Deficiency in SI protein results in osmotic diarrhea due to an inability to hydrolyze intestinal disaccharides into component monosaccharides. Therefore, we conclude that the newly established cell line is comprised of small intestine-derived epithelial cells, which in turn suggests that this cell line can be used in future research on disease inducing porcine diarrhea. Typically, differentiated cells were taken for representing the intestinal villus tip cells, while the undifferentiated cells were used to mimic the basilar crypt cells of the intestines. Thus, the prototypical characteristics of differentiated porcine intestinal epithelial cells are the presence of tight junctions and distinct microvilli on their apical surfaces. Here, we demonstrated that porcine intestinal epithelial cells expressed markers typical of differentiated enterocytes, specifically, ZYM-SIEC02 cells were positive for E-cadherin, ZO-1, Occludin, villin, and sucrose isomaltase, which is the most reliable indicator of intestinal cell differentiation in vitro, indicating the presence of differentiated villus cells in the culture. IPEC-J2 cells were cultured for 1 days or 21 days, representing undifferentiated proliferating and highly differentiated IPEC-J2 cells, respectively. However, in our experiment, all the ZYM-SIEC02 cells we used were cultured to have a confluent monolayer within 3 or 6 days according to the density of cell cultures. In this regard, the characteristics of differentiated ZYM-SIEC02 cell were evidently due to the time that isolated and purified the primary porcine intestinal epithelial cells was long enough for the differentiation of pSIECs, which prepared for the lipofection. Thus, ZYM-SIEC02 cells remain the characteristic of differentiated pSIECs, and markers typical of differentiated Cycloheximide enterocytes were detected. Moreover, we used tissue culture method to isolate epithelial cells from small intestine, it was unavoidable that the growth of fibroblasts over cultured primary intestinal epithelial cells, however, on the other side of the coin, the intestinal epithelial cell differentiation need the heterologous cell-cell contacts for denovo synthesis to trigger cell polarity and differentiation. The addition of EGF may be another important inducement for differentiation as it plays a pivotal role in the regulation of intestinal epithelial proliferation and differentiation. Over time in culture, cells maintained their epithelial morphology as well as expression of markers. These characteristics were not affected by cryogenic freezing and retrieval. An early study by Hayflick et al demonstrated that normal human somatic cells have a finite replicative potential in vitro, and will stop dividing after a finite number of population doublings and enter senescense.

Cisplatin nephrotoxicity has been recognized as a complex multifactorial process that includes oxidative stress pathways involved in apoptosis. Increased oxidative stress is one of the earliest features associated with the development of cisplatininduced nephropathy. Several investigators have demonstrated that Paclitaxel interaction of cisplatin with SH-groups leads to glutathione depletion, along with a decline of cellular antioxidant system and accumulation of reactive oxygen species or their products. Mitochondrial injury seems to play an important role in cisplatin-induced nephrotoxicity. Several functional and structural alterations of the mitochondria have been observed in cell cultures and in vivo animal models of cisplatin nephrotoxicity. This was evidenced by decreased mitochondrial mass with reduction of activities of oxidative phosphorylation complexes and manganese superoxide dismutase. This selectivity for mitochondria is probably caused by the accumulation of positively charged aquated complexes of cisplatin in the negatively charged inner space of the mitochondria. Thus, increased oxidative stress in cisplatin nephrotoxicity may be simply a consequence of disrupted respiratory chain and decreased antioxidant activity since mitochondria are a major source and target for damage by ROS. Oxidative stress and mitochondrial damage have been proposed as important factors that are involved in the activation of apoptotic pathway and cisplatin-induced cell death in vitro as well as in vivo. These events, together, result in the loss of renal function during cisplatin nephrotoxicity, triggering acute renal failure and tubular injury. A wide variety of antioxidants have been reported to exhibit protective effects against the deleterious effects of cisplatin-induced nephrotoxicity. As superoxide anions are the major injurious oxidant species generated by mitochondria, earlier studies have focused on the protective role for mitochondrial localized MnSOD in several models of free radicals-mediated cell injury. Tempol is a membrane-permeable radical scavenger which has SOD and catalase activities. Tempol has been reported experimentally to ameliorate oxidative stressmediated renal dysfunction and glomerular injury. Moreover, tempol ameliorated endothelial cell dysfunction in diabetic rats and reduced infarct size in an experimental model of regional myocardial ischemia/reperfusion. A phase I clinical trial in patients receiving whole brain radiotherapy suggested that tempol may be effective at preventing radiation-induced alopecia with only mild toxicity. Tempol, as an antioxidant, has been previously demonstrated to prevent injury induced by cisplatin using established renal epithelial cell line, LLC-PK1. However, no study has investigated the effect of tempol in an in vivo experimental model of cisplatin-induced nephrotoxicity in addition to mitochondrial role in its possible mediated protection. Therefore, the goal of the present study was directed to examine the implication of this membrane-permeable SOD-mimetic agent, tempol, in the prevention of mitochondrial dysfunction in cisplatin-induced nephrotoxicity. Moreover, the effect of tempol on cisplatin antineoplastic efficacy was investigated.

