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tight junction [keywords]
- Safeguarding Stem Cell-Based Regenerative Therapy against Iatrogenic Cancerogenesis: Transgenic Expression of DNASE1, DNASE1L3, DNASE2, DFFB Controlled By POLA1 Promoter in Proliferating and Directed Differentiation Resisting Human Autologous Pluripotent Induced Stem Cells Leads to their Death. [JOURNAL ARTICLE]
- J Stem Cell Res Ther 2013 Jul 22; Suppl 9(5)
The worst possible complication of using stem cells for regenerative therapy is iatrogenic cancerogenesis. The ultimate goal of our work is to develop a self-triggering feedback mechanism aimed at causing death of all stem cells, which resist directed differentiation, keep proliferating, and can grow into tumors.The specific aim was threefold: (1) to genetically engineer the DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter; (2) to bioengineer anti-SSEA-4 antibody guided vectors delivering transgenes to human undifferentiated and proliferating pluripotent stem cells; (3) to cause death of proliferating and directed differentiation resisting stem cells by transgenic expression of the human recombinant the DNases (hrDNases).The DNA constructs for the human, recombinant DNASE1, DNASE1L3, DNASE2, DFFB controlled by POLA promoter were genetically engineered. The vectors targeting specifically SSEA-4 expressing stem cells were bioengineered. The healthy volunteers' bone marrow mononuclear cells (BMMCs) were induced into human, autologous, pluripotent stem cells with non-integrating plasmids. Directed differentiation of the induced stem cells into endothelial cells was accomplished with EGF and BMP. The anti-SSEA 4 antibodies' guided DNA vectors delivered the transgenes for the human recombinant DNases' into proliferating stem cells.Differentiation of the pluripotent induced stem cells into the endothelial cells was verified by highlighting formation of tight and adherens junctions through transgenic expression of recombinant fluorescent fusion proteins: VE cadherin, claudin, zona occludens 1, and catenin. Proliferation of the stem cells was determined through highlighting transgenic expression of recombinant fluorescent proteins controlled by POLA promoter, while also reporting expression of the transgenes for the hrDNases. Expression of the transgenes for the DNases resulted in complete collapse of the chromatin architecture and degradation of the proliferating cells' genomic DNA. The proliferating stem cells, but not the differentiating ones, were effectively induced to die.Herein, we describe attaining the proof-of-concept for the strategy, whereby transgenic expression of the genetically engineered human recombinant DNases in proliferating and directed differentiation resisting stem cells leads to their death. This novel strategy reduces the risk of iatrogenic neoplasms in stem cell therapy.
- Recruitment and retention of human autologous CD34+ CD117+ CD133+ bone marrow stem cells to infarcted myocardium followed by directed vasculogenesis: Novel strategy for cardiac regeneration. [JOURNAL ARTICLE]
- Mol Cell Ther 2013 Dec 13.
Ongoing clinical trials, in regenerative therapy of patients suffering from myocardial infarctions, rely primarily upon administration of bone marrow stem cells to the infarcted zones. Unfortunately, low retention of these cells, to the therapeutic delivery sites, reduces effectiveness of this strategy; thus it has been identified as the most critical problem for advancement of cardiac regenerative medicine.The specific aim of this work was three-fold: (1) to isolate highly viable populations of human, autologous CD34+, CD117+, and CD133+ bone marrow stem cells; (2) to bioengineer heterospecific, tetravalent antibodies and to use them for recruiting of the stem cells to regenerated zones of infarcted myocardium; (3) to direct vasculogenesis of the retained stem cells with the defined factors.Cardiac tissue was biopsied from the hearts of the patients, who were receiving orthotopic heart transplants after multiple cardiac infarctions. This tissue was used to engineer fully human in vitro models of infarcted myocardium. Bone marrow was acquired from these patients. The marrow cells were sorted into populations of cells displaying CD34, CD117, and CD133. Heterospecific, tetravalent antibodies were bioengineered to bridge CD34, CD117, CD133 displayed on the stem cells with cardiac myosin of the infarcted myocardium. The sorted stem cells were administered to the infarcted myocardium in the in vitro models.Administration of the bioengineered, heterospecific antibodies preceding administration of the stem cells greatly improved the stem cells' recruitment and retention to the infarcted myocardium. Treatment of the retained stem cells with vascular endothelial growth factor and angiopoietin efficiently directed their differentiation into endothelial cells, which expressed vascular endothelial cadherin, platelet / endothelial cell adhesion molecule, claudin, and occludin, while forming tight and adherens junctions.This novel strategy improved retention of the patients' autologous bone marrow stem cells to the infarcted myocardium followed by directed vasculogenesis. Therefore, it is worth pursuing it in support of the ongoing clinical trials of cardiac regenerative therapy.
