Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse β-defensins-2, -3 and -4 (mBD2–4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that β-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.     

Induced pluripotent stem cells (iPSCs) as game-changing tools in the treatment of neurodegenerative disease: Mirage or reality?

Based on investigations, there exist tight correlations between neurodegenerative diseases' incidence and progression and aberrant protein aggregreferates in nervous tissue. However, the pathology of these diseases is not well known, leading to an inability to find an appropriate therapeutic approach to delay occurrence or slow many neurodegenerative diseases' development. The accessibility of induced pluripotent stem cells (iPSCs) in mimicking the phenotypes of various late-onset neurodegenerative diseases presents a novel strategy for in vitro disease modeling. The iPSCs provide a valuable and well-identified resource to clarify neurodegenerative disease mechanisms, as well as prepare a promising human stem cell platform for drug screening. Undoubtedly, neurodegenerative disease modeling using iPSCs has established innovative opportunities for both mechanistic types of research and recognition of novel disease treatments. Most important, the iPSCs have been considered as a novel autologous cell origin for cell-based therapy of neurodegenerative diseases following differentiation to varied types of neural lineage cells (e.g. GABAergic neurons, dopamine neurons, cortical neurons, and motor neurons). In this review, we summarize iPSC-based disease modeling in neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, and Huntington's disease. Moreover, we discuss the efficacy of cell-replacement therapies for neurodegenerative disease.     

Mesenchymal stem cells as the game-changing tools in the treatment of various organs disorders: Mirage or reality?

Recently a growing attention in scientific community has been gathered on potential application of mesenchymal stem cells (MSCs) in various fields of medicine. Owing to the fact that they can be easily isolated from different sources, and simply proliferated in large quantities while keeping their original biological characteristics, they can be successfully used as cell-based therapeutics. Engineering MSCs and other type of stem cells to be carriers of therapeutic agents is a new tactic in the targeted gene and cell therapy of cancers and degenerative diseases. Various useful properties of MSCs including tropism toward tumor/injury site(s), weakly immunogenic, production of anti-inflammatory molecules, and safety against normal tissues have made them prone for regenerative medicine, targeted therapy and treating injured tissues, and immunological abnormalities. In this review, we introduce latest advances, methods, and applications of MSCs in gene therapy of various malignant organ disorders. Additionally, we will cover the problems and challenges which researchers have faced with when trying to translate their basic experimental findings in MSCs research to clinically applicable therapeutics.     

From bench to bed: the potential of stem cells for the treatment of Parkinson’s disease

Parkinson’s disease (PD) is the most common movement disorder. The neuropathology is characterized by the loss of dopamine neurons in the substantia nigra pars compacta. Transplants of fetal/embryonic midbrain tissue have exhibited some beneficial clinical effects in open-label trials. Neural grafting has, however, not become a standard treatment for several reasons. First, the supply of donor cells is limited, and therefore, surgery is accompanied by difficult logistics. Second, the extent of beneficial effects has varied in a partly unpredictable manner. Third, some patients have exhibited graft-related side effects in the form of involuntary movements. Fourth, in two major double-blind placebo-controlled trials, there was no effect of the transplants on the primary endpoints. Nevertheless, neural transplantation continues to receive a great deal of interest, and now, attention is shifting to the idea of using stem cells as starting donor material. In the context of stem cell therapy for PD, stem cells can be divided into three categories: neural stem cells, embryonic stem cells, and other tissue-specific types of stem cells, e.g., bone marrow stem cells. Each type of stem cell is associated with advantages and disadvantages. In this article, we review recent advances of stem cell research of direct relevance to clinical application in PD and highlight the pros and cons of the different sources of cells. We draw special attention to some key problems that face the translation of stem cell technology into the clinical arena.     

Roles of Wnt/β-catenin signaling in the gastric cancer stem cells proliferation and salinomycin treatment

The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells'' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and β-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.     

