Dual Delivery of Gemcitabine and Paclitaxel by Wet-Spun Coaxial Fibers Induces Pancreatic Ductal Adenocarcinoma Cell Death, Reduces Tumor Volume, and Sensitizes Cells to Radiation.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, with surgical resection of the tumor in conjunction with systemic chemotherapy the only potential curative therapy. Up to 80% of diagnosed cases are deemed unresectable, prompting the need for alternative treatment approaches. Herein, coaxial polymeric fibers loaded with two chemotherapeutic agents, gemcitabine (Gem) and paclitaxel (Ptx), are fabricated to investigate the effect of local drug delivery on PDAC cell growth in vitro and in vivo. A wet-spinning fabrication method to form a coaxial fiber with a polycaprolactone shell and alginate core loaded with Ptx and Gem, respectively, is used. In vitro, Gem+Ptx fibers display significant cytotoxicity as well as radiosensitizing properties toward PDAC cell lines greater than the equivalent free drugs, which may be attributed to a radiosensitizing effect of the polymers. In vivo studies assessing Gem+Ptx fiber efficacy found that Gem+Ptx fibers reduce tumor volume in a xenograft mouse model of PDAC. Importantly, no difference in mouse weight, circulating cytokines, or liver function is observed in mice treated with Gem+Ptx fibers compared to the empty fiber controls confirming the safety of the implant approach. With further development, Gem+Ptx fibers can improve the treatment of unresectable PDAC in the future.

Characterization of colony-forming cells in adult human articular cartilage.

Recent studies have shown that adult human articular cartilage contains stem-like cells within the native structure. In this study, we aimed to determine the localization of putative stem cell markers such as CD90, STRO-1, OCT-3/4, CD105 and CD166 in adult human articular cartilage tissue sections and demonstrate the expression of these markers within the expanded surface zone colony-forming (CF) cells and evaluate their differentiation potential. Biopsy samples were either fixed immediately for immunohistochemical analyses or processed for in vitro cell culture. Immunohistochemical and flow cytometry analyses were performed by using CD90, STRO-1, OCT-3/4, CD105 and CD166 antibodies. Isolated colony-forming (CF) cells were further stimulated, by using the appropriate growth factors in their pellet culture, to obtain cartilage, bone and adipose lineages. We observed that the expression of the stem cell markers were in various zones of the human adult cartilage. Flow cytometry results showed that in CF cells the expression of CD90 and CD166 was high, while OCT-3/4 was low. We also determined that CF cells could be stimulated towards cartilage, bone and adipose lineages. The results of this research support the idea that the resident stem-like cells in adult human articular cartilage express these putative stem cell markers, but further experimental investigations are needed to determine the precise localization of these cells.

Comparison of the effects of desflurane and bupivacaine on Th1 and Th2 responses.

BACKGROUND: Anesthesia and surgical trauma are known to affect various functions of the immune system. Alterations reported in the immune system, such as imbalance of Th1 (IFN-gamma) and Th2 (IL-4, 5, 10) cytokines, may result from a number of factors, including pre-medication, type of anesthetic drug, and modality of anesthesia. In this study, we investigated the effects of spinal and general anesthesia with desflurane and bupivakain, respecttively, on Th1 (IFN-gamma) and Th2 (IL-10) cytokines, absolute lymphocyte and natural regulatory T cell numbers (Treg). METHODS: Peripheral Blood Mononuclear cells from 24 patients with Benign Prostate Hyperplasia (BPH), undergoing transuretheral prostatectomy under spinal (n = 12) and general (n = 12) anesthesia were analyzed before and 24 hours after surgery. Intracellular cytokine production in response to mitogen stimulation and absolute numbers of Tregs and lymphocytes were determined by using flow cytometry. RESULTS: In patients who received spinal anesthesia, while the frequency of IFN-gamma (1.68% +/- 0.74 vs. 1.03% +/- 0.74) and IL-10 producing CD4+ T cells decreased (2.62% +/- 2.24 vs. 1.04% +/- 1.06; p < 0.05), the ratio of Th1/Th2 remained similar (1.14 +/- 0.7 vs. 1.52 +/- 1.02; p > 0.05) after surgery. In contrast, in the general anesthesia group the frequency of CD4 IFN-gamma+ T cells increased (1.3% +/- 0.7 vs. 2.5% +/- 1.2; p < 0.05) and the frequency of CD4+ IL-10+ T cells decreased (1.1% +/- 0.68 vs. 0.67% +/- 0.47), resulting in an increased Th1/Th2 ratio (1.61 +/- 1.1 vs. 4.77 +/- 3.95; p < 0.05). Absolute lymphocyte and Treg numbers did not change significantly in both groups following surgery. CONCLUSIONS: Our results support the notion that general anesthesia, rather than spinal anesthesia, alters the balance of Th1 and Th2 in favor of Th1 responses. However, whether this has any effect on the susceptibility to postsurgery-related infections remains to be determined.