- The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells. [JOURNAL ARTICLE]
- Food Chem Toxicol 2014 Jul 17.
Actinidin, a kiwifruit cysteine protease, is a marker allergen for genuine sensitization to this food allergen source. Inhalatory cysteine proteases have the capacity for disruption of tight junctions (TJs) enhancing the permeability of the bronchial epithelium. No such properties have been reported for allergenic food proteases so far. The aim was to determine the effect of actinidin on the integrity of T84 monolayers by evaluating its action on the TJ protein occludin. Immunoblot and immunofluorescence were employed for the detection of occludin protein alterations. Gene expression was evaluated by RT-PCR. Breach of occludin network was assessed by measuring transepithelial resistance, blue dextran leakage and passage of allergens from the apical to basolateral compartment. Actinidin exerted direct proteolytic cleavage of occludin; no alteration of occludin gene expression was detected. There was a reduction of occludin staining upon actinidin treatment as a consequence of its degradation and dispersion within the membrane. There was an increase in permeability of the T84 monolayer resulting in reduced transepithelial resistance, blue dextran leakage and passage of allergens actinidin and thaumatin-like protein from the apical to basolateral compartment. Opening of TJs by actinidin may increase intestinal permeability and contribute to the proces of sensitization in kiwifruit allergy.
- Sucrose Esters Increase Drug Penetration, But Do Not Inhibit P-Glycoprotein in Caco-2 Intestinal Epithelial Cells. [JOURNAL ARTICLE]
- J Pharm Sci 2014 Jul 16.
Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and β-catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P-gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
- Loss of tight junction proteins (Claudin 1, 4, and 7) correlates with aggressive behavior in colorectal carcinoma. [JOURNAL ARTICLE]
- Med Sci Monit 2014.:1255-1262.
Background Tight junction proteins in the cell organize paracellular permeability and they play a critical role in apical cell-to-cell adhesion and epithelial polarity. Claudins are major integral membrane proteins of tight junctions, especially Claudin 1, 4, and 7, which are known as the impermeability Claudins. In this study, we investigated the importance of loss of Claudin 1, 4, and 7 expression, and their relation to tumor progression in colorectal cancer patients. Material and Methods Loss of Claudin 1, 4, and 7 expression was examined by immunohistochemical method in 70 patients diagnosed with colorectal cancer. Cases with loss of Claudin expression in <1/3 of tumor cells were classified as mild loss, whereas cases with loss of Claudin expression ³1/3 of tumor cells were classified as moderate-to-marked loss in order to evaluate the relation between loss of Claudin 1, 4, and 7 expression and clinicopathologic data. Results The severe suppression of Claudin 1, 4, and 7 expression was found to be significantly related to the depth of tumor invasion, positive regional lymph nodes, histological grade, lymphovascular invasion, perineural invasion, and lymphocytic response. Additionally, severity of loss in Claudin 4 expression was found to have a relation with distant metastasis. Conclusions Claudin 1, 4, and 7 are important building blocks of paracellular adhesion molecules. Their decreased expression in colorectal cancer seems to have critical effects on cell proliferation, motility, invasion, and immune response against the tumor.
- Cell-Cell Communication in the Tumor Microenvironment, Carcinogenesis, and Anticancer Treatment. [JOURNAL ARTICLE]
- Cell Physiol Biochem 2014 Jul 8; 34(2):213-243.
The delineation of key molecular pathways has enhanced our knowledge of the biology of tumor microenvironment, tumor dissemination, and carcinogenesis. The complexities of cell-cell communication and the possibilities for modulation provide new opportunities for treating cancers. Cells communicate by direct and indirect signaling. Direct cell-cell communication involves both, self-self-communication (intracrine and autocrine), and adjacent communication with nearby cells (juxtacrine), which themselves are regulated by distinct pathways. Indirect intercellular communication involves local communication over short distances (paracrine and synaptic signaling) or over large distances via hormones (endocrine). The essential components of cell-cell communication involve communication junctions (Connexins, Plasmodesmata, Ion Channels, Chemical Synapses, and Pannexins), occluding junctions (Tight Junctions), and anchoring junctions (Adherens, Desmosomes, Focal Adhesions, and Hemidesmosomes). The communication pathways pass through junctions at physical cell-cell attachments, and they go, as well, through the extracellular matrix (ECM) via the different transmembrane adhesion proteins (Cadherins and Integrins). We have here reviewed cell-cell communication involving (1) the components of junctions and their dynamic interplay with the other aspects of communication, including (2) the tumor microenvironment and carcinogenesis, (3) coupling and migration, (4) the underlying cell-cell and sub-cellular communication mechanisms (signaling) of anticancer treatments, and finally, (5) aspects of recent research on cell-cell communication. © 2014 S. Karger AG, Basel.
