Showing papers on "Tissue culture published in 1993"

Quantitation of human immunodeficiency virus type 1 infection kinetics.

Abstract: Tissue culture infections of CD4-positive human T cells by human immunodeficiency virus type 1 (HIV-1) proceed in three stages: (i) a period following the initiation of an infection during which no detectable virus is produced; (ii) a phase in which a sharp increase followed by a peak of released progeny virions can be measured; and (iii) a final period when virus production declines. In this study, we have derived equations describing the kinetics of HIV-1 accumulation in cell culture supernatants during multiple rounds of infection. Our analyses indicated that the critical parameter affecting the kinetics of HIV-1 infection is the infection rate constant k = Inn/ti, where n is the number of infectious virions produced by one cell (about 10(2)) and ti is the time required for one complete cycle of virus infection (typically 3 to 4 days). Of particular note was our finding that the infectivity of HIV-1 during cell-to-cell transmission is 10(2) to 10(3) times greater than the infectivity of cell-free virus stocks, the inocula commonly used to initiate tissue culture infections. We also demonstrated that the slow infection kinetics of an HIV-1 tat mutant is not due to a longer replication time but reflects the small number of infectious particles produced per cycle.

Activation of tobacco retrotransposons during tissue culture.

Abstract: Sequences of at least three new families of retrotransposons (Tto1-Tto3) were amplified by PCR from cDNA prepared from protoplasts of an established tobacco cell line, based on the fact that certain amino acids are highly conserved in the reverse transcriptases encoded by retrotransposons. Structural analysis indicates that Tto1 is 5.5 kb long and has features typical of retrotransposons. Transcription of Tto1 starting in the long terminal repeat was active only in cultured cells. Protoplast formation enhanced the transcription. The copy number of Tto1 increased 10-fold in established cell lines; it also increased in plants regenerated from tissue cultures and in transgenic plants. These results indicate that Tto1 is activated during tissue culture. This is the first demonstration of activation of a plant retrotransposon by tissue culture. The copy number of Tto2 and a previously isolated transposon, Tnt1, also increased in established cell lines, indicating that these two retrotransposons may also be activated by tissue culture. These three retrotransposons are cryptic in normally propagated plants: no difference in the copy number was observed between individuals of the same cultivars or even between different cultivars.

Establishment of conditionally immortalized epithelial-cell lines from both colon and small-intestine of adult h-2kb-tsa58 transgenic mice

Abstract: Intestinal mucosal cells have proved difficult to culture in vitro. Many attempts have been made to develop long-term cultures of these cells either by direct culturing or by attempting to immortalize these cells by using a range of transforming viral genes, but with little success. The recent development of a transgenic mouse bearing a temperature-sensitive mutation of the simian virus 40 large tumor antigen gene (tsA58) has enabled us to initiate conditionally immortalized cultures of epithelial cells from both small intestinal and colonic mucosa of adult mice. Crypts were isolated from either the small intestines or colons of young adult mice and cultured at the permissive temperature (33 degrees C) in medium containing conditioned medium from a human colon carcinoma cell line, LIM1863. Crypts from both tissues yielded cultures of epithelial cells that have now been in culture for more than 12 months with regular passaging. The epithelial nature of the cells has been confirmed by staining with anti-keratin antibodies. The intestinal origin of the cells was demonstrated by the ability of the cells to synthesize low levels of both brush border peptidases and a disaccharidase. The levels of expression of these enzymes were modulated by the addition of sodium butyrate or phorbol myristate acetate to the medium, which resulted in an increase in the synthesis of the peptidases and a decrease in the synthesis of the disaccharidase. The cells proliferate continuously at the permissive temperature (33 degrees C), but proliferation ceases at the nonpermissive temperature (39.5 degrees C). To our knowledge, this is the first description of the establishment of epithelial cell lines from both small intestine and colon of the same mouse strain. The success reported here indicates that this transgenic mouse will be a useful source of tissue for the study of the mechanisms that control the proliferation and eventual differentiation and senescence of the cells of the intestinal mucosa. These mice will also be a useful source of cells for attempts to culture cells from other tissues that have proved difficult to culture in vitro.

Tissue inhibitor of metalloproteinases‐2 inhibits bFGF‐induced human microvascular endothelial cell proliferation

Abstract: Tissue inhibitor of metalloproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of 3H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 +/- 12 nM (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture plastic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo.

