Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

The use of polymerase chain reaction for early diagnosis of tuberculosis in Mycobacterium tuberculosis culture

Early diagnosis plays a vital role in controlling tuberculosis. The conventional methodology is slow, with results taking several weeks, in addition to having low sensitivity, especially in clinical paucibacillary samples. The objective of this study was to evaluate the use of polymerase chain reaction (PCR) on solid medium culture for a rapid diagnosis of tuberculosis, mainly in cases of negative sputum smears. Forty sputum samples were collected from inpatients with tuberculosis treated for less than 2 days. Bacilloscopy, PCR for sputum, culture on Löwestein-Jensen (LJ) solid medium, and daily PCR from culture were performed on each sample. DNA extracted from the BCG vaccine, which contains attenuated bacillus Calmette-Guérin, was used as the positive control. Smear microscopy showed 68.6% sensitivity, 80% specificity, 96% positive predictive value, and 26.7% negative predictive value, with culture on LJ medium as the gold standard. Culture at day 28 showed 74.3% sensitivity and 100% specificity. PCR of DNA extracted from sputum amplified a 1027-bp fragment of the 16s RNA gene, showing 22.9% sensitivity and 60% specificity. PCR performed with DNA extracted from daily culture showed that, from the 17th to the 40th day, the sensitivity (85.7%) and specificity (60%) were constant. We conclude that a 17-day culture is a good choice for rapid diagnosis and to interfere with the transmission chain of tuberculosis.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 x 10(7) CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.

Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.

A spheroid-based 3-D culture model for pancreatic cancer drug testing, using the acid phosphatase assay

Current therapy for pancreatic cancer is multimodal, involving surgery and chemotherapy. However, development of pancreatic cancer therapies requires a thorough evaluation of drug efficacy in vitro before animal testing and subsequent clinical trials. Compared to two-dimensional culture of cell monolayer, three-dimensional (3-D) models more closely mimic native tissues, since the tumor microenvironment established in 3-D models often plays a significant role in cancer progression and cellular responses to the drugs. Accumulating evidence has highlighted the benefits of 3-D in vitro models of various cancers. In the present study, we have developed a spheroid-based, 3-D culture of pancreatic cancer cell lines MIAPaCa-2 and PANC-1 for pancreatic drug testing, using the acid phosphatase assay. Drug efficacy testing showed that spheroids had much higher drug resistance than monolayers. This model, which is characteristically reproducible and easy and offers rapid handling, is the preferred choice for filling the gap between monolayer cell cultures and in vivo models in the process of drug development and testing for pancreatic cancer.

Characterization of bacteriophages infecting clinical isolates of Pseudomonas aeruginosa stored in a culture collection

Some clinical isolates of Pseudomonas aeruginosa stored in our culture collection did not grow or grew poorly and showed lysis on the culture plates when removed from the collection and inoculated on MacConkey agar. One hypothesis was that bacteriophages had infected and killed those clinical isolates. To check the best storage conditions to maintain viable P. aeruginosa for a longer time, clinical isolates were stored at various temperatures and were grown monthly. We investigated the presence of phage in 10 clinical isolates of P. aeruginosa stored in our culture collection. Four strains of P. aeruginosa were infected by phages that were characterized by electron microscopy and isolated to assess their ability to infect. The best condition to maintain the viability of the strains during storage was in water at room temperature. Three Siphoviridae and two Myoviridae phages were visualized and characterized by morphology. We confirmed the presence of bacteriophages infecting clinical isolates, and their ability to infect and lyse alternative hosts. Strain PAO1, however, did not show lysis to any phage. Mucoid and multidrug resistant strains of P. aeruginosa showed lysis to 50% of the phages tested.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Nichesourcing The Uralic Languages For The Benefit Of Linguistic Research And Lingual Societies