The role of conjunctival epithelial cell xanthine oxidoreductase/xanthine oxidase in oxidative reactions on the ocular surface of dry eye patients with Sjögren's syndrome

Previous papers examined lipid peroxidase levels and myeloperoxidase activity as products of oxidative and inflammatory reactions in the tear fluid of patients suffering from dry eye. The aim of the present paper was to investigate whether the enzymes xanthine oxidoreductase/xanthine oxidase known to generate reactive oxygen species contribute to oxidative reactions on the ocular surface. Xanthine oxidoreductase/xanthine oxidase were examined immunohistochemically as well as histochemically in conjunctival epithelial cells of patients suffering from dry eye. Patients with verified autoimmune dry eye (Sj?gren's syndrome) participated in our study; normal eyes served as controls. Conjunctival epithelial cells were obtained by the method of impression cytology using Millicell membranes. The results revealed a pronounced expression, as well as activity of xanthine oxidoreductase/xanthine oxidase in the conjunctival epithelium of dry eye. It is suggested that reactive oxygen species which are generated by this enzymatic system, contribute to oxidative reactions on the eye surface of patients with ocular manifestations of autoimmune disease (Sj?gren's syndrome).     

Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Background  

The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.     

The structure of the Golgi apparatus: a sperm’s eye view in principal epithelial cells of the rat epididymis

 The Golgi apparatus of epididymal principal cells shares many structural features with other cell types. Saccular regions are arranged in a cis-Golgi network, eight flattened saccules, and several trans-Golgi networks (TGNs). Dilated tubules form intersaccular connecting regions which joint together saccules at the same or different levels between adjacent stacks. Wells exist as large perforations in register with the four cis-most saccules and serve as areas of vesicular interactions. TGNs are variable and can appear to peel off the stack or to be detached from it in the form of an anastomotic tubular network with pale dilated areas corresponding to prosecretory granules connected by short narrow bridges. Elongated or discoid dilated cisternae of endoplasmic reticulum (ER) (sparsely granulated) lie over the cis face of the stack, from which they are separated by an intermediate compartment filled with vesicles and tubules. The ER is also closely juxtaposed to the TGNs and the eighth saccule but interconnections are never seen between them. Vesicles of the COP variety reside at all levels of the stack and appear to bud off the cis-located ER and the edges of the saccules, while clathrin-coated vesicles appear mainly on the trans face of the stack and next to lysosomes. In the supranuclear cytoplasm, clusters of vesicles and tubules, at times budding off enveloping ER, appear to radiate toward the Golgi stacks where they fuse with cis Golgi elements. Taken together, these observations suggest dynamic functions and interactions for the various Golgi elements, associated vesicles, ER, and vesicular tubular clusters. Accepted: 29 January 1998     

MiRNA-26b inhibits the proliferation,migration, and epithelial–mesenchymal transition of lens epithelial cells

In the present study we investigated the effects of lung injury on energy metabolism (succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels), respiratory mechanics (dynamic and static compliance, elastance and respiratory system resistance) in the lungs of rats, as well as on phospholipids in bronchoalveolar lavage fluid. The protective effect of physical exercise on the alterations caused by lung injury, including lung edema was also evaluated. Wistar rats were submitted to 2 months of physical exercise. After this period the lung injury was induced by intratracheal instillation of lipopolysaccharide. Adult Wistar rats were submitted to 2 months of physical exercise and after this period the lung injury was induced by intratracheal instillation of lipopolysaccharide in dose 100 μg/100 g body weight. The sham group received isotonic saline instillation. Twelve hours after the injury was performed the respiratory mechanical and after the rats were decapitated and samples were collected. The rats subjected to lung injury presented a decrease in activities of the enzymes of the electron transport chain and ATP levels in lung, as well as the formation of pulmonary edema. A decreased lung dynamic and static compliance, as well as an increase in respiratory system resistance, and a decrease in phospholipids content were observed. Physical exercise was able to totally prevent the decrease in succinate dehydrogenase and complex II activities and the formation of pulmonary edema. It also partially prevented the increase in respiratory system resistance, but did not prevent the decrease in dynamic and static compliance, as well as in phospholipids content. These findings suggest that the mitochondrial dysfunction may be one of the important contributors to lung damage and that physical exercise may be beneficial in this pathology, although it did not prevent all changes present in lung injury.     

Imaging the boundaries—innovative tools for microscopy of living cells and real-time imaging

Recently, light microscopy moved back into the spotlight, which is mainly due to the development of revolutionary technologies for imaging real-time events in living cells. It is truly fascinating to see enzymes “at work” and optically acquired images certainly help us to understand biological processes better than any abstract measurements. This review aims to point out elegant examples of recent cell-biological imaging applications that have been developed with a chemical approach. The discussed technologies include nanoscale fluorescence microscopy, imaging of model membranes, automated high-throughput microscopy control and analysis, and fluorescent probes with a special focus on visualizing enzyme activity, free radicals, and protein–protein interaction designed for use in living cells.     