Detection of enteroviruses in groundwater with the polymerase chain reaction.

Abstract: Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.

Replicative senescence of human fibroblast-like cells in culture

Abstract: The life history of fibroblast and fibroblast-like cells includes an initial stage of outgrowth and establishment in culture; a period of vigorous proliferation which has a variable length, depending on the tissue of origin, age of the donor, etc.; a period of declining proliferative vigor which includes substantial cell death; and finally, the emergence of an (apparently) long-lived population which is unable to proliferate in response to growth factors. During the phase of declining proliferative vigor, the cells acquire characteristics, some of which are similar to the characteristics of cells in older individuals. Eventually the culture completely loses proliferative capacity. A comparable life history has been described for glial cells, keratinocytes, vascular smooth muscle cells, endothelial cells, and lymphocytes which suggests that this life history is characteristic of those cell types that, in vivo, retain the capacity for proliferation throughout the life span. Numerous studies have shown a correlation between the age of the tissue donor and the replicative life span of the cells in culture. In addition, for a small sample of species, there is a direct correlation between fibroblast replicative life span in vitro and maximum life span potential of the species. The period in the life history that is usually referred to as the "senescent phase" is probably more complicated than was originally thought, since studies with life span modulators suggest that there is a "conditionally" senescent state from which cells can be rescued for one or more additional rounds of DNA synthesis. Finally, the cells enter an "obligatory" arrested state in which only SV40 infection can reverse the block to DNA synthesis but not the block to mitosis. The modern era of aging research in tissue culture is just over 30 years old. The inception of the field really began with the recognition by Hayflick and Moorhead (109) that the phenomenon of senescence in vitro paralleled, in some of its characteristics, cell aging in vivo and thus provided a model that could be used to study the cellular mechanisms underlying senescence in controlled environmental conditions. The research in this area began with a detailed characterization and comparison of young versus senescent cell morphology and physiology. These studies provided the basis for a wide variety of subsequent studies that addressed possible mechanisms underlying cell senescence. These included studies on DNA repair, protein synthetic errors, chromatin structure and function, and mechanisms for modulating replicative life span.(ABSTRACT TRUNCATED AT 400 WORDS)

Early events in higher-plant embryogenesis.

Abstract: In this review several recent findings in plant embryogenesis will be described. The emphasis will be on a number of selected studies that deal with events in the first and crucial steps of the development of the zygotic embryo and with events in the transition of somatic cells into embryogenic cells. In the first section, early zygotic embryo mutants of Arabidopsis will be highlighted. In the second section, essential steps in the formation of embryogenic cells and somatic embryos will be discussed. Based on these studies, the question will be raised which cellular mechanisms control early zygotic embryogenesis and whether analogous mechanisms are involved in the formation of embryogenic cells in tissue culture.

Transforming growth factor beta 1 can induce estrogen-independent tumorigenicity of human breast cancer cells in athymic mice.

Abstract: We have examined the effect of transforming growth factor beta 1 (TGF-beta 1) overexpression in human breast cancer cell tumorigenicity in athymic mice. Estrogen-dependent MCF-7 cells were stably transfected with pSVTGF beta 1. A clone was isolated which overexpressed TGF-beta 1 mRNA and secreted > 10-fold more TGF-beta activity into the tissue culture medium. Similar to the parent line, the MCF-7/TGF-beta 1 cells were relatively insensitive to exogenous TGF-beta 1 and exhibited low levels of TGF-beta receptors. Clonogenicity in soft agarose, doubling time, morphology, and sensitivity to 17 beta-estradiol and the antiestrogen tamoxifen were not altered in the transfected cells. Inoculation s.c. of MCF-7/TGF-beta 1 cells in ovariectomized nude mice resulted in 100% tumor formation which was totally abrogated by i.p. administration of the neutralizing anti-TGF-beta 2G7 IgG2B. The parent cells formed tumors only after estrogen supplementation. By immunohistochemistry, higher levels of TGF-beta 1 protein were detected in MCF-7/TGF-beta 1 tumors than in estrogen-induced parent MCF-7 tumors. Administration of 1 microgram TGF-beta 1 i.p. daily for 3 weeks after tumor cell inoculation transiently supported estrogen-independent growth of parent MCF-7 tumors in castrated nude mice. These data indicate that overexpression of TGF-beta 1 in human breast cancer cells can contribute to their escape from hormone dependence.

