Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer

Resistance to chemotherapy and metastases are the major causes of breast cancer-related mortality. Moreover, cancer stem cells (CSC) play critical roles in cancer progression and treatment resistance. Previously, it was found that CSC-like cells can be generated by aberrant activation of epithelial–mesenchymal transition (EMT), thereby making anti-EMT strategies a novel therapeutic option for treatment of aggressive breast cancers. Here, we report that the transcription factor FOXC2 induced in response to multiple EMT signaling pathways as well as elevated in stem cell-enriched factions is a critical determinant of mesenchymal and stem cell properties, in cells induced to undergo EMT- and CSC-enriched breast cancer cell lines. More specifically, attenuation of FOXC2 expression using lentiviral short hairpin RNA led to inhibition of the mesenchymal phenotype and associated invasive and stem cell properties, which included reduced mammosphere-forming ability and tumor initiation. Whereas, overexpression of FOXC2 was sufficient to induce CSC properties and spontaneous metastasis in transformed human mammary epithelial cells. Furthermore, a FOXC2-induced gene expression signature was enriched in the claudin-low/basal B breast tumor subtype that contains EMT and CSC features. Having identified PDGFR-β to be regulated by FOXC2, we show that the U.S. Food and Drug Administration-approved PDGFR inhibitor, sunitinib, targets FOXC2-expressing tumor cells leading to reduced CSC and metastatic properties. Thus, FOXC2 or its associated gene expression program may provide an effective target for anti-EMT-based therapies for the treatment of claudin-low/basal B breast tumors or other EMT-/CSC-enriched tumors.

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75(NTR) in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75NTR was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75NTR, CD24 antigens and ALDH activity (ALDEFLUOR® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

Markers of cutaneous human papillomavirus infection in individuals with tumor-free skin, actinic keratoses, and squamous cell carcinoma

Separately, actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) have been associated with cutaneous human papillomavirus (HPV) infections. To further explore the association between HPV infection and SCC development, we determined markers of cutaneous HPV infection within a single population in persons with precursor lesions (AK), cancerous lesions (SCC), and without. Serum and plucked eyebrow hairs were collected from 57 tumor-free controls, 126 AK, and 64 SCC cases. Presence of HPV L1 and E6 seroreactivity and viral DNA were determined for HPV types 5, 8, 15, 16, 20, 24, and 38. Significant positive associations with increasing severity of the lesions (controls, AK, and SCC, respectively) were observed for overall HPV L1 seropositivity (13%, 26%, and 37%) and for HPV8 (4%, 17%, and 30%). In parallel, the proportion of L1 seropositive individuals against multiple HPV types increased from 14% to 39% and 45%. The overall E6 seroreactivity, however, tended to decline with AK and SCC, especially for HPV8 (21%, 11%, and 2%). HPV DNA positivity was most prevalent in the AK cases (54%) compared with the SCC cases (44%) and the tumor-free controls (40%). Among all participants, there was a positive trend between overall HPV DNA positivity and L1 seropositivity, but not E6 seropositivity. Taken together, our data suggest that cutaneous HPV infections accompanied by detectable HPV DNA in eyebrow hairs and HPV L1 seropositivity, but not E6 seropositivity, are associated with an increased risk of AK and SCC.

Re: Human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin

Human papillomaviruses (HPVs) cause cervical cancer and some other types of epithelial cancers. HPV types from the phylogenic beta genus (beta-PVs), formerly known as epidermodysplasia verruciformis–associated HPV types, are frequently detected in nonmelanoma skin cancers, especially in squamous cell carcinomas (SCCs). An etiologic relationship with beta-PV infection is suspected...

Quantifying the roles of cell motility and cell proliferation in a circular barrier assay

Moving fronts of cells are essential features of embryonic development, wound repair and cancer metastasis. This paper describes a set of experiments to investigate the roles of random motility and proliferation in driving the spread of an initially confined cell population. The experiments include an analysis of cell spreading when proliferation was inhibited. Our data have been analysed using two mathematical models: a lattice-based discrete model and a related continuum partial differential equation model. We obtain independent estimates of the random motility parameter, D, and the intrinsic proliferation rate, λ, and we confirm that these estimates lead to accurate modelling predictions of the position of the leading edge of the moving front as well as the evolution of the cell density profiles. Previous work suggests that systems with a high λ/D ratio will be characterized by steep fronts, whereas systems with a low λ/D ratio will lead to shallow diffuse fronts and this is confirmed in the present study. Our results provide evidence that continuum models, based on the Fisher–Kolmogorov equation, are a reliable platform upon which we can interpret and predict such experimental observations.

