatment, identifying these factors as candidate components for mediating the late-induced expression pattern. Regulation of early-induced genes within the first 6 h after administration of T3 on transcript levels correlates with altered protein levels after 24 and 48 h in vivo.

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Functional in vivo and in vitro effects of 20q11.21 genetic aberrations on hPSC differentiation

Scientific Reports ◽

10.1038/s41598-020-75657-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hye-Yeong Jo ◽

Youngsun Lee ◽

Hongryul Ahn ◽

Hyeong-Jun Han ◽

Ara Kwon ◽

...

Keyword(s):

Cell Fate ◽

Early Stage ◽

Expression Patterns ◽

Transcriptome Profiling ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Negative Effect ◽

In Vitro Effects

Abstract Human pluripotent stem cells (hPSCs) have promising therapeutic applications due to their infinite capacity for self-renewal and pluripotency. Genomic stability is imperative for the clinical use of hPSCs; however, copy number variation (CNV), especially recurrent CNV at 20q11.21, may contribute genomic instability of hPSCs. Furthermore, the effects of CNVs in hPSCs at the whole-transcriptome scale are poorly understood. This study aimed to examine the functional in vivo and in vitro effects of frequently detected CNVs at 20q11.21 during early-stage differentiation of hPSCs. Comprehensive transcriptome profiling of abnormal hPSCs revealed that the differential gene expression patterns had a negative effect on differentiation potential. Transcriptional heterogeneity identified by single-cell RNA sequencing (scRNA-seq) of embryoid bodies from two different isogenic lines of hPSCs revealed alterations in differentiated cell distributions compared with that of normal cells. RNA-seq analysis of 22 teratomas identified several differentially expressed lineage-specific markers in hPSCs with CNVs, consistent with the histological results of the altered ecto/meso/endodermal ratio due to CNVs. Our results suggest that CNV amplification contributes to cell proliferation, apoptosis, and cell fate specification. This work shows the functional consequences of recurrent genetic abnormalities and thereby provides evidence to support the development of cell-based applications.

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PDTM-35. MODELLING MEDULLOBLASTOMA WITH MOUSE MODELS AND HUMAN CEREBELLAR ORGANOIDS

Neuro-Oncology ◽

10.1093/neuonc/noz175.811 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi195-vi195

Author(s):

Luca Tiberi

Keyword(s):

Mouse Models ◽

Drug Testing ◽

New Drugs ◽

Driver Gene ◽

Humanized Mouse Model ◽

Molecular Features ◽

Group 3 ◽

In Vivo Screen

Abstract Malignant medulloblastoma (MB) is the most common brain tumor affecting children and it remains responsible for a high percentage of morbidity and mortality among cancer patients. During the past few years, studies on human MB and mouse models have uncovered the existence of four major MB groups, with specific pathological and molecular features: WNT, SHH, Group 3 and Group 4. Of the four groups, patients with Group 3 MB have the worst outcome with nearly 50% of the tumors metastatic at the time of diagnosis. Therefore, developing “humanized “ mouse model of Group3 Medulloblastoma would be of paramount importance for the identification and testing of new drugs for pediatric patients, tailored on the genetic condition of the patient itself. We performed an in-vivo screen to identify new cancer-inducing genes starting from the published genome-wide analyses of MB patients. This screen led to the identification of novel driver gene combinations required for tumorigenesis. To study Medulloblastoma in human cells, we generated human cerebellar organoids. Organoids are cellular structures that resemble whole organs and can be generated from pluripotent stem cells or isolated organ progenitors. We were able to induce cerebellar progenitors to self-form neuro-epithelial structures that mimic early human cerebellar plate, composed by cerebellar progenitors, cerebellar neurons (interneurons, Purkinje cells, and granule neurons) and glial cells. Notably, we have generated human putative iPSC-derived cancer organoids with gene combinations mimicking human Group3 MB. Indeed, organoids that mimic cancer can be used as an alternative system for drug testing that better recapitulate effects in human patients as compared to other in-vitro and in-vivo systems.