Detection and localization of cytokine immunoreactivity in retro-ocular connective tissue in Graves' ophthalmopathy

Abstract: Paracrine interactions between fibroblasts residing in the retro-ocular space and infiltrating lymphocytes/macrophages are thought to be of central importance in the pathogenesis of Graves' ophthalmopathy (GO). Although various roles have been suggested for interferon-gamma (IFN gamma), tumour necrosis factor-alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) in GO, their actual presence in Graves' retro-ocular connective tissue has not been demonstrated. We examined surgical specimens obtained during orbital decompression from patients with severe GO (n = 6), and from normal individuals (n = 5), for the presence of IFN gamma, TNF alpha and IL-1 alpha. We used immunohistochemical methods on frozen tissue sections and primary fibroblast cultures, and sodium dodecylsulfate polyacrylamide-gel electrophoresis of tissue extracts and tissue culture supernatants. In addition, immunohistochemical staining of tissues for characterization of the mononuclear cell infiltrates was performed. Aggregates of mononuclear cells in retro-ocular connective and fatty tissue were found in five of six GO tissue specimens, but in none of the control specimens. We detected immunoreactivity for the three cytokines (IFN gamma, TNF alpha and IL-1 alpha) in the five GO tissue specimens that contained mononuclear cell aggregates. In addition, IL-1 alpha immunoreactivity was demonstrable in primary and subsequent GO fibroblast cultures and in their supernatants. In contrast, no immunoreactivity for any of these cytokines was detected in tissue specimens, primary cultures or culture supernatants derived from normal individuals. The presence of mononuclear cell infiltrates and associated immunoreactivity for IFN gamma, TNF alpha, IL-1 alpha in retro-ocular connective tissue derived from patients with GO suggests that the previously demonstrated in vitro functions of these cytokines may indeed be operative in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of Matrix Metalloproteinases and Their Inhibitors (Tissue Inhibitor of Metalloproteinases) by Human Prostate in Explant Cultures: Reduced Tissue Inhibitor of Metalloproteinase Secretion by Malignant Tissues

Abstract: Unregulated secretion of matrix metalloproteinases (MMPs) or their endogenous protein inhibitors (tissue inhibitor of metalloproteinases, TIMPs) has been implicated in tumor invasion and metastasis. Species of MMPs and TIMPs secreted by epithelial cultures of normal, benign, and malignant prostate were identified and their levels were compared. Fragments of fresh tissue were cultured in a serum-free medium that supported the outgrowth of prostatic epithelial cells. Biochemical analysis of the conditioned media by gelatin zymography and enzyme assays showed that both normal and neoplastic tissues secreted latent and active forms of both M(r) 72,000 type IV collagenase (MMP-2) and M(r) 92,000 gelatinase (MMP-9). However, conditioned media from malignant prostate explants contained a higher proportion of the active form of MMP-2. Significant amounts of free TIMPs were secreted by normal juvenile and adult prostates, but they were either markedly reduced or not detectable in conditioned media from neoplastic tissues. These findings suggest that there is an imbalance of secretion between MMPs and TIMPs in prostatic carcinoma.

De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene

Abstract: Role of ethylene in de novo shoot morphogenesis from explants and plant growth of mustard (Brassica juncea cv. India Mustard) in vitro was investigated, by culturing explants or plants in the presence of the ethylene inhibitors aminoethoxyvinylglycine (AVG) and AgNO3. The presence of 20 μM AgNO3 or 5 μM AVG in culture medium containing 5 μM naphthaleneacetic acid and 10 μM benzyladenine were equally effective in promoting shoot regeneration from leaf disc and petiole explants. However, AgNO3 greatly enhanced ethylene production which reached a maximum after 14 days, whereas ethylene levels in the presence of AVG remained low during 3 weeks of culture. The promotive effect of AVG on shoot regeneration was overcome by exogenous application of 25 μM 2-chloroethylphosphonic acid (CEPA), but AgNO3-induced regeneration was less affected by CEPA. For whole plant culture, AVG did not affect plant growth, although it decreased ethylene production by 80% and both endogenous levels of 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC by 70–80%. In contrast, AgNO3 stimulated all 3 parameters of ethylene synthesis. Both AgNO3 and CEPA were inhibitory to plant growth, with more severe inhibition occuring in AgNO3. Leaf discs derived from plants grown with AVG or AgNO3 were highly regenerative on shoot regeneration medium without ethylene inhibitor, but the presence of AgNO3 in the medium was inhibitory to regeneration of those derived from plants grown with AgNO3.

