Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair

Rationale: Chronic lung disease characterized by loss of lung tissue,inflammation, and fibrosis represents a major global health burden. Cellular therapies that could restore pneumocytes and reduce inflammation and fibrosis would be a major advance in management. Objectives: To determine whether human amnion epithelial cells (hAECs), isolated from term placenta and having stem cell–like and antiinflammatory properties, could adopt an alveolar epithelial phenotype and repair a murine model of bleomycin-induced lung injury. Methods: Primary hAECs were cultured in small airway growth medium to determine whether the cells could adopt an alveolar epithelial phenotype. Undifferentiated primary hAECs were also injected parenterally into SCID mice after bleomycin-induced lung injury and analyzed for production of surfactant protein (SP)-A, SP-B, SP-C, and SP-D. Mouse lungs were also analyzed for inflammation and collagen deposition. Measurements and Main Results: hAECs grown in small airway growth medium developed an alveolar epithelial phenotype with lamellar body formation, production of SPs A–D, and SP-D secretion. Although hAECs injected into mice lacked SPs, hAECs recovered from mouse lungs 2 weeks posttransplantation produced SPs. hAECs remained engrafted over the 4-week test period. hAEC administration reduced inflammation in association with decreased monocyte chemoattractant protein-1, tumor necrosis factor-a, IL-1 and -6, and profibrotic transforming growth factor-b in mouse lungs. In addition,lung collagen content was significantly reduced by hAEC treatment as a possible consequence of increased degradation by matrix metalloproteinase-2 and down-regulation of the tissue inhibitors f matrix metalloproteinase-1 and 2. Conclusions: hAECs offer promise as a cellular therapy for alveolar restitution and to reduce lung inflammation and fibrosis.

Expression and regulation of vascular endothelial growth factor ligands and receptors during menstruation and post-menstrual repair of human endometrium

Regeneration and growth of the human endometrium after shedding of the functional layer during menstruation depends on an adequate angiogenic response. We analysed the mRNA expression levels of all known vascular endothelial growth factor (VEGF) ligands and receptors in human endometrium collected in the menstrual and proliferative phases of the menstrual cycle. In addition, we evaluated the expression of VEGF-A, VEGF-R2 and NRP-1 at the protein level. Two periods of elevated mRNA expression of ligands and receptors were observed, separated by a distinct drop at cycle days (CDs) 9 and 10. Immunohistochemical staining showed that VEGF and VEGF-R2 were expressed in epithelial, stromal and endothelial cells. NRP-1 was mainly confined to stroma and blood vessels; only in late-proliferative endometrium, epithelial staining was also observed. Except for endothelial VEGF-R2 expression in CDs 6-8, there were no significant differences in the expression of VEGF, VEGF-R2 or NRP-1 in any of the cell compartments. In contrast, VEGF release by cultured human endometrium explants decreased during the proliferative phase. This output was significantly reduced in menstrual and early-proliferative endometrium by estradiol (E2) treatment. Western blot analysis indicated that part of the VEGF-A was trapped in the extracellular matrix (ECM). Changes in VEGF ligands and receptors were associated with elevated expression of the hypoxia markers HIF1 alpha and CA-IX in the menstrual and early proliferative phases. HIF1 alpha was also detected in late-proliferative phase endometrium. Our findings indicate that VEGF-A exerts its actions mostly during the first half of the proliferative phase. Furthermore, VEGF-A production appears to be triggered by hypoxia in the menstrual phase and subsequently suppressed toy estrogen during the late proliferative phase.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Fully automated, semiautomated, and manual morphometric analysis of corneal subbasal nerve plexus in individuals with and without diabetes

Purpose The aim of the study was to determine the association, agreement, and detection capability of manual, semiautomated, and fully automated methods of corneal nerve fiber length (CNFL) quantification of the human corneal subbasal nerve plexus (SNP). Methods Thirty-three participants with diabetes and 17 healthy controls underwent laser scanning corneal confocal microscopy. Eight central images of the SNP were selected for each participant and analyzed using manual (CCMetrics), semiautomated (NeuronJ), and fully automated (ACCMetrics) software to quantify the CNFL. Results For the entire cohort, mean CNFL values quantified by CCMetrics, NeuronJ, and ACCMetrics were 17.4 ± 4.3 mm/mm2, 16.0 ± 3.9 mm/mm2, and 16.5 ± 3.6 mm/mm2, respectively (P < 0.01). CNFL quantified using CCMetrics was significantly higher than those obtained by NeuronJ and ACCMetrics (P < 0.05). The 3 methods were highly correlated (correlation coefficients 0.87–0.98, P < 0.01). The intraclass correlation coefficients were 0.87 for ACCMetrics versus NeuronJ and 0.86 for ACCMetrics versus CCMetrics. Bland–Altman plots showed good agreement between the manual, semiautomated, and fully automated analyses of CNFL. A small underestimation of CNFL was observed using ACCMetrics with increasing the amount of nerve tissue. All 3 methods were able to detect CNFL depletion in diabetic participants (P < 0.05) and in those with peripheral neuropathy as defined by the Toronto criteria, compared with healthy controls (P < 0.05). Conclusions Automated quantification of CNFL provides comparable neuropathy detection ability to manual and semiautomated methods. Because of its speed, objectivity, and consistency, fully automated analysis of CNFL might be advantageous in studies of diabetic neuropathy.