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Role of the TNF pathway in the progression of diabetic nephropathy in KK-Ay mice

AJP Renal Physiology ◽

10.1152/ajprenal.00509.2013 ◽

2014 ◽

Vol 306 (11) ◽

pp. F1335-F1347 ◽

Cited By ~ 32

Author(s):

Keisuke Omote ◽

Tomohito Gohda ◽

Maki Murakoshi ◽

Yu Sasaki ◽

Saiko Kazuno ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Early Stage ◽

Mrna Levels ◽

Monocyte Chemoattractant Protein 1 ◽

Protein Levels ◽

Tnf Α ◽

Soluble Tnf Receptor

Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNF-α remains unclear. The objectives of the present study were to examine whether TNF-α inhibition with a soluble TNF receptor (TNFR)2 fusion protein, i.e., etanercept (ETN), improves the early stage of DN in the type 2 diabetic model of the KK-Ay mouse and to also investigate which TNF pathway, TNFR1 or TNFR2, is predominantly involved in the progression of this disease. ETN was injected intraperitoneally into mice for 8 wk. Renal damage was evaluated by immunohistochemistry, Western blot analysis, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNF-α and/or high glucose (HG) and treated with ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNF-α and TNFR1, in ETN-treated KK-Ay mice were significantly decreased compared with untreated KK-Ay mice. mRNA levels of ICAM-1, VCAM-1, and monocyte chemoattractant protein-1 and the number of F4/80-positive cells were all decreased after treatment. Numbers of cleaved caspase-3- and TUNEL-positive cells in untreated mice were very few and were not different from ETN-treated mice. In vitro, stimulation with TNF-α or HG markedly increased both mRNA levels of TNFRs, unlike in the in vivo case. Furthermore, ETN partly recovered TNF-α-induced but not HG-induced TNFR mRNA levels. In conclusion, it appears that ETN may improve the progression of the early stage of DN predominantly through inhibition of the anti-inflammatory action of the TNF-α-TNFR2 pathway.

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Establishment of Colorectal Cancer Organoids in Microfluidic-Based System

Micromachines ◽

10.3390/mi12050497 ◽

2021 ◽

Vol 12 (5) ◽

pp. 497

Author(s):

Diana Pinho ◽

Denis Santos ◽

Ana Vila ◽

Sandra Carvalho

Keyword(s):

Colorectal Cancer ◽

Drug Screening ◽

Molecular Mechanisms ◽

Early Stage ◽

Low Cost ◽

New Drugs ◽

Individual Level ◽

Conventional Culture ◽

On Chip

Colorectal cancer is the second leading cause of cancer death worldwide. Significant advances in the molecular mechanisms underlying colorectal cancer have been made; however, the clinical approval of new drugs faces many challenges. Drug discovery is a lengthy process causing a rapid increase in global health care costs. Patient-derived tumour organoids are considered preclinical models with the potential for preclinical drug screening, prediction of patient outcomes, and guiding optimized therapy strategies at an individual level. Combining microfluidic technology with 3D tumour organoid models to recapitulate tumour organization and in vivo functions led to the development of an appropriate preclinical tumour model, organoid-on-a-chip, paving the way for personalized cancer medicine. Herein, a low-cost microfluidic device suitable for culturing and expanding organoids, OrganoidChip, was developed. Patient-derived colorectal cancer organoids were cultured within OrganoidChip, and their viability and proliferative activity increased significantly. No significant differences were verified in the organoids’ response to 5-fluorouracil (5-FU) treatment on-chip and on-plate. However, the culture within the OrganoidChip led to a significant increase in colorectal cancer organoid-forming efficiency and overall size compared with conventional culture on a 24-well plate. Interestingly, early-stage and late-stage organoids were predominantly observed on-plate and within the OrganoidChip, respectively. The OrganoidChip thus has the potential to generate in vivo-like organotypic structures for disease modelling and drug screening applications.