Re-use of domain knowledge to provide confidence for adoption of off-site manufacturing for construction in Australia

Many construction industry decision-makers believe there is a lack of off-site manufacture (OSM) adoption for non-residential construction in Australia. Identification of construction business process was considered imperative in order to assist decision-makers to increase OSM utilisation. The premise that domain knowledge can be re-used to provide an intervention point in the construction process led a team of researchers to construct simple base-line process models for the complete construction process, segmented into six phases. Sixteen domain knowledge industry experts were asked to review the construction phase base-line models to answer the question “Where in the process illustrated by this base-line model phase is an OSM task?”. Through an iterative and generative process a number of off-site manufacture intervention points were identified and integrated into the process models. The re-use of industry expert domain knowledge provided suggestions for new ways to do basic tasks thus facilitating changes to current practice. It is expected that implementation of the new processes will lead to systemic industry change and thus a growth in productivity due to increased adoption of OSM.

The moderating effect of knowledge sharing on the relationship between manufacturing activities and business performance

This paper investigates the critical role of knowledge sharing (KS) in leveraging manufacturing activities, namely integrated supplier management (ISM) and new product development (NPD) to improve business performance (BP) within the context of Taiwanese electronic manufacturing companies. The research adopted a sequential mixed method research design, which provided both quantitative empirical evidence as well as qualitative insights, into the moderating effect of KS on the relationships between these two core manufacturing activities and BP. First, a questionnaire survey was administered, which resulted in a sample of 170 managerial and technical professionals providing their opinions on KS, NPD and ISM activities and the BP level within their respective companies. On the basis of the collected data, factor analysis was used to verify the measurement model, followed by correlation analysis to explore factor interrelationships, and finally moderated regression analyses to extract the moderating effects of KS on the relationships of NPD and ISM with BP. Following the quantitative study, six semi-structured interviews were conducted to provide qualitative in-depth insights into the value added from KS practices to the targeted manufacturing activities and the extent of its leveraging power. Results from quantitative statistical analysis indicated that KS, NPD and ISM all have a significant positive impact on BP. Specifically, IT infrastructure and open communication were identified as the two types of KS practices that could facilitate enriched supplier evaluation and selection, empower active employee involvement in the design process, and provide support for product simplification and the modular design process, thereby improving manufacturing performance and strengthening company competitiveness. The interviews authenticated many of the empirical findings, suggesting that in the contemporary manufacturing context KS has become an integral part of many ISM and NPD activities and when embedded properly can lead to an improvement in BP. The paper also highlights a number of useful implications for manufacturing companies seeking to leverage their BP through innovative and sustained KS practices.

Identifying key enablers to improve business performance in Taiwanese electronic manufacturing companies

Purpose – Integrated supplier management (ISM), new product development (NPD) and knowledge sharing (KS) practices are three primary business activities utilised to enhance manufacturers' business performance (BP). The purpose of this paper is to empirically investigate the relationships between these three business activities (i.e. ISM, NPD, KS) and BP in a Taiwanese electronics manufacturing context. Design/methodology/approach – A questionnaire survey is first administered to a sample of electronic manufacturing companies operating in Taiwan to elicit the opinions of technical and managerial professionals regarding business activities and BP within their companies. A total of 170 respondents from 83 companies respond to the survey. Factor, correlation and path analysis are undertaken on this quantitative data set to derive the key factors which leverage business outcomes in these companies. Following empirical analysis, six semi-structured interviews are undertaken with manufacturing executives to provide qualitative insights into the underlying reasons why certain business activity factors are the strongest predictors of BP. Findings – The investigation shows that the ISM, NPD and KS constructs all play an important role in the success of company operations and creating business outcomes. Specifically, the key factors within these constructs which influenced BP are: supplier evaluation and selection; design simplification and modular design; information technology infrastructure and systems and open communication. Accordingly, sufficient financial and human resources should be allocated to these important activities to derive accelerated rates of improved BP. These findings are supported by the qualitative interviews with manufacturing executives. Originality/value – The paper depicts the pathways to improved manufacturing BP, through targeting efforts into the above-mentioned factors within the ISM, NPD and KS constructs. Based on the empirical path model, and the specific insights derived from the explanatory interviews with manufacturing executives, the paper also provides a number of practical implications for manufacturing companies seeking to enhance their BP through improved operational activities.