The goal of the present study was directed to examine the potential role of a membranepermeable SOD-mimetic agent, tempol, in alleviating mitochondrial dysfunction in cisplatin-induced nephrotoxicity and to evaluate the effect of tempol on cisplatin antineoplastic efficacy. Cisplatin induced weight loss might be due to gastrointestinal toxicity and reduction of food ingestion. The present data show that administration of cisplatin to mice caused marked elevation of serum creatinine and urea levels as well as significant glucosuria and proteinuria. Cisplatin causes acute renal failure due to its preferential accumulation within the proximal tubular cells in the outer medulla of the kidney. The alterations in glomerular function in cisplatin-treated mice may be secondary to the oxidative stress status observed in the present study, which induces mesangial cells contraction, alteration of filtration surface area and modification of ultrafiltration coefficient factors that decrease the glomerular filtration rate. Pretreatment with tempol normalized serum creatinine level and caused significant reduction of serum urea, glucosuria and proteinuria compared to cisplatin group, indicating improvement of glomerular and tubular functions. The present results clearly indicate a significant degree of oxidative stress in both mitochondrial and postmitochondrial fractions in renal tissues of cisplatin-treated mice. This was demonstrated by a significant elevation of TBARS content and a significant reduction of GSH content along with inhibition of SOD and catalase activities in both mitochondrial and postmitochondrial fractions. The enhanced state of oxidative stress in cisplatin-treated group was associated with significant reduction of mitochondrial oxidative phosphorylation capacity and complexes I and III activities together with significant elevation of mNOS protein expression and caspase-3 activity. This was correlated with a pronounced reduction of ATP content indicating marked deterioration of mitochondrial respiration and energy production. GSH is one of the essential components for maintaining cell integrity because of its reducing properties and participation in the cell metabolism. The depletion of the renal GSH level has been observed in response to oxidative stress caused by cisplatin treatment. Platinum was shown to preferentially bind to GSH and protein thiol in kidneys after cisplatin treatment. The concentration of platinum-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Formation of these complexes limits the amount of drug available for DNA binding and therefore, a positive correlation has been AB1010 reported between GSH levels and resistance to cisplatin. Cisplatin affects many enzymes that protect the cells from oxidative damage, among which Cu, Zn-SOD, Mn-SOD and catalase. SOD plays an important role in the dismutation of superoxide anions. Decreased SOD activity as observed in this study could lead to incomplete scavenging of superoxide anions that are produced during the normal metabolic process with further initiation and propagation.