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Modeling Endometrial Cancer: Past, Present, and Future

International Journal of Molecular Sciences ◽

10.3390/ijms19082348 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2348 ◽

Cited By ~ 16

Author(s):

Tom Van Nyen ◽

Cristian P. Moiola ◽

Eva Colas ◽

Daniela Annibali ◽

Frédéric Amant

Keyword(s):

Endometrial Cancer ◽

Reproductive Tract ◽

Early Stage ◽

Female Reproductive Tract ◽

New Drugs ◽

Low Grade ◽

In Vivo Models ◽

Molecular Determinants

Endometrial cancer is the most common type of cancer of the female reproductive tract. Although prognosis is generally good for patients with low-grade and early-stage diseases, the outcomes for high-grade and metastatic/recurrent cases remain poor, since traditional chemotherapy regimens based on platinum and taxanes have limited effects. No targeted agents have been approved so far, although several new drugs have been tested without striking results in clinical trials. Over the last decades, many efforts have been made towards the establishment and development of preclinical models, aiming at recapitulating the structural and molecular determinants of the disease. Here, we present an overview of the most commonly used in vitro and in vivo models and discuss their peculiar features, describing their main applications and the value in the advancement of both fundamental and translational endometrial cancer research.

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vsp Gene Expression byGiardia lamblia Clone GS/M-83-H7 during Antigenic Variation In Vivo and In Vitro

Infection and Immunity ◽

10.1128/iai.69.9.5278-5285.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5278-5285 ◽

Cited By ~ 25

Author(s):

M. Bienz ◽

M. Siles-Lucas ◽

P. Wittwer ◽

N. Müller

Keyword(s):

Gene Expression ◽

Antigenic Variation ◽

Expression Patterns ◽

Amino Acid Level ◽

Late Time ◽

Continuous Change ◽

Ancestral Gene ◽

Time Points

ABSTRACT Giardia lamblia infections are associated with antigenic variation of the parasite, which is generated by a continuous change of the variant-specific surface proteins (VSPs). Many investigations on the process of antigenic variation were based on the use of G. lamblia clone GS/M-83-H7, which expresses VSP H7 as its major surface antigen. In the present study, mice were infected with the aforementioned clonal line to investigatevsp gene expression during the complex process of antigenic variation of the parasite. Trophozoites collected from the intestines of individual animals at different time points postinfection (p.i.) were analyzed directly for their vsp gene expression patterns, i.e., without cultivating the recovered parasites in vitro. Because few trophozoites were recovered at late time points p.i., a combined 5′ rapid amplification of cDNA ends–reverse transcription-PCR approach was utilized. This allowed detection and subsequent sequence analysis of vsp gene transcripts upon generation of amplified cDNA analogues. The same PCR approach was applied for analysis of vsp gene expression in variants obtained after negative selection of axenic GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. In an overall view of the entire panel of in vivo- and in vitro-derived parasite populations, expression of 29 different vspgene sequences could be demonstrated. In vivo antigenic variation ofG. lamblia clone GS/M-83-H7 was shown to be a continuous process involving the consecutive appearance of relatively distinct sets of vsp transcripts. During the 42-day infection period investigated, this process activated at least 22 differentvsp genes. Comparative molecular analyses of the amino acid level demonstrated that all cDNA segments identified encode structural elements typical of the terminal segment ofGiardia VSP. The similarity of most of the GS/M-83-H7 VSP sequences identified in the present study supports previous suggestions that vsp gene diversification in G. lamblia is the result of ancestral gene duplication, mutation, and/or recombination events.

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Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation

Biochemical Journal ◽

10.1042/bj20112062 ◽

2012 ◽

Vol 443 (3) ◽

pp. 789-798 ◽

Cited By ~ 19

Author(s):

Seung-Won Choi ◽

Da-Un Jeong ◽

Jeong-Ah Kim ◽

Boyoung Lee ◽

Kyu Sang Joeng ◽

...

Keyword(s):

Early Stage ◽

Skeletal Development ◽

Low Density Lipoprotein ◽

Expression Patterns ◽

Density Lipoprotein ◽

Indian Hedgehog ◽

Related Protein ◽

Chondrocyte Hypertrophy ◽

Protein Levels ◽

Chondrocyte Maturation

The Ihh (Indian Hedgehog) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 (NK3 homeobox 2) is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in the present study, we investigated whether Nkx3.2, an early-stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. We show that Ihh signalling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (low-density-lipoprotein-receptor-related protein) (Wnt co-receptor) and Sfrp (secreted frizzled-related protein) (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocytes. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signalling by deletion of either Ihh or smoothened. Thus these results suggest that Ihh/Wnt5a signalling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis.

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