Morphology-based model for predicting osteocyte-like cell line (MLO-Y4) growth process

This work has led to the development of empirical mathematical models to quantitatively predicate the changes of morphology in osteocyte-like cell lines (MLO-Y4) in culture. MLO-Y4 cells were cultured at low density and the changes in morphology recorded over 11 hours. Cell area and three dimensional shape features including aspect ratio, circularity and solidity were then determined using widely accepted image analysis software (ImageJTM). Based on the data obtained from the imaging analysis, mathematical models were developed using the non-linear regression method. The developed mathematical models accurately predict the morphology of MLO-Y4 cells for different culture times and can, therefore, be used as a reference model for analyzing MLO-Y4 cell morphology changes within various biological/mechanical studies, as necessary.

Empirical study to identify the key business activities contributing to manufacturing business performance

Purpose: Within the context of high global competitiveness, knowledge management (KM) has proven to be one of the major factors contributing to enhanced business outcomes. Furthermore, knowledge sharing (KS) is one of the most critical of all KM activities. From a manufacturing industry perspective, supply chain management (SCM) and product development process (PDP) activities, require a high proportion of company resources such as budget and manpower. Therefore, manufacturing companies are striving to strengthen SCM, PDP and KS activities in order to accelerate rates of manufacturing process improvement, ultimately resulting in higher levels of business performance (BP). A theoretical framework along with a number of hypotheses are proposed and empirically tested through correlation, factor and path analyses. Design/methodology/approach: A questionnaire survey was administered to a sample of electronic manufacturing companies operating in Taiwan to facilitate testing the proposed relationships. More than 170 respondents from 83 organisations responded to the survey. The study identified top management commitment and employee empowerment, supplier evaluation and selection, and design simplification and modular design as the key business activities that are strongly associated with the business performance. Findings: The empirical study supports that key manufacturing business activities (i.e., SCM, PDP, and KS) are positively associated with BP. The findings also evealed that some specific business activities such as SCMF1,PDPF2, and KSF1 have the strongest influencing power on particular business outcomes (i.e., BPF1 and BPF2) within the context of electronic manufacturing companies operating in Taiwan. Practical implications: The finding regarding the relationship between SCM and BP identified the essential role of supplier evaluation and selection in improving business competitiveness and long term performance. The process of forming knowledge in companies, such as creation, storage/retrieval, and transfer do not necessarily lead to enhanced business performance; only through effectively applying knowledge to the right person at the right time does. Originality/value: Based on this finding it is recommended that companies should involve suppliers in partnerships to continuously improve operations and enhance product design efforts, which would ultimately enhance business performance. Business performance depends more on an employee’s ability to turn knowledge into effective action.

An in-vitro 3-D cell culture model for studying pathomechanisms in AMD

Purpose To develop a novel 3-D cell culture model with the view to studying the pathomechanisms underlying the development of age-related macular degeneration (AMD). Our central hypothesis is that the silk structural protein fibroin used in conjunction with cultured human cells can be used to mimic the structural relationships between the RPE and choriocapillaris in health and disease. Methods Co-cultures of human RPE cells (ARPE-19 cells grown in Miller’s medium) and microvascular endothelial cells (HMEC-1 cells grown in endothelial culture medium) were established on opposing sides of a synthetic Bruch’s membrane (3 microns thick) constructed from B mori silk fibroin. Cell attachment was facilitated by pre-coating the fibroin membrane with vitronectin (for ARPE-19 cells) and gelatin (for HMEC-1 cells) respectively. The effects of tropoelastin on attachment of ARPE-19 cells was also examined. Barrier function was examined by measurement of trans-epithelial resistance (TER) using a voltohmmeter (EVOM-2). The phagocytic activity of the synthetic RPE was tested using vitronectin-coated microspheres (2 micron diameter FluoSpheres). In some cultures, membrane defects were created by puncturing within a 24 G needle. The architecture of the synthetic tissue before and after wounding was examined by confocal microscopy after staining for ZO-1 and F-actin. Results The RPE layer of the 3D model developed a cobblestoned morphology (validated by staining for ZO-1 and F-actin), displayed barrier function (validated by measurement of TER) and demonstrated cytoplasmic uptake of vitronectin-coated microspheres. Attachment of ARPE-19 cells to fibroin was unaffected by tropoelastin. Microvascular endothelial cells attached well to the gelatin-coated surface of the fibroin membrane and remained physically separated from the overlaying RPE layer. The fibroin membranes were amenable to puncturing without collapse thus providing the opportunity to study transmembrane migration of the endothelial cells. Conclusions Synthetic Bruch’s membranes constructed from silk fibroin, vitronectin and gelatin, support the co-cultivation of RPE cells and microvascular endothelial cells. The resulting RPE layer displays functions similar to that of native RPE and the entire tri-layered structure displays potential to be used as an in vitro model of choroidal neovascularization.