Similar to the effect of co-culture with neonatal mouse retinal cells, Taurine and RA promoted upregulation of retinal progenitor markers in human LNS. This suggests that defined culture conditions may replace the use of animal tissue in the future. We did not observe LNS cell migration or integration into the host retina following sub-retinal transplantation into neonatal mice. Cell integration into the retina remains challenging. Despite being derived from the same origin as neural retina, iris or CB derived cells have also shown limited ability for retinal integration. The proportions of cells which integrate into embryonic retinal explants or retina from degenerate animal models are small. Studies using retinal progenitor cells from embryonic retina have also shown little integration into host retina, although mature retinal phenotypes have been observed following sub-retinal transplantation. MacLaren et al. investigated the optimal cell resource for functional integration into adult retina. The cells which migrated and integrated were shown to be post-mitotic rod precursor cells. Therefore, the ontogenetic stage of transplanted cells is important for successful cell integration. Grafted LNS cells in this study were not fully committed post-mitotic cells. This may explain why cell integration was not observed. The host Cycloheximide microenvironment is also essential for inducing cell differentiation and migration. In a study involving transplantation of IPE derived cells, the grafted cells expressed the photoreceptor specific marker rhodopsin when they were transplanted into the SRS of embryonic chicken eyes. On the contrary, they did not express rhodopsin or other neural markers when they were transplanted into the vitreous cavity. This is in accordance with our observation that the LNS cells transplanted into the vitreous do not express photoreceptor markers. LNS display plasticity, the potential to cross the tissue/germ layer boundary and generate cells other than their origin. However, LNS have limited potential to generate photoreceptorlike cells. The highest rhodopsin expression level noted using LNS derived cells was,3% of that observed compared to using neonatal mouse retinal tissue. Reports on other ocular stem-like/ progenitor cells also show limited success in the generation of photoreceptor regardless of cell origin. Recently two independent groups showed CE-derived cells failed to give rise to photoreceptor cells. Retinal neurosphere cells derived from neonatal mice also had a low efficiency in generation of rhodopsin positive cells during spontaneous differentiation. It has been suggested that cell reprogramming is likely to be needed for robust photoreceptor cell production. LNS cells would also be an optimal cell resource for reprogramming and/or transdifferentiation and subsequent retinal repair. They are readily accessible, highly proliferative and multipotent ocular stem cells. iPSCs have been generated from mouse and human somatic cells by ectopic expression of four transcription factors including OCT4, SOX2, c-Myc and KLF4. Due to risks such as insertional mutagenesis or tumour formation, it is desirable to use the minimal number of transcription factors and to eliminate oncogenic factors.

In injured epithelium, fibronectin deposits at the SB to aid in synthesis of a new connective tissue matrix, and to provide substrate for keratinocyte migration to repair wounds. Proximal to the injury, activated keratinocytes, expressing keratin 6, migrate to close the wound and provide resistance to mechanical stress. SB GW786034 keratinocytes also replicate rapidly to refill the wound ; the accelerated differentiation can cause parakeratosis, with nuclei intercalated in the SC. These classical markers of epithelial integrity have not been assessed in the foreskin of sexually active males, but could help determine if foreskin microtrauma is a factor in enhancing STI transmission. Multiple studies have aimed to identify differences among foreskin anatomical sites, to test the hypothesis that the inner foreskin provides a permeable site for pathogen entry. HIV risk has been associated with sub-prepucial wetness, where the inner foreskin is in contact with the penile shaft. However, the epithelial structures associated with foreskin permeability have not been fully explored. Most studies aiming to document the barrier functions of inner foreskin have focused on SC thickness. The SC is composed of consecutive filaments of cross-linked keratin 1, keratin 10, filaggrin, involucrin, cornified envelope proteins, and lipids, conferring strength, elasticity and protection. SC thickness studies have provided contradicting results: one study reported thickening of inner foreskin relative to outer in men with a history of penile infections, two reported no differences, and three documented thinning. In addition to keratinocyte homeostasis and SC integrity, recent evidence indicates that skin permeability is further regulated by the structure of tight junctions in the SG. Involucrin, a terminal marker of keratinocyte maturation, is synthesized in the SS and cross-linked in the SG to provide structural support for TJs. TJ proteins, such as claudins and occludin, form homotypic interactions among adjacent cells and regulate paracellular transport of water, ions, and large molecules. Studies indicate that occludin and claudin 1 form a barrier for extracellular biotin in the healthy human SG, and leakage correlates with their disappearance from the cell membrane. TJ proteins also respond to acute and chronic inflammatory signals that regulate trans-epithelial resistance. Occludin is believed to play its mayor role in the leak transport pathway, which responds to inflammatory stimuli such as IFN-c and TNFa by endocytosis of TJ components and regulates the passage of larger molecules, such as HIV and bacterial products in reconstructed monolayers. The overexpression of occludin has been shown to increase sensitivity of the leak pathway in response to inflammatory cytokines, but its potential role in the entry of pathogens at the pluri-stratified foreskin is unknown. To further examine reasons behind increased STI risk in uncircumcised men, we explored differences among skin factors in the inner and outer foreskin of sexually active men who have sex with men at risk of HIV. We describe differences in the cornified envelope, in TJ proteins in the SG and SS, in secretion of inflammatory cytokines, and in the density of CCR5.