Natural history of corneal nerve morphology in mild neuropathy associated with type 1 diabetes : Development of a potential measure of diabetic peripheral neuropathy

Purpose To investigate longitudinal changes of subbasal nerve plexus (SNP) morphology and its relationship with conventional measures of neuropathy in individuals with diabetes. Methods A cohort of 147 individuals with type 1 diabetes and 60 age-balanced controls underwent detailed assessment of clinical and metabolic factors, neurologic deficits, quantitative sensory testing, nerve conduction studies and corneal confocal microscopy at baseline and four subsequent annual visits. The SNP parameters included corneal nerve fiber density (CNFD), branch density (CNBD) and fiber length (CNFL) and were quantified using a fully-automated algorithm. Linear mixed models were fitted to examine the changes in corneal nerve parameters over time. Results At baseline, 27% of the participants had mild diabetic neuropathy. All SNP parameters were significantly lower in the neuropathy group compared to controls (P<0.05). Overall, 89% of participants examined at baseline also completed the final visit. There was no clinically significant change to health and metabolic parameters and neuropathy measures from baseline to the final visit. Linear mixed model revealed a significant linear decline of CNFD (annual change rate, -0.9 nerve/mm2, P=0.01) in the neuropathy group compared to controls, which was associated with age (β=-0.06, P=0.04) and duration of diabetes (β=-0.08, P=0.03). In the neuropathy group, absolute changes of CNBD and CNFL showed moderate correlations with peroneal conduction velocity and cold sensation threshold, respectively (rs, 0.38 and 0.40, P<0.05). Conclusion This study demonstrates dynamic small fiber damage at the SNP, thus providing justification for our ongoing efforts to establish corneal nerve morphology as an appropriate adjunct to conventional measures of DPN.

Differing phagocytic capacities of accessory and main olfactory ensheathing cells and the implication for olfactory glia transplantation therapies

The rodent olfactory systems comprise the main olfactory system for the detection of odours and the accessory olfactory system which detects pheromones. In both systems, olfactory axon fascicles are ensheathed by olfactory glia, termed olfactory ensheathing cells (OECs), which are crucial for the growth and maintenance of the olfactory nerve. The growth-promoting and phagocytic characteristics of OECs make them potential candidates for neural repair therapies such as transplantation to repair the injured spinal cord. However, transplanting mixed populations of glia with unknown properties may lead to variations in outcomes for neural repair. As the phagocytic capacity of the accessory OECs has not yet been determined, we compared the phagocytic capacity of accessory and main OECs in vivo and in vitro. In normal healthy animals, the accessory OECs accumulated considerably less axon debris than main OECs in vivo. Analysis of freshly dissected OECs showed that accessory OECs contained 20% less fluorescent axon debris than main OECs. However, when assayed in vitro with exogenous axon debris added to the culture, the accessory OECs phagocytosed almost 20% more debris than main OECs. After surgical removal of one olfactory bulb which induced the degradation of main and accessory olfactory sensory axons, the accessory OECs responded by phagocytosing the axon debris. We conclude that while accessory OECs have the capacity to phagocytose axon debris, there are distinct differences in their phagocytic capacity compared to main OECs. These distinct differences may be of importance when preparing OECs for neural transplant repair therapies.

Disorganising principles: Corporeal fragmentation and the possibilities for repair

Dancers investigate ever-expanding relationships to embodiment through the variety of unique choreographic signatures that are continually erupting in professional practice. They live fragmented lineages that are interrupted and redirected as they traverse between various projects led by different choreographers or the same choreographer pursuing different creative goals. As contemporary dance continues to reconceive ways of moving, the dominant lineages of dance training are less useful as reference points through which dancers can recalibrate bodily activity and thus rebalance. In this chapter, I examine the impulse towards fragmentation in contemporary dance and explore how moments of agency for dancers might arise and be seized within the complexities of this environment. These issues are discussed in relation to my encounter with a bodywork therapy of Japanese origin, Amatsu, which I studied throughout 2012, and through the teaching principles of Gill Clarke as illuminated through the Minding Motion project, which explored Clarke’s pedagogy for Tanzplan, Germany 2010 (Diehl and Lampert, 2011). Moments from performance and bodywork practice are offered as examples throughout the chapter.

