Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges.

This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different ('A' to 'D') blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages 'B' to 'D' (but not stage 'A') started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages 'B' and 'C' buds develop (3-20 days) one or more large (1 mm diameter) spheres. Stage 'D' buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage 'A' bud cultures. Spheres grew at a rate of 60 microm in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 microm in diameter per day, for 3-15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.     

Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri

The present paper describes the immune role played by a recently identified (Gasparini et al. 2008) member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri.B. schlosseri RBL (BsRBL) can activate phagocytes through: (i) induction of their directional movement towards the source of the molecule; (ii) modification of cytoskeleton, required for shape changes; (iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and (iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognised by anti-IL1α and anti-TNFα antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three-dimensional model for BsRBL is presented.     

Epithelial hyperplasia in organotypical aggregate cultures from mouse embryonal lung cells

The incidence of epithelial hyperplasia is studied in cultures of aggregates from epithelial and mesenchymal cells of embryonal lungs of intact and urethrane-treated Asn and C57B1 mice (transplacental urethrane, 3 g/kg). In the control, epithelial hyperplasia was detected in 5.9% Asn and in 9.9% C57B1 mice. In experimental aggregates epithelial hyperplasia was 54.3% in Asn and 39.8% in C57B1 mice (p<0.001). In aggregates with only one tissue component from experimental and another from control embryos, the incidence of epithelial hyperplasia was lower than in experimental animals: 27.1–28.3% (p<0.001) in Asn and 16.7–35.1% (p<0.05) in C57B1 mice. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 6, pp. 673–676, June, 1998     

Progress in the development of shrimp cell cultures in Thailand

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 ± 10 mmol/kg, a temperature range of 25--28 °C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic-like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.     

Primary cell cultures isolated from Penaeus monodon prawns

We have devised a cell culture system for Penaeus monodon prawn cells that uses a defined synthetic medium. Organs were removed from adult prawns ranging in size from 13--19cm rostrum-to-telson length. Cultures consisted of either a blend of hematopoietic and lymphoid cells or ovarian cells. The cells divide rapidly in culture, doubling on average once per week for 5 to 6 weeks. These cultures continue to survive for at least 5 months but the rates of cell division are low after the first 5--6 weeks. In the literature, unicellular eukaryotic marine organisms such as chytrids may contaminate marine cell cultures. In some cases these eukaryotic contaminants may be difficult to distinguish from prawn cells unless detailed ultrastructure or characteristic developmental stages such as zoospores can be observed. Alternatively, we prepared molecular probes from repeated DNA sequences 100--400 bp in length in the P. monodon genome. These species-specific probes were hybridised to genomic DNA from cell culture to confirm proliferation of P. monodon cells in our cultures.     

Morphological and functional characteristics of human pituitary lactotrophic adenomas in cell cultures

Some features of the morphological cellular structure of prolactin secreting human pituitary adenomas and their secretion of prolactin and somatotropic hormone in primary suspension cultures were investigated. A possiblein vitro proliferation of lactotrophs was established. The inhibitory effect of somatostatin and its synthetic analog sandostatin, on prolactin secretion in prolactinomas was found to be less than in somatotropic hormone-secreting pituitary tumors. Presented by A. N. Konovalov, Member of the Russian Academy of Medical Sciences Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 11, pp. 543–546, November, 1994     

Secretory activity of lactotrophs and its regulation by hypothalamic hormones in primary pituitary cell cultures from rats of different ages

Laboratory of Biological Research into Hormonal Compounds, Institute of Experimental Endocrinology, All-Union Endocrinologic Research Center, Academy of Medical Sciences, Moscow. (Presented by Academician Yu. A. Pankov, Academy of Medical Sciences.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 4, pp. 402–404, April, 1992.     