- Associated changes in the transcription levels of IL-17A and tight junction-associated genes in the duodenal mucosa of rhesus macaques repeatedly exposed to simian/human immunodeficiency virus. [JOURNAL ARTICLE]
- Exp Mol Pathol 2014 Jul 14.
Mucosal barrier dysfunction might play a key role in HIV/AIDS, yet the early effects of HIV-1 on intestinal mucosal barrier, especially tight junctions (TJ) have not been well addressed.To investigate the effects of acute HIV-1 infection on the expression of intestinal IL-17A and TJ-associated genes using an NHP-AIDS model.TaqMan probe real-time RT-PCR methods were established and claudin-1, claudin-3, occludin and zonula occluden-1 (ZO-1) mRNA levels in the duodenal biopsies of rhesus macaques collected before and after rectal exposures to SHIV-SF162P4 were examined and compared with that of IL-17A, IL-6, TGF-β, RORγt, T-bet, Foxp3 and GATA-3.The mRNA levels of TJ-associated genes were statistically significantly reduced soon after viral exposures and the mRNA levels of claudin-1, occludin and ZO-1 in viral positive tissues (from Group I) were lower than that in viral negative tissues (from Group II) after viral exposure. IL-17A mRNA levels were also decreased and positively correlated with the mRNA levels of the TJ-associated genes after viral exposure or infection, although the levels of IL-6, TGF-β and RORγt mRNA showed no statistical difference. The levels of GATA-3 mRNA in tissues collected before viral exposure were statistically different between Group I and Group II animals. The balance between T-bet and GATA-3 mRNA levels in Group II was markedly altered and statistically significantly different from that in Group I.Acute SHIV, and by extension HIV infection could affect the expression of TJ-associated genes, probably through IL-17A and other immune alterations.
- Proinflammatory cytokine-induced Tight Junction remodeling through dynamic self-assembly of claudins. [JOURNAL ARTICLE]
- Mol Biol Cell 2014 Jul 16.
Tight Junctions (TJs) are dynamic, mutiprotein intercellular adhesive contacts that provide a vital barrier function in epithelial tissues. TJs are remodeled during physiologic development and pathological mucosal inflammation, and differential expression of the claudin family of TJ proteins determines epithelial barrier properties. However, the molecular mechanisms involved in TJ remodeling are incompletely understood. Using acGFP-claudin 4 as a bio-sensor of TJ remodeling, we observed increased claudin 4 Fluorescence Recovery after Photobleaching (FRAP) dynamics in response to inflammatory cytokines. Interferon gamma (IFN-γ) and Tumor Necrosis Factor alpha (TNF-α) increased the proportion of mobile claudin 4 in the TJ. Upregulation of claudin 4 protein rescued these mobility defects and cytokine-induced barrier compromise. Furthermore, claudins 2 and 4 have reciprocal effects on epithelial barrier function, exhibit differential FRAP dynamics, and compete for residency within the TJ. These findings establish a model of TJs as self-assembling systems that undergo remodeling in response to proinflammatory cytokines through a mechanism of heterotypic claudin binding incompatibility.
- Fluid therapy in patients with brain injury: what does physiology tell us? [JOURNAL ARTICLE]
- Crit Care 2014 Mar 12; 18(2):119.
- P42 Ebp1 functions as a tumor suppressor in non-small cell lung cancer. [JOURNAL ARTICLE]
- BMB Rep 2014 Jul 16.
Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGFβ-stimulated lung cancer cells, A549. SB431542, a type I TGFβ receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGFβ on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGFβ in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-shTJP1), wound healing was much lower compared with cells infected with control viral particles. Taken together, these data suggest that TGFβ enhances TJP1 expression which may play a role beyond structural support in tight junctions during cancer development.