Utility of assessing nerve morphology in central cornea versus whorl area for diagnosing diabetic peripheral neuropathy

Purpose To compare small nerve fiber damage in the central cornea and whorl area in participants with diabetic peripheral neuropathy (DPN) and to examine the accuracy of evaluating these 2 anatomical sites for the diagnosis of DPN. Methods A cohort of 187 participants (107 with type 1 diabetes and 80 controls) was enrolled. The neuropathy disability score (NDS) was used for the identification of DPN. The corneal nerve fiber length at the central cornea (CNFLcenter) and whorl (CNFLwhorl) was quantified using corneal confocal microscopy and a fully automated morphometric technique and compared according to the DPN status. Receiver operating characteristic analyses were used to compare the accuracy of the 2 corneal locations for the diagnosis of DPN. Results CNFLcenter and CNFLwhorl were able to differentiate all 3 groups (diabetic participants with and without DPN and controls) (P < 0.001). There was a weak but significant linear relationship for CNFLcenter and CNFLwhorl versus NDS (P < 0.001); however, the corneal location x NDS interaction was not statistically significant (P = 0.17). The area under the receiver operating characteristic curve was similar for CNFLcenter and CNFLwhorl (0.76 and 0.77, respectively, P = 0.98). The sensitivity and specificity of the cutoff points were 0.9 and 0.5 for CNFLcenter and 0.8 and 0.6 for CNFLwhorl. Conclusions Small nerve fiber pathology is comparable at the central and whorl anatomical sites of the cornea. Quantification of CNFL from the corneal center is as accurate as CNFL quantification of the whorl area for the diagnosis of DPN.

The role of inflammation in cutaneous repair

Inflammation is a fundamental component of the normal adult wound healing response occurring even in the absence of infection. It performs many beneficial roles such as the clearing of damaged cells and extracellular matrix (ECM), the removal of pathogens that might other wise multiply and spread, and the secretion of mediators that regulate other aspects of wound healing such as proliferation, re-epithelialisation and wound remodelling. Yet, excess and/or prolonged inflammation is detrimental to wound healing and leads to increased fibrosis and scarring, which can be disfiguring and, in cases such as contractures, can lead to disability. Furthermore, excessive inflammation is a major contributing factor to the persistence of chronic non-healing wounds, which are “stuck” in the inflammatory phase of healing and fail to reepithelialise. Current research suggest that the type of immune cells, their timing and the level of inflammation in a wound could have dramatic effect on whether a wound heals in a timely fashion and the final quality of the repaired tissue. Studies suggest that altering the level of inflammation might be beneficial in terms of reducing scarring and improving the rate of healing in chronic wounds. This review looks at the role of the major immune cells in normal and impaired wound healing and strategies that might be used to reduce inflammation in wounds.

Mechanisms of cisplatin resistance: DNA repair and cellular implications

Cisplatin (cis-diamminedichloroplatinum (II)), is a platinum based chemotherapeutic employed in the clinic to treat patients with lung, ovarian, colorectal or head and neck cancers. Cisplatin acts to induce tumor cell death via multiple mechanisms. The best characterized mode of action is through irreversible DNA cross-links which activate DNA damage signals leading to cell death via the intrinsic mitochondrial apoptosis pathway. However, the primary issue with cisplatin is that while patients initially respond favorably, sustained cisplatin therapy often yields chemoresistance resulting in therapeutic failure. In this chapter, we review the DNA damage and repair pathways that contribute to cisplatin resistance. We also examine the cellular implications of cisplatin resistance that may lead to selection of subpopulations of cells within a tumor. In better understanding the mechanisms conferring cisplatin resistance, novel targets may be identified to restore drug sensitivity.

The role of DNA repair pathways in cisplatin resistant lung cancer

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.

Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

DNA repair pathways and their therapeutic potential in lung cancer

Lung cancer is the leading cause of cancer-related mortality. According to WHO, 1.37 million deaths occur globally each year as a result of this disease. More than 70% of these cases are associated with prior tobacco consumption and/or cigarette smoking, suggesting a direct causal relationship. The development and progression of lung cancer and other malignancies involves the loss of genetic stability, resulting in acquisition of cumulative genetic changes; this affords the cell increased malignant potential. As such, an understanding of the mechanisms through which these events may occur will potentially allow for development of new anticancer therapies. This review will address the association between lung cancer and genetic instability, with a central focus on genetic mutations in the DNA damage repair pathways. In addition, we will discuss the potential clinical exploitation of these pathways, both in terms of biomarker staging, as well as through direct therapeutic targeting.

Longitudinal assessment of corneal subbasal nerve morphology as a potential measure of diabetic peripheral neuropathy

This thesis represents a significant step forward in developing a validated measure for diabetic peripheral neuropathy – a debilitating and prevalent complication of diabetes. The candidate investigated corneal nerve structure in healthy people as well as in type 1 diabetic individuals in a 4-year longitudinal study. The outcomes of stability of the corneal small nerve fibre in healthy people and evidence of significant decline in diabetic individuals with peripheral neuropathy over time provide justification for the ongoing efforts to establish corneal nerve structure as an objective and appropriate adjunct to conventional measures of peripheral neuropathy.