Cryopreservation technology for plant cell cultures

Summary Described in detail is a technique for the cryopreservation, long-term storage, thawing, recovery, and regrowth of embryogenic suspension cultures of maize (Zea mays L.) With this system we have successfully preserved many different culture lines, and recovered living material after more than 2 yr storage in liquid nitrogen. The recovered tissue is not apparently different from the starting material. With minor variations, the technique has also been used forDactylis glomerata and other suspension culture cells, as well as callus cultures. Approaches for the cryopreservation of other plant parts, such as seeds, embryos and meristems are discussed.     

Relevance of allergens from cats and dogs to asthma in the northernmost province of Sweden: Schools as a major site of exposure

BACKGROUND: The prevalence of asthma in the northernmost region of Sweden has been estimated at 6% to 8% in spite of the very dry climate. The causes of the increase in asthma are not clear, but conditions are unfavorable for dust mite growth, and domestic animals are thought to be the primary source of indoor allergens. OBJECTIVES: We sought to investigate the relationship between asthma, exposure, and sensitization in Northern Sweden, with a focus on the role of schools. METHODS: Serum was collected from 110 asthmatic children, 55 children with symptoms of asthma but no established diagnosis, and 63 control children (age, 7 and 8 years). Total IgE and specific IgE to 7 allergens were measured. Dust samples were collected from the classrooms of 7- and 8-year-old children in 22 schools from Kiruna and Lulea, Sweden. For comparison, dust was also collected from 24 homes in Kiruna and 2 schools in Virginia in the United States. RESULTS: Serum IgE antibody assays on 165 children with respiratory symptoms confirmed that there was a high degree of sensitization to cat, dog, and birch in Northern Sweden. Cat and dog allergens were present in almost all of the school samples in Sweden. By contrast, dust mite and cockroach allergens were generally unmeasurable. The highest levels of cat and dog allergens were found in samples from desks and chairs. Cat and dog allergen levels in the schools were comparable with but higher than those in the homes without pets. The schools in Virginia had similar allergen levels, except that samples from this humid region also had significant mite allergen. CONCLUSIONS: In this climate the primary sensitization associated with asthma is to cat dander and dog dander but also to birch pollen. Mite and cockroach allergens were not present in the dust samples, and sensitization to these allergens was not significant. The schools appear to be a major site of exposure to cat and dog allergens. These results are relevant both to an understanding of the reasons for the increase in asthma in this region and to any proposal to reduce exposure to allergens.     

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2× L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.     

NK cells: new issues and challenges. Preface

NK cells are innate lymphocytes crucial for surveillance against pathogens and tumors. Although the basic mechanisms through which NK cells operate have been established, many questions are still unresolved. Are all NK cells equal or can we identify subsets with distinct developmental origin and function? How do NK cells interact with other components of the immune system, such as DC, effector T cells, and Treg cells to elicit effective immune responses? How do NK cells become tolerant to self and preclude autoimmunity? Finally, can we design novel therapeutic avenues for cancer treatment based on NK cells? The editorial team of the European Journal of Immunology asked the opinion of some of the leading experts in the NK‐cell field regarding the new challenges and opportunities facing this area of research and the thoughts of the experts are presented in this viewpoint series. This introduction to the series brings together some of the novel concepts emerging from the experts' discussions.     

Food allergy. Part 2: Diagnosis and management

Patients with food-induced allergic disorders may be first seen with a variety of symptoms affecting the skin, respiratory tract, gastrointestinal tract, and/or cardiovascular system. The skin and respiratory tract are most often affected by IgE-mediated food-induced allergic reactions, whereas isolated gastrointestinal disorders are most often caused by non-IgE-mediated reactions. When evaluating possible food-induced allergic disorders, it is often useful to categorize disorders into IgE- and non-IgE-mediated syndromes. The initial history and physical examination are essentially identical for IgE- and non-IgE-mediated disorders, but the subsequent evaluation differs substantially. Proper diagnoses often require screening tests for evidence of food-specific IgE and proof of reactivity through elimination diets and oral food challenges. Once properly diagnosed, strict avoidance of the implicated food or foods is the only proven form of treatment. Clinical tolerance to food allergens will develop in many patients over time, and therefore follow-up food challenges are often indicated. However, a number of novel immunomodulatory strategies are in the developmental stage and should provide more definitive treatment for some of these food-induced allergic disorders in the next several years.     