Distribution of CD105 and CD166 positive cells in the proximal epiphysis of developing rat humerus.

The expression of cell surface receptors, CD105 and CD166, are characteristic of mesenchymal stem cells in cartilage. However, there is limited data regarding their immunolocalization in the cartilage of developing rat epiphysis. The purpose of this study was to determine the presence of CD105 and CD 166 positive cells in the proximal epiphysis of developing rat humerus and specify their zonal distribution with age. The tissues of rat humerus were taken on embryonic day 15 (E15), embryonic day 19 (E19), postnatal day 10 (PN10), postnatal day 20 (PN20) and adult rats and studied for the immunolocalization of CD105 and CD166. Our results showed that CD105 and CD166 positive cells were scattered in early stages of development of humerus epiphysis. For E15, only the hypertrophic zone was positive, whereas for E19 almost all zones of the epiphysis were positively stained for these markers. For PN10 and PN20, the CD105 and CD166 positive cells were mainly localized on the surface of the articular cartilage. In adult articular cartilage the CD105 and CD166 positive cells were localized in the superficial and transitional zones and in the upper regions of the deep zone. Our study provides evidence that in the developing cartilage tissue the localization of CD105 and CD166 positive cells is both dynamic and stage dependent, which may imply the existence of stem cell-like cells in cartilage from an early age to adult.

Regulated production of SnoN2 is a feature of testicular differentiation.

Transforming growth factor betas (TGF beta s) and activins are key regulators of male fertility, affecting somatic and germ cell proliferation and differentiation in the developing and adult testis. Several studies have shown that these ligands influence discrete developmental stages, suggesting that temporal expression of modifying factors may determine their specific signaling outcomes. Upon binding to cell surface receptors, TGFbeta and activin signals are transduced intracellularly by the phosphorylation and nuclear accumulation of SMAD2 and SMAD3 transcription factors. The objective of this study was to determine the cellular localization of phosphorylated SMAD2/3 and the transcriptional repressor SnoN (Ski-like), a modifier of SMAD2/3 transcriptional activity, in mouse testes. Western blot established that only the smaller SnoN isoform, SnoN2, is produced in the testis. By immunohistochemistry, widespread phospho-SMAD2/3 distribution was observed in somatic and germ cells at all ages. In contrast, SnoN2 production was highly regulated, being detected only in gonocytes and interstitial cells at birth and in pachytene spermatocytes at puberty. In the adult, SnoN2 expression differed to that during the first wave, being ubiquitously expressed but exhibiting regulated nuclear localization. In another model of spermatogenic differentiation, the irradiated rat testis, widespread phospho-SMAD2/3 contrasted with restricted SnoN2 expression. SnoN2 was limited to interstitial cells, with reduced staining intensity observed associated with the timing of spermatogenesis resumption. We conclude that somatic and germ cells at all differentiation stages are actively transducing TGFbeta superfamily signals but that responses to these ligands may be selectively modulated by controlled production and nuclear localization of SnoN2.

Effects of formaldehyde inhalation on the junctional proteins of nasal respiratory mucosa of rats.

