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1.
Int J Dev Biol ; 2024 May 13.
Article in English | MEDLINE | ID: mdl-38770834

ABSTRACT

The development of skin appendages, including hair follicles, teeth and mammary glands is initiated through the formation of the placode, a local thickening of the epithelium. The Wnt/ß-catenin signaling cascade is an evolutionary conserved pathway with an essential role in placode morphogenesis, but its downstream targets and their exact functions remain ill defined. In this study, we identify Achaete-scute complex-like 4 (Ascl4) as a novel target of the Wnt/ß-catenin pathway and demonstrate its expression pattern in the signaling centers of developing hair follicles and teeth. Ascl transcription factors belong to the superfamily of basic helix-loop-helix transcriptional regulators involved in cell fate determination in many tissues. However, their specific role in the developing skin remains largely unknown. We report that Ascl4 null mice have no overt phenotype. Absence of Ascl4 did not impair hair follicle morphogenesis or hair shaft formation suggesting that it is non-essential for hair follicle development. No tooth or mammary gland abnormalities were detected either. We suggest that other transcription factors may functionally compensate for the absence of Ascl4, but further research is warranted to assess this possibility.

2.
Cell Rep ; 42(6): 112643, 2023 06 27.
Article in English | MEDLINE | ID: mdl-37318953

ABSTRACT

Morphogenesis of ectodermal organs, such as hair, tooth, and mammary gland, starts with the formation of local epithelial thickenings, or placodes, but it remains to be determined how distinct cell types and differentiation programs are established during ontogeny. Here, we use bulk and single-cell transcriptomics and pseudotime modeling to address these questions in developing hair follicles and epidermis and produce a comprehensive transcriptomic profile of cellular populations in the hair placode and interplacodal epithelium. We report previously unknown cell populations and marker genes, including early suprabasal and genuine interfollicular basal markers, and propose the identity of suprabasal progenitors. By uncovering four different hair placode cell populations organized in three spatially distinct areas, with fine gene expression gradients between them, we posit early biases in cell fate establishment. This work is accompanied by a readily accessible online tool to stimulate further research on skin appendages and their progenitors.


Subject(s)
Hair Follicle , Transcriptome , Mice , Animals , Hair Follicle/metabolism , Transcriptome/genetics , Mice, Transgenic , Epidermis/metabolism , Hair
3.
J Exp Zool B Mol Dev Evol ; 336(1): 7-17, 2021 01.
Article in English | MEDLINE | ID: mdl-33128445

ABSTRACT

When a null mutation of a gene causes a complete developmental arrest, the gene is typically considered essential for life. Yet, in most cases, null mutations have more subtle effects on the phenotype. Here we used the phenotypic severity of mutations as a tool to examine system-level dynamics of gene expression. We classify genes required for the normal development of the mouse molar into different categories that range from essential to subtle modification of the phenotype. Collectively, we call these the developmental keystone genes. Transcriptome profiling using microarray and RNAseq analyses of patterning stage mouse molars show highly elevated expression levels for genes essential for the progression of tooth development, a result reminiscent of essential genes in single-cell organisms. Elevated expression levels of progression genes were also detected in developing rat molars, suggesting evolutionary conservation of this system-level dynamics. Single-cell RNAseq analyses of developing mouse molars reveal that even though the size of the expression domain, measured in the number of cells, is the main driver of organ-level expression, progression genes show high cell-level transcript abundances. Progression genes are also upregulated within their pathways, which themselves are highly expressed. In contrast, a high proportion of the genes required for normal tooth patterning are secreted ligands that are expressed in fewer cells than their receptors and intracellular components. Overall, even though expression patterns of individual genes can be highly different, conserved system-level principles of gene expression can be detected using phenotypically defined gene categories.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Odontogenesis/genetics , Odontogenesis/physiology , Tooth/growth & development , Animals , Biological Evolution , Gene Expression Profiling , Single-Cell Analysis , Up-Regulation
4.
Acta Dermatovenerol Croat ; 26(2): 105-108, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29989865

ABSTRACT

The aim of this study was to investigate how many patients with atopic dermatitis applied hypoallergenic cosmetics compared with topical corticosteroids. The study included 41 patients with atopic dermatitis (AD) who were treated from June 1 to July 1, 2017 at the Department of Dermatology and Venerology, Clinical Hospital Mostar. According to the data analyzed, 46.34% of patients used hypoallergenic cosmetics while 24.39% used them periodically. 32.7% patients used topical corticosteroids and 17.07 % used them periodically. 19.51 % of the patients with atopic dermatitis used alternative products, while 17.07% used them periodically. Out of the total number of patients, most of the patients used neutral products. 78.05% used neutral cosmetic products, and 19.51 % used them periodically. 65.85 % patients used sunscreens and 24.40 % used them periodically. The majority of patients used neutral cosmetic products and sunscreens. There was no statistically significant difference in the frequency of the use hypoallergenic cosmetics compared with topical corticosteroids (χ2 = 1.802; df = 2; P=0.405). Women used sunscreens more often (χ2 = 16,02; df = 1; p = <0,05).