The emerging technologies have recently challenged the libraries to reconsider their role as a mere mediator between the collections, researchers, and wider audiences (Sula, 2013), and libraries, especially the nationwide institutions like national libraries, haven’t always managed to face the challenge (Nygren et al., 2014). In the Digitization Project of Kindred Languages, the National Library of Finland has become a node that connects the partners to interplay and work for shared goals and objectives. In this paper, I will be drawing a picture of the crowdsourcing methods that have been established during the project to support both linguistic research and lingual diversity. The National Library of Finland has been executing the Digitization Project of Kindred Languages since 2012. The project seeks to digitize and publish approximately 1,200 monograph titles and more than 100 newspapers titles in various, and in some cases endangered Uralic languages. Once the digitization has been completed in 2015, the Fenno-Ugrica online collection will consist of 110,000 monograph pages and around 90,000 newspaper pages to which all users will have open access regardless of their place of residence. The majority of the digitized literature was originally published in the 1920s and 1930s in the Soviet Union, and it was the genesis and consolidation period of literary languages. This was the era when many Uralic languages were converted into media of popular education, enlightenment, and dissemination of information pertinent to the developing political agenda of the Soviet state. The ‘deluge’ of popular literature in the 1920s to 1930s suddenly challenged the lexical orthographic norms of the limited ecclesiastical publications from the 1880s onward. Newspapers were now written in orthographies and in word forms that the locals would understand. Textbooks were written to address the separate needs of both adults and children. New concepts were introduced in the language. This was the beginning of a renaissance and period of enlightenment (Rueter, 2013). The linguistically oriented population can also find writings to their delight, especially lexical items specific to a given publication, and orthographically documented specifics of phonetics. The project is financially supported by the Kone Foundation in Helsinki and is part of the Foundation’s Language Programme. One of the key objectives of the Kone Foundation Language Programme is to support a culture of openness and interaction in linguistic research, but also to promote citizen science as a tool for the participation of the language community in research. In addition to sharing this aspiration, our objective within the Language Programme is to make sure that old and new corpora in Uralic languages are made available for the open and interactive use of the academic community as well as the language societies. Wordlists are available in 17 languages, but without tokenization, lemmatization, and so on. This approach was verified with the scholars, and we consider the wordlists as raw data for linguists. Our data is used for creating the morphological analyzers and online dictionaries at the Helsinki and Tromsø Universities, for instance. In order to reach the targets, we will produce not only the digitized materials but also their development tools for supporting linguistic research and citizen science. The Digitization Project of Kindred Languages is thus linked with the research of language technology. The mission is to improve the usage and usability of digitized content. During the project, we have advanced methods that will refine the raw data for further use, especially in the linguistic research. How does the library meet the objectives, which appears to be beyond its traditional playground? The written materials from this period are a gold mine, so how could we retrieve these hidden treasures of languages out of the stack that contains more than 200,000 pages of literature in various Uralic languages? The problem is that the machined-encoded text (OCR) contains often too many mistakes to be used as such in research. The mistakes in OCRed texts must be corrected. For enhancing the OCRed texts, the National Library of Finland developed an open-source code OCR editor that enabled the editing of machine-encoded text for the benefit of linguistic research. This tool was necessary to implement, since these rare and peripheral prints did often include already perished characters, which are sadly neglected by the modern OCR software developers, but belong to the historical context of kindred languages and thus are an essential part of the linguistic heritage (van Hemel, 2014). Our crowdsourcing tool application is essentially an editor of Alto XML format. It consists of a back-end for managing users, permissions, and files, communicating through a REST API with a front-end interface—that is, the actual editor for correcting the OCRed text. The enhanced XML files can be retrieved from the Fenno-Ugrica collection for further purposes. Could the crowd do this work to support the academic research? The challenge in crowdsourcing lies in its nature. The targets in the traditional crowdsourcing have often been split into several microtasks that do not require any special skills from the anonymous people, a faceless crowd. This way of crowdsourcing may produce quantitative results, but from the research’s point of view, there is a danger that the needs of linguists are not necessarily met. Also, the remarkable downside is the lack of shared goal or the social affinity. There is no reward in the traditional methods of crowdsourcing (de Boer et al., 2012). Also, there has been criticism that digital humanities makes the humanities too data-driven and oriented towards quantitative methods, losing the values of critical qualitative methods (Fish, 2012). And on top of that, the downsides of the traditional crowdsourcing become more imminent when you leave the Anglophone world. Our potential crowd is geographically scattered in Russia. This crowd is linguistically heterogeneous, speaking 17 different languages. In many cases languages are close to extinction or longing for language revitalization, and the native speakers do not always have Internet access, so an open call for crowdsourcing would not have produced appeasing results for linguists. Thus, one has to identify carefully the potential niches to complete the needed tasks. When using the help of a crowd in a project that is aiming to support both linguistic research and survival of endangered languages, the approach has to be a different one. In nichesourcing, the tasks are distributed amongst a small crowd of citizen scientists (communities). Although communities provide smaller pools to draw resources, their specific richness in skill is suited for complex tasks with high-quality product expectations found in nichesourcing. Communities have a purpose and identity, and their regular interaction engenders social trust and reputation. These communities can correspond to research more precisely (de Boer et al., 2012). Instead of repetitive and rather trivial tasks, we are trying to utilize the knowledge and skills of citizen scientists to provide qualitative results. In nichesourcing, we hand in such assignments that would precisely fill the gaps in linguistic research. A typical task would be editing and collecting the words in such fields of vocabularies where the researchers do require more information. For instance, there is lack of Hill Mari words and terminology in anatomy. We have digitized the books in medicine, and we could try to track the words related to human organs by assigning the citizen scientists to edit and collect words with the OCR editor. From the nichesourcing’s perspective, it is essential that altruism play a central role when the language communities are involved. In nichesourcing, our goal is to reach a certain level of interplay, where the language communities would benefit from the results. For instance, the corrected words in Ingrian will be added to an online dictionary, which is made freely available for the public, so the society can benefit, too. This objective of interplay can be understood as an aspiration to support the endangered languages and the maintenance of lingual diversity, but also as a servant of ‘two masters’: research and society.