Tissue culture of the organ of Corti.

Abstract: In 1975, Sobkowicz et al. (1) described long-term organotypic cultures of the organ of Corti of the newborn mouse. This paper provides detailed methods for dissection and maintenance of the isolated organ of Corti with its corresponding segment of spiral ganglion in culture. Descriptions and illustrations of cellular characteristics of the developing organ are carefully documented. The work is based on 19 years of experience and over one thousand cultures. Review of the literature and the application of the technique to research on the inner ear are provided.

To do tissue culture in two or three dimensions? That is the question.

Abstract: Alexis Carrel introduced the in vitro culture of tissues in the beginning of the century utilizing a culture system that allowed the three-dimensional growth of tissues. Leighton improved upon this system by developing a substrate of sponge matrices. Other methods of three-dimensional culture include collagen gels and what are known as organ culture systems on filters or meshes. In addition, cell suspensions can be converted into multicellular spheroids, another form of three-dimensional culture. Comparison of the three-dimensional culture methods with two-dimensional culture methods has shown critical differences in the behavior of biological systems in culture. For example, in vivo-like drug responses are observed in three-dimensional but frequently not in two-dimensional cultures, indicating that drug response may be a function of tissue architecture. The in vivo mechanism of drug resistance may involve alterations in cell-cell interaction which may occur in three-dimensional culture as opposed to monolayer culture. Practical applications of three-dimensional culture include the development of a drug-response assay that correlates not only with drug resistance but also with drug sensitivity and survival of cancer patients. It has been shown that gene expression may be more in vivo-like in three-dimensional cultures than in two-dimensional monolayer cultures. For example, tumor antigens may be expressed in three-dimensional culture and not in monolayer culture. Thus, future studies utilizing three-dimensional cultures may significantly enhance our understanding of gene expression and resistance to drugs and enhance the efficacy of cancer chemotherapy by correctly predicting active drug regimens for individual patients.

Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.

Abstract: A novel insect cell line from Trichoplusia ni, BTI-Tn 5B1-4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein-Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28-fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5-fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment-dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1 L scales was optimized with respect to cell density using standard techniques for the growth of attachment-dependent cells. Roller bottles precoated with DEAE-based microcarriers and suspension cultures employing collagen-coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier-coated roller bottles or in suspension cultures using collagen-coated microcarriers at concentrations close to those observed in tissue culture flasks.

Dna methylation and tissue culture-induced variation in plants'

Abstract: Plant cells growing in an artificial culture environment make numerous genetic mistakes. These alterations are manifested as increased frequencies of single-gene mutations, chromosome breakages, transposable element activations, quantitative trait variations, and modifications of normal DNA methylation patterns. Evidence is presented that indicates a high frequency of DNA hypomethylation as the result of the tissue culture process. Fifteen percent of the methylation changes appear to have been homozygous in the original regenerated plants. A hypothesis is advanced that relates DNA methylation to the variety of genetic alterations found among maize tissue culture regenerants and their progenies. The epigenetic nature of DNA methylation raises questions concerning the stability of tissue culture-induced changes in self-pollinations and crosses.

Breast Carcinoma and the Role of Iron Metabolism

Abstract: Transferrin receptors on proliferating and malignant cells are well documented. Iron is an essential micronutrient for cell growth that plays an important role in energy metabolism and DNA synthesis. Malignant cells requiring more iron modulate a transferrin receptor. Iron-bound transferrin interacts with this receptor, facilitating the transport of iron across the cell membrane. Transferrin is a glycoprotein and is the chief iron transport protein in mammalian blood. The more aggressive the tumor, the higher the transferrin receptor levels and the greater the proliferative index. We have found by cytochemical and ultrastructural studies that ferritin, an iron storage protein, is increased in breast cancer tissue. Anaplastic tumors have higher tissue ferritin levels. Tissue ferritin concentration may be an indirect method of measuring transferrin receptors and thus might be an index of proliferation and a prognostic indicator. Transferrin may be used as a carrier to target toxic therapy selectively to tumor tissue. A platinum transferrin complex (MPTC-63) has been developed and shown to be cytostatic in tissue culture, animal, and human studies. It also sensitizes tissue to agents that produce free radicals, such as adriamycin, and thus is synergistic with other drugs and radiation. Other transferrin complexes and conjugates of gallium, indium, and daunorubicin have also shown growth inhibition in tissue culture and animals. Human studies are in progress. By studying iron metabolism in breast cancer, we may be able to selectively inhibit tumor growth without toxic effects, and with other tumor biologic data be better able to select the stage I patient for adjuvant therapy.

Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin "comets".

Abstract: The ActA protein of the gram-positive pathogen Listeria monocytogenes is a 90-kDa polypeptide required for interaction of the bacteria with components of the host cell microfilament system to generate intra- and intercellular movement. To study the localization, distribution, and expression of the ActA polypeptide in L. monocytogenes grown either in broth culture or in infected tissue culture cells, we first isolated ActA by monoclonal antibody-based immunoaffinity chromatography. Polyclonal rabbit antisera raised against purified ActA revealed that ActA was associated with the cell wall and exposed on the surface of the bacteria, readily accessible to ActA antibodies. In contrast, a C-terminally truncated ActA1 polypeptide expressed by the isogenic actA1 mutant was detected only in the supernatant fluids. Immunofluorescence microscopy and electron microscopic studies using immunogold labeling showed that ActA was present on the surface of the bacteria infecting PtK2 and J774 cells at all stages of the infection cycle and was not found to be associated with the actin "tail" of individual bacteria. For the isogenic actA1 mutant strain, which grew as microcolonies within infected cells, only diffuse staining of the secreted ActA1 polypeptide in the host cytoplasm was observed. The ActA polypeptide therefore appears to be required in the initiation of actin accumulation by the bacterium and is apparently not directly involved in the generation of the actin tail. Analysis of strains of several L. monocytogenes serotypes indicated microheterogeneity in the molecular weights of the ActA polypeptides of individual strains and led to the detection of a serotype 3a strain that does not produce ActA.

Cell culture models for lead toxicity in neuronal and glial cells

Abstract: Two goals of lead (Pb) neurotoxicity research are to identify molecular and cellular alterations that underlie behavioral deficits and to define mechanisms of Pb uptake and tolerance in cells that accumulate Pb. Cell and tissue cultures are practical tools with which to pursue these goals, offering such advantages over in vivo methods as defined cell types, an extracellular environment that can be precisely manipulated, and direct observation. On the other hand, toxicity studies with cultured cells also present new challenges of design and interpretation. If a living vertebrate is like an orchestra playing a Beethoven symphony, then tissue culture is like two of the violinists playing their part alone. Historically, Pb toxicity studies with cell and tissue culture can be divided into an exploratory phase, an expansion phase, and a newly emerging intensification phase. In the exploratory phase, gross cytotoxic effects from massive Pb exposure (50-500 microM) were characterized. The collective data suggest differential sensitivity to Pb toxicity among various types of cultured neural cells, ranked as follows from most to least sensitive: myelinating cells, neurons, and astroglia. In addition, astroglia were shown to take up and store large amounts of Pb intracellularly, a phenomenon resembling the Pb-sequestering ability hypothesized for mature astroglia in vivo. The mechanisms of Pb entry may involve an anion exchanger, Ca2+ channels, or some other transport process. Three ingrained problems concerning the use of cell cultures began to emerge: appropriate dose regimens, biologically relevant forms of Pb (i.e. ionized or complexed with other molecules), and suitable measurements of Pb effects. These problems received scrutiny in the expansion phase, during which subcellular targets of Pb-induced damage were examined, specifically membranes, enzymes, and Ca-mediated cellular processes. Investigators attempted to define a biologically relevant dose regimen in vitro, as well as a threshold dose below which Pb had no biological effect. Effects of Pb at nanomolar concentrations in intact cells and tissue homogenates stimulated the metamorphosis of Pb toxicity studies in cell culture into a new phase, the intensification phase. Alterations in discrete molecular targets, particularly those effects in the cell that may be metabolically amplified, will be a major focus of this phase. Critical molecular targets for Pb-induced injury appear to be present during neuritogenesis and/or synaptogenesis. With the availability of cell culture models for neurite extension and synapse formation, this area may be another focus for innovative Pb neurotoxicity research.(ABSTRACT TRUNCATED AT 400 WORDS)