Subject(s)
Cosmetics/therapeutic use , Dermatitis, Atopic/drug therapy , Dermatologic Agents/therapeutic use , Administration, Topical , Adolescent , Adrenal Cortex Hormones/therapeutic use , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Young Adult
5.
Amino Acids ; 49(3): 529-540, 2017 03.
Article in English | MEDLINE | ID: mdl-27503559

ABSTRACT

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Autoantibodies/biosynthesis , Celiac Disease/immunology , GTP-Binding Proteins/immunology , Immunocompromised Host , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Transglutaminases/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/genetics , CHO Cells , Celiac Disease/genetics , Celiac Disease/pathology , Cricetulus , Female , GTP-Binding Proteins/genetics , Gene Expression , Glutens/chemistry , Glutens/immunology , Humans , Immunoglobulin A/biosynthesis , Immunohistochemistry , Inflammation , Injections, Intraperitoneal , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Intestine, Small/immunology , Intestine, Small/pathology , Mice , Mice, Nude , Protein Glutamine gamma Glutamyltransferase 2 , Recombinant Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Transglutaminases/genetics
6.
Expert Opin Ther Targets ; 19(3): 335-48, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25410283

ABSTRACT

INTRODUCTION: The only current treatment for celiac disease is a strict gluten-free diet. The ubiquitous presence of gluten in groceries, however, makes the diet burdensome and difficult to maintain, and alternative treatment options are thus needed. Here, the important role of transglutaminase 2 (TG2) in the pathogenesis of celiac disease makes it an attractive target for drug development. AREAS COVERED: The present paper gives an overview of TG2 and addresses its significance in the pathogenesis of celiac disease. Moreover, the article summarizes preclinical studies performed with TG2 inhibitors and scrutinizes issues related to this therapeutic approach. EXPERT OPINION: Activation of TG2 in the intestinal mucosa is central in celiac disease pathogenesis and researchers have therefore suggested TG2 inhibitors as a potential therapeutic approach. However, a prerequisite for such a drug is that it should be specific for TG2 and not affect the activity of other members of the transglutaminase family. Such compounds have already been introduced and tested in vitro, but a major obstacle to further development is the lack of a well-defined animal model for celiac disease. Nonetheless, with encouraging results in preclinical studies clinical trials with TG2 inhibitors are eagerly awaited.


Subject(s)
Celiac Disease/drug therapy , GTP-Binding Proteins/antagonists & inhibitors , Molecular Targeted Therapy , Transglutaminases/antagonists & inhibitors , Animals , Celiac Disease/enzymology , Celiac Disease/physiopathology , Diet, Gluten-Free , Drug Design , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , GTP-Binding Proteins/metabolism , Humans , Intestinal Mucosa/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , Transglutaminases/metabolism
7.
Expert Opin Investig Drugs ; 23(8): 1079-91, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24806736

ABSTRACT

INTRODUCTION: Gluten is the main trigger for celiac disease, and the current treatment is based on its elimination from the diet. Although the symptoms usually disappear during the diet, it is restrictive and difficult to maintain. Further, despite a strict treatment the small-bowel mucosal damage does now always heal. Consequently, adherence is often poor and new treatment approaches are needed. With an increased understanding of the disease pathogenesis, several novel treatments have been suggested, and some of them have already entered Phase II clinical trials. AREAS COVERED: This article reviews the latest status of the drugs in development for celiac disease. The article focuses mainly on synthetic drugs currently entering in clinical trials. EXPERT OPINION: It is anticipated that some of the treatments under investigation will soon enter Phase III clinical trials, although challenges remain. For instance, histological studies are problematic in wide-scale clinical studies. On the other hand, the existing non-invasive serological methods and clinical outcome measures might be too insensitive for monitoring responses to the possible drug candidates. There is also no animal model which would accurately reflect celiac disease. Well-conducted basic and clinical research is required to develop better non-invasive surrogate markers and patient-related outcomes for future pharmacological studies.


Subject(s)
Celiac Disease/drug therapy , Diet, Gluten-Free , Glutens/metabolism , Animals , Celiac Disease/diet therapy , Drug Design , Humans , Intestinal Mucosa/pathology
8.
PLoS One ; 8(6): e65887, 2013.
Article in English | MEDLINE | ID: mdl-23824706

ABSTRACT

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.


Subject(s)
Autoantibodies/immunology , Celiac Disease/immunology , GTP-Binding Proteins/immunology , Neovascularization, Pathologic/immunology , Transglutaminases/immunology , Animals , Endothelial Cells/immunology , Female , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Protein Glutamine gamma Glutamyltransferase 2
9.
J Mol Med (Berl) ; 90(7): 817-26, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22223195

ABSTRACT

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies.