Temporal summation of intrinsically photosensitive retinal ganglion cell (ipRGC) contributions to the human pupil.

Purpose: IpRGCs mediate non-image forming functions including photoentrainment and the pupil light reflex (PLR). Temporal summation increases visual sensitivity and decreases temporal resolution for image forming vision, but the summation properties of nonimage forming vision are unknown. We investigated the temporal summation of inner (ipRGC) and outer (rod/cone) retinal inputs to the PLR. Method: The consensual PLR of the left eye was measured in six participants with normal vision using a Maxwellian view infrared pupillometer. Temporal summation was investigated using a double-pulse protocol (100 ms stimulus pairs; 0–1024 ms inter-stimulus interval, ISI) presented to the dilated fellow right eye (Tropicamide 1%). Stimulus lights (blue λmax = 460 nm; red λmax = 638 nm) biased activity to inneror outer retinal inputs to non-image forming vision. Temporal summation was measured suprathreshold (15.2 log photons.cm−2.s−1 at the cornea) and subthreshold (11.4 log photons.cm−2.s−1 at the cornea). Results: RM-ANOVAs showed the suprathreshold and subthreshold 6 second post illumination pupil response (PIPR: expressed as percentage baseline diameter) did not significantly vary for red or blue stimuli (p > .05). The PIPR for a subthreshold red 16 ms double-pulse control condition did not significantly differ with ISI (p > .05). The maximum constriction amplitude for red and blue 100 ms double- pulse stimuli did not significantly vary with ISI (p > .05). Conclusion: The non-significant changes in suprathreshold PIPR and subthreshold maximum pupil constriction indicate that inner retinal ipRGC inputs and outer retinal photoreceptor inputs to the PLR do not show temporal summation. The results suggest a fundamental difference between the temporal summation characteristics of image forming and non-image forming vision.

Knowledge sharing for strengthening manufacturing operation and business performance

Manufacturing companies have strived to enhance managerial and technical capabilities to improve business performance. Building these capabilities requires effective share of knowledge - the strategic resource. Specifically, knowledge sharing (KS) between different manufacturing departments can improve manufacturing processes since leveraging organisational knowledge plays an enssential role in achieving competitive advantage. This paper presents an empirical investigation into the impact of KS on the effectiveness of supply chain management (SCM) and the product development process (PDP) in achieving desired business performance (BP). A questionnaire survey was administered from electronic manufacturing companies operating in Taiwan. 168 valid responses were received and used to statistically examine the relationships between the concepts (SCM, PDP, KS, BP). The study findings reveal that within the Taiwanese electronic manufacturing companies KS is an essential enabler for facilitating the effectiveness of SCM and PDP in achieving desired BP.

Regulating for corporate human rights abuses : the emergence of corporate reporting on the ILO’s human rights standards within the global garment manufacturing and retail industry

Despite the ubiquitous nature of the discourse on human rights there is currently little research on the emergence of disclosure by multinational corporations on their human rights obligations or the regulatory dynamic that may lie behind this trend. In an attempt to begin to explore the extent to which, if any, the language of human rights has entered the discourse of corporate accountability, this paper investigates the adoption of the International Labour Organisation's (ILO) human rights standards by major multinational garment retail companies that source products from developing countries, as disclosed through their reporting media. The paper has three objectives. Firstly, to empirically explore the extent to which a group of multinational garment retailers invoke the language of human rights when disclosing their corporate responsibilities. The paper reviews corporate reporting media including social responsibility codes of conduct, annual reports and stand-alone social responsibility reports released by 18 major global clothing and retail companies during a period from 1990 to 2007. We find that the number of companies adopting and disclosing on the ILO's workplace human rights standards has significantly increased since 1998 – the year in which the ILO's standards were endorsed and accepted by the global community (ILO, 1998). Secondly, drawing on a combination of Responsive Regulation theory and neo-institutional theory, we tentatively seek to understand the regulatory space that may have influenced these large corporations to adopt the language of human rights obligations. In particular, we study the role that International Governmental Organisation's (IGO) such as ILO may have played in these disclosures. Finally, we provide some critical reflections on the power and potential within the corporate adoption of the language of human rights.