"Rakkaat poikaiset!". Simon ja Wilhelmina Janssonin perhekirjeet egodokumentteina (1858-1887)

Kustavilainen talonpoikaislaivuri Simon Jansson ja hänen vaimonsa Wilhelmina o.s. Widbom kävivät kolmenkymmenen vuoden ajan kirjeenvaihtoa kouluun ja yliopistoon lähetettyjen poikiensa Waldemar, Evald ja Emil Jahnssonin kanssa. Lähes 150 suomen- ja ruotsinkielistä kirjettä on säilynyt Suomalaisen Kirjallisuuden Seuran kirjallisuusarkistossa ja Turun maakunta-arkistossa. Lähiluvun kautta kirjeistä muodostuu yksityiskohtainen lähde saaristolaisperheen elämäntapaan, arkeen ja työhön sekä saaristoyhteisön sosiaalisen kanssakäymisen muotoihin. Ne kertovat myös ensimmäisen polven oppisivistyneistön synnystä ja niistä resursseista, joita hyödyntäen koulua käymättömät vanhemmat saattoivat kouluttaa lapsensa. Tutkimus liittyy suomalaisten 1800-luvun itseoppineiden kirjoittajien tekstien ja maaseudun kirjallistumisen tutkimukseen. Keskeinen käsite on egodokumentti, jolla tarkoitetaan kirjeitä ja muita tekstejä, joissa kirjoittaja kertoo itse omasta elämästään. Hollantilainen ja ranskalainen egodokumenttien tutkimus on pyrkinyt haastamaan uudenlaisten lähdeaineistojen kautta käsityksiä menneisyydestä sekä nostamaan esiin muun muassa yksilöllisen kokemuksen, kirjoittamisen kulttuurin ja dokumenttien materiaalisuuden. Janssonin perheen kirjeet poikkeavat monin tavoin 1800-luvun säätyläisten kirjeistä. Niiden funktio oli hyvin käytännöllinen ja arkinen, mutta ne olivat myös vanhempien keino tukea perheen yhteistä sosiaalisen nousun projektia. Kustavia koskevan tiheän uutisoinnin kautta ne pyrkivät pitämään kaupunkilaistuvat pojat osana vanhaa yhteisöään. Perheen isä vastasi suurelta osin kirjeenvaihdosta ja siihen liittyvästä huolenpidosta, mikä kertoo sukupuolittuneen työnjaon joustavuudesta saaristossa. Tutkimus osoittaa, ettei isän ammattiin perustuva yliopisto-opiskelijoiden tutkimus ole tavoittanut sitä naisten kautta välittyvää sosiaalista ja kulttuurista pääomaa, joka saattoi olla ratkaiseva perheiden kouluttaessa lapsiaan. Kirjeiden analyysi tarkentaa kuvaa perinteisen talonpoikaispurjehduksen sopeutumisesta vuosisadan loppupuolen uudistuksiin.

Sustainable Development – the Key to Competitiveness CSR as an added value to Alstom’s corporate performance

Corporate social responsibility or CSR is today a widely recognized concept which is receiving in- creasing popularity extremely rapidly, especially in the business world. The pressure on companies to carry out their business practices in ethical manners, which promote the wellbeing of the environment and society, is coming from all directions and all stakeholders. Alstom, a French multinational conglomerate operating in the rail transport and energy industry, is no exception to this norm. This company, which will be used as the case example in this thesis, is being brought to bay in terms of engaging in CSR practices and practicing business with high ethics. It is surely not a negatively conceived phenomenon that CSR is being put on a pedestal – quite the opposite. Instead of corporations practicing CSR only to meet their stakeholder requirements through practicing window dressing, many corporations actually strive to benefit from the practice of corporate social business. In addition to bringing benefit to externals a corporation such as Alstom itself can benefit from being involved in CSR. The purpose of this thesis is to evaluate the current strategic values and the future perspectives of CSR at Alstom and moreover the added value which the practice of CSR could bring Alstom as a business. A set of perspectives from a futures studies viewpoint is looked at, with critical examination of the company’s current corporate practices as well as the CSR related studies and theories written for corporations. Through this, some solutions and practices will be suggested to Alstom in order for it to fully utilize the potential of corporate social business and the value it can bring in the most probable futures that the company is expected to face. By utilizing the Soft Systems Methodology (SSM), a method mainly used in organizations to solve problematic issues in management and policy contexts, a process is developed to see what improvements could be of help in improving Alstom and its way towards involving CSR in its business practices even more than it currently does. Alstom is already deeply involved in the practicing of CSR and its vision has a strong emphasis on this popular concept of today. In order to stay in the game and to use CSR as a competitive advantage to the company, Alstom ought to embed corporate social practices even deeper in its organizational culture by using them as a tool to reduce risk and costs, increasing employee commitment and customer loyalty and to attract socially responsible investors, just to name a few. CSR as a concept is seen to have great potential in the future, an opportunity Alstom will not miss.