Subject(s)
Autoantibodies/immunology , Celiac Disease/immunology , Celiac Disease/metabolism , GTP-Binding Proteins/immunology , Transglutaminases/immunology , rhoB GTP-Binding Protein/metabolism , Angiogenesis Inhibitors/immunology , Angiogenesis Inhibitors/pharmacology , Animals , Autoantibodies/pharmacology , Celiac Disease/genetics , Cluster Analysis , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Silencing , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Immunoglobulin A/immunology , Immunoglobulin A/pharmacology , Mice , Mice, Inbred BALB C , Protein Glutamine gamma Glutamyltransferase 2 , RNA Interference , rhoB GTP-Binding Protein/genetics
10.
Gut ; 60(11): 1487-93, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21471568

ABSTRACT

BACKGROUND AND OBJECTIVE: Antitransglutaminase (anti-TG2) antibodies are synthesised in the intestine and their presence seems predictive of future coeliac disease (CD). This study investigates whether mucosal antibodies represent an early stage of gluten intolerance even in the absence of intestinal damage and serum anti-TG2 antibodies. METHODS: This study investigated 22 relatives of patients with CD genetically predisposed to gluten intolerance but negative for both serum anti-TG2 antibodies and intestinal abnormalities. Fifteen subjects were symptomatic and seven were asymptomatic. The presence of immunoglobulin A anti-TG2 antibodies in the intestine was studied by creating phage-antibody libraries against TG-2. The presence of intestinal anti-TG2 antibodies was compared with the serum concentration of the intestinal fatty acid-binding protein (I-FABP), a marker for early intestinal mucosal damage. The effects of a 12-month gluten-free diet on anti-TG2 antibody production and the subjects' clinical condition was monitored. Twelve subjects entered the study as controls. RESULTS: The intestinal mucosa appeared normal in 18/22; 4 had a slight increase in intraepithelial lymphocytes. Mucosal anti-TG2 antibodies were isolated in 15/22 subjects (68%); in particular symptomatic subjects were positive in 13/15 cases and asymptomatic subjects in 2/7 cases (p=0.01). No mucosal antibodies were selected from the controls' biopsies. There was significant correlation between the presence of intestinal anti-TG2 antibodies and positive concentrations of I-FABP (p=0.0008). After a gluten-free diet, 19/22 subjects underwent a second intestinal biopsy, which showed that anti-TG2 antibodies had disappeared in 12/15 (p=0.002), while I-FABP decreased significantly (p<0.0001). The diet resolved both extraintestinal and intestinal symptoms. CONCLUSIONS: A new form of genetic-dependent gluten intolerance has been described in which none of the usual diagnostic markers is present. Symptoms and intestinal anti-TG2 antibodies respond to a gluten free-diet. The detection of intestinal anti-TG2 antibodies by the phage-antibody libraries has an important diagnostic and therapeutic impact for the subjects with gluten-dependent intestinal or extraintestinal symptoms. Clinical trial number NCT00677495.


Subject(s)
Antibodies, Anti-Idiotypic/blood , Celiac Disease/diet therapy , Celiac Disease/diagnosis , GTP-Binding Proteins/immunology , Intestinal Mucosa/immunology , Transglutaminases/immunology , Adolescent , Adult , Asymptomatic Diseases , Celiac Disease/genetics , Celiac Disease/immunology , Child , Child, Preschool , Diet, Gluten-Free , Fatty Acid-Binding Proteins/blood , Female , Genetic Predisposition to Disease , Health Status , Humans , Male , Middle Aged , Peptide Library , Protein Glutamine gamma Glutamyltransferase 2 , Young Adult
11.
Nucleic Acids Res ; 38(9): e110, 2010 May.
Article in English | MEDLINE | ID: mdl-20144949

ABSTRACT

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.


Subject(s)
Protein Interaction Mapping/methods , Sequence Analysis, DNA/methods , DNA, Complementary/chemistry , GTP-Binding Proteins/metabolism , Humans , Open Reading Frames , Peptide Library , Protein Glutamine gamma Glutamyltransferase 2 , Protein Interaction Domains and Motifs , Transglutaminases/metabolism
12.
Scand J Gastroenterol ; 45(4): 421-7, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20095873

ABSTRACT

OBJECTIVE: Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. MATERIAL AND METHODS: The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. RESULTS: Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. CONCLUSIONS: Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.


Subject(s)
Antibodies, Monoclonal/immunology , Autoantibodies/physiology , Celiac Disease/enzymology , Celiac Disease/immunology , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/immunology , Neovascularization, Pathologic/immunology , Transglutaminases/antagonists & inhibitors , Transglutaminases/immunology , Analysis of Variance , Biopsy , Blotting, Western , Cell Culture Techniques , Endothelial Cells/immunology , Endothelium, Vascular/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin A/immunology , Protein Glutamine gamma Glutamyltransferase 2
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