Misfolding of membrane proteins in health and disease: the lady or the tiger?

Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated, such that the bilayer may play roles in normal and aberrant folding. It can be argued that the in vivo partitioning of eukaryotic membrane proteins between folding and misfolding pathways is under kinetic control. Moreover, the balance between these pathways can be surprisingly delicate.     

Proteome analysis of the transformation potential of the Epstein–Barr virus-encoded latent membrane protein 1 in nasopharyngeal epithelial cells NP69

Latent membrane protein 1 (LMP1) of Epstein-Barr virus has been identified to be crucial in inducing cell transformation. However, the mechanism of LMP1-mediated epithelial cell transformation remains unclear. In this study, nasopharyngeal epithelial cells NP69 were infected with retrovirus with gene encoding wild type LMP1 or mutational LMP1 defective in binding to tumor necrosis factor receptor-associated death domain (TRADD). The NP69-LMP1(TRADD) lost some malignant phenotypes compared with the NP69-LMP1(WT). We performed proteomic approach to gain the differential protein expression profile associated with LMP1-mediated epithelial cell transformation. Furthermore, the differential expressional levels of partial identified proteins were confirmed by Western blot and real-time RT-PCR. Some were known to be related to the development of LMP1-induced transformation, and some were new LMP1-associated proteins. These data are valuable for further study of the mechanism of LMP1 in human nasopharyngeal carcinoma and provide some new clues for investigating other LMP1-associated tumors.     

Decreased expression of antioxidant enzymes in the conjunctival epithelium of dry eye (Sjögren's syndrome) and its possible contribution to the development of ocular surface oxidative injuries

Previous studies have described elevated lipid peroxidase, myeloperoxidase and xanthine oxidoreductase/xanthine oxidase levels on the ocular surface of patients suffering from autoimmune dry eye (Sj?gren's syndrome, SS). Reactive oxygen species generated by various enzymatic systems may be dangerous to the eye if they are not sufficiently cleaved by antioxidants. Because antioxidants have not been investigated in dry eye, the aim of this study was to examine the expression of antioxidant enzymes that cleave reactive oxygen species and play a key role in antioxidant protection. Conjunctival epithelial cells of dry eye (SS) patients were obtained by the method of impression cytology using Millicell membranes. Normal eyes served as controls. In the conjunctival epithelium superoxide dismutase, catalase and glutathione peroxidase were examined immunohistochemically. The enzyme expression levels were determined by image analysis and statistical evaluation. In contrast to normal eyes, where antioxidant enzymes were highly expressed in the conjunctival epithelium, in dry eye their expression was much less pronounced in correlation with the increasing severity of dry eye symptoms. Our study suggests that the decreased expression of antioxidant enzymes in dry eye disease (SS) contributes to the development of anterior eye surface oxidative injuries.     

Peripheral blood-derived autologous stem cell therapy for the treatment of patients with late-stage peripheral artery disease—results of the short- and long-term follow-up

Background aimsRegeneration of the occluded peripheral arteries by autologous stem cell therapy is an emerging treatment modality for no-option patients with peripheral artery disease (PAD). The purpose of this study was to assess safety and efficacy of in vitro–expanded, peripheral blood-derived, autologous stem cells (VesCell) in no-option patients with PAD.MethodsA phase II, open-label, randomized clinical study was performed on 20 patients to investigate the safety and efficacy of VesCell therapy at 1 and 3 months of follow-up. The long-term (2 years) efficacy of the therapy was also evaluated.ResultsNo side effects of VesCell therapy were found. During the 3 month follow-up in the control group, one death occurred and six major amputations were performed; in the treated group, there were no deaths or major amputations. The difference of limb loss is significant between the two groups. At 2-year follow-up in the control group, two deaths and six major amputations occurred; in the treated group, there were three major amputations. At 3-month follow-up, the change in hemodynamic parameters showed a significant increase in the treated group over the control group; in the treated group, further improvement was detected at 2 years. As the result of the VesCell treatment, change in pain score, wound healing and walking ability test showed an improvement compared with the control group; at 2 years, incremental improvement was observed.ConclusionsPeripheral blood-derived, in vitro–expanded autologous angiogenic precursor therapy appears to be a safe, promising and effective adjuvant therapy for PAD patients.