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12 h after the challenge, which was characterized by the early local secretion of TNF-α and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12 h after the challenge and no pneumococci could be recovered after 36 h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-α and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30 h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.     

Preparation of metaphase chromosomes in cell cultures derived from an aquatic reptile

Summary Cell cultures (GTS) of epithelioid nature derived from the skin of a green sea turtle,Chelonia mydas, were treated with colchicine at a final concentration of 0.5 g/ml for 16 h. Mitotic cells were harvested by brief treatment with trypsin-Versene, subjected to hypotonic solution (1% sodium citrate) and fixed in (13) acetic acid: methanol. Giemsa stained preparations were photographed on High Contrast Copy film under phase contrast optics using a bluegreen filter. The result was significant enhancement of the microchromosome portion of the complement morphologically characteristic of reptilian metaphase chromosomes. By this method it was determined that the GTS cell line retains the female diploid number of the Chelonia species.     

Influence of the TP53 codon 72 polymorphism on the cellular responses to X-irradiation in fibroblasts from nonagenarians

In mice, genetic modification of the gene encoding p53 affects both cancer incidence and longevity. In humans, we recently found that a TP53 codon 72 Arginine (Arg) to Proline (Pro) polymorphism affected both cancer incidence and longevity as well. The TP53 codon 72 polymorphism has previously been shown to influence the apoptotic potential of human cells in response to oxidative stress. Here, we studied the influence of this polymorphism on the cellular responses to X-irradiation of fibroblasts obtained from nonagenarians. We found that the average clonogenic survival after X-irradiation was similar for the three TP53 codon 72 genotype groups. As described before, X-irradiation did not induce an appreciable degree of apoptosis in human fibroblasts. However, percentages of senescence-associated (SA)-β-galactosidase positive cells (p < 0.001), micronucleated cells (p < 0.001) and cells displaying abnormal nuclear morphologies (p < 0.001) significantly increased with the radiation dose. Compared to Arg/Arg fibroblasts, Pro/Pro fibroblasts exhibited higher irradiation dose-dependent increases in SA-β-galactosidase positive cells (p_interaction = 0.018), micronucleated cells (p_interaction = 0.005) and cells displaying abnormal nuclear morphologies (p_interaction = 0.029) at 3 days after irradiation. Possibly, these differences in cellular responses to stress between the TP53 codon 72 genotypes contribute to the differences in cancer incidence and longevity observed earlier for these genotypes.     

Interval based fuzzy systems for identification of important genes from microarray gene expression data: Application to carcinogenic development

In the present article, we develop two interval based fuzzy systems for identification of some possible genes mediating the carcinogenic development in various tissues. The methodology involves dimensionality reduction, classifying the genes through incorporation of the notion of linguistic fuzzy sets low, medium and high, and finally selection of some possible genes mediating a particular disease, obtained by a rule generation/grouping technique. The effectiveness of the proposed methodology, is demonstrated using five microarray gene expression datasets dealing with human lung, colon, sarcoma, breast cancer and leukemia. Moreover, the superior capability of the methodology in selecting important genes, over five other existing gene selection methods, viz., Significance Analysis of Microarrays (SAM), Signal-to-Noise Ratio (SNR), Neighborhood analysis (NA), Bayesian Regularization (BR) and Data-adaptive (DA) is demonstrated, in terms of the enrichment of each GO category of the important genes based on P-values. The results are appropriately validated by earlier investigations, gene expression profiles and t-test. The proposed methodology has been able to select genes that are more biologically significant in mediating the development of a disease than those obtained by the others.