Exposure to formaldehyde, which is an organic compound, disturbs the integrity of nasal mucosa. In this study, we aimed to clarify the protein changes in the junctional complex of nasal mucosa of Wistar rats exposed to formaldehyde inhalation. The study was performed in 20 female Wistar rats. Rats were divided into two groups randomly. Control rats were allowed free access to standard rat chaw and tap water (n:10). Experimental group was exposed to formaldehyde vapor at 15ppm, 6h/day, 5 days/week for 12 weeks (n:10). Histological evaluation of the experimental model was determined by hematoxylin-eosin (HE) and periodic acid Schiff (PAS) stainings of paraffin-embedded nasal mucosa tissues and by electron microscopy. The effects of formaldehyde inhalation on the distribution of occludin, E-cadherin, and gamma-catenin were assessed by immunohistochemistry. The nasal mucosa of the experimental group was correlated with hypertrophy in goblet cell, degeneration in basal lamina, stratification of epithelium, and proliferation. Thickness of basal lamina and also local degenerative regions, vacuole increase in cytoplasmic areas, irregular forms of kinocilium and loss of sharpness in the kinocilium membrane were the findings at the ultrastructural level. The expressions of E-cadherin, occludin, gamma-catenin proteins in intercellular junctional complexes of rat nasal mucosa were also decreased in experimental group compared to control group. The findings of the present study indicated that formaldehyde vapor inhalation in the concentrations and duration of exposure used in the present experiment significantly decreased the density of structural proteins of the junctional complex in the nasoepithelium. It was suggested that, the formaldehyde inhalation could cause complete impairment of intercellular junctional complexes and disturb the tissue integrity in nasal mucosa at higher concentrations.

The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells.

The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.

The Fas system may have a role in male reproduction.

OBJECTIVE: To assess what the distributions of Fas system proteins are in normal rat testicular tissue; to assess whether there is a change in these distributions and in expression levels with experimentally-induced varicocele of 9, 11, and 13 weeks; and to assess whether there is a relationship between apoptosis and the Fas system in varicocele-induced rat testis. DESIGN: Comparative and controlled study. SETTING: University animal care and operation unit. ANIMAL(S): Wistar male rats for experimental and control groups. INTERVENTION(S): The control group underwent sham operation (n = 6). Rats in experimental groups underwent partial ligation of the renal vein to induce an experimental varicocele and then were killed at 9 (n = 6), 11 (n = 6), and 13 (n = 6) weeks after induction of varicocele. MAIN OUTCOME MEASURE(S): Tissues were fixed and processed for paraffin and Araldite embedding, and subsequently immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, and transmission electron microscopy were performed. In addition, Western blotting was applied. RESULT(S): In control testis, we detected the expression of FasL in spermatids, interestingly at the progressing stages of acrosome formation and in the heads of the spermatozoa being released to lumen. Varicocele induction revealed a significant down-regulation of this protein, especially 11 weeks after the operation, without altering its distribution. Fas protein was present in cytoplasmic extrusions of the elongated spermatids and evidently in Leydig cells of the interstitial tissue. The expression of Fas protein was diminished after 11 weeks of varicocele induction, both in Leydig cells and in cytoplasmic extrusions. The decrease of Fas was significant in the 13-week-old varicocele group, whereas that of FasL was significant in the 11-week-old varicocele group. Compared with sham-operated animals, a minor increase in the number of apoptotic germ cells in varicocele groups was detected. CONCLUSION(S): Our results exposed other possible important roles of the Fas system in addition to than apoptosis in male reproduction. We suggest that the role of the Fas system needs further investigation both in animal models and in human male infertility.

Effect of experimental varicocele on the expressions of Notch 1, 2, and 3 in rat testes: an immunohistochemical study.

OBJECTIVE: To study expressions of Notch receptor isoforms (Notch 1, 2, and 3) in normal and varicocele-induced rat testes to examine their possible functions in cell fate. DESIGN: Comparative and controlled study. SETTING: Animal Care and Operation Unit, Akdeniz University. ANIMAL(S): Wistar male rats for experimental and control groups. INTERVENTION(S): The control group underwent a sham operation (n = 6). The experimental groups underwent partial ligation of the renal vein to induce an experimental varicocele and then were killed 9 (n = 6), 11 (n = 6), and 13 (n = 6) weeks after the induction of varicocele. MAIN OUTCOME MEASURE(S): All tissues were fixed and routinely processed for paraffin embedding. Subsequent immunohistochemical studies were performed. RESULT(S): In the sham-operation rat testes, Leydig cells and elongated spermatids were immunopositive for Notch 1. Notch-2 expression was present in Leydig cells, spermatogonia, and primary spermatocytes. Notch-3 expression was limited to Leydig cells. Varicocele formation diminished the expression of both Notch-1 and Notch-2 receptors as the varicocele formation progressed over time. CONCLUSION(S): The present study suggests that Notch 1 is related to the maturation of spermatids. Notch 2 is related to both proliferation and maturation of spermatogenic cells, whereas Notch 3 seems to be related to Leydig cell functions. The decrease of both Notch-1 and Notch-2 expression depended on the degree of varicocele development over time, indicating a potential role in varicocele-associated testicular dysfunction.