Trigeminal ganglion neurons affect corneal epithelial phenotype. Influence on type VII collagen expression in vitro

Abstract: Purpose To examine whether trigeminal ganglion (TG) neurons in tissue culture influence expression of Type VII collagen (a major component of the anchoring fibrils involved in the attachment of the epithelium to the underlying stroma) by cultured corneal epithelial cells. Methods A two-chambered coculture system was used. Fetal rabbit TG neurons were cultured into a central chamber on collagen- or laminin-coated tissue culture dishes. After good neurite outgrowth (average 7 days), rabbit corneal epithelial explants were placed into the outer chamber. Once neurite-epithelial cell interaction occurred, the cultures were immunostained for Type VII collagen. Direct coculture of TG neurons onto confluent passaged rabbit corneal epithelium also was studied. Results Neurites in contact with the epithelial cells in the outer chamber formed branching complexes, but staining for Type VII collagen was negative. In cocultures of TG neurons onto confluent passaged rabbit corneal epithelium, there was extensive neurite branching on and around the epithelial cells within a week. Scattered epithelial cells, many in clusters, were found to express Type VII collagen, as determined by immunofluorescence staining. Conclusions Based on the finding from this study that TG neurons influence production of Type VII collagen by rabbit corneal epithelium in vitro, it is likely that TG neurons influence corneal epithelial phenotypic characteristics that are critical to the maintenance of healthy epithelium in vivo.

Exogenous IL-7 promotes the growth of CD3-CD4-CD8-CD44+CD25+/- precursor cells and blocks the differentiation pathway of TCR-alpha beta cells in fetal thymus organ culture.

Abstract: Addition of human rIL-7 to fetal thymic organ culture started at day 13, 14, or 15 did not influence the number of cells generated during a 12-day culture period. However, the IL-7 treatment resulted in a preferential expansion of cells with a phenotype characteristic for cells at an early step of differentiation. The cells were CD4-CD8-CD3-CD2- and SCA-1+. Analysis of the coordinate expression of CD44 and CD25 on these cells showed that the majority of the cells were either CD44+CD25- or CD44+CD25intermediate. TCR-alpha beta cells were present but in a significantly lower number as compared to the control cultures. The cell number of TCR-gamma delta cells was increased. All these effects were moderate after 6 days, but unequivocal after 12 days of culture. Treatment of the fetal organ culture with mAb-neutralizing murine IL-7 resulted in an inhibition of the proliferation of the fetal thymocytes. No particular subset studied was preferentially inhibited. By using a model of reconstitution of 14-day embryonic thymuses depleted of thymocytes by deoxyguanosine and reconstituted with fetal day 13 liver cells and set up in organ culture with or without IL-7, it was shown in a clear cut way that IL-7 indeed promotes expansion of the early precursor cells and TCR-gamma delta cells, but prevents the generation of TCR-alpha beta cells. In addition, reconstitution experiments were set up in the presence of mAb-neutralizing murine IL-7. This treatment resulted in the inhibition of the growth of the fetal thymocytes without inhibiting preferentially a particular subset. These data indicate that IL-7 acts at an early step of T cell differentiation and plays a role to expand precursor cells, but prevents this population from additional differentiation towards the TCR-alpha beta pathways, whereas the differentiation towards TCR-gamma delta cells is not influenced or even enhanced.

Autocrine control of wound repair by insulin-like growth factor I in cultured endothelial cells.

Abstract: The repair process of the vascular endothelium is modulated by growth factors from both endogenous (within the vessel wall) and exogenous (blood borne) sources. We utilized a tissue culture model of endothelial wounding to gain further insight into the potential autocrine control of proliferation during wound repair. Cultured porcine aortic endothelial monolayers were mechanically wounded by passing a 7-mm sterile glass rod over the surface of the culture. Proliferation at the wound edge was quantified using [3H]thymidine autoradiography. In wounded cultures incubated in media supplemented with 10% fetal calf serum, 81 +/- 2% of the nuclei at the wound edge were labeled. When the cultures were incubated in serum-free media, proliferation at the wound edge was only slightly diminished with 65 +/- 3% (P < 0.05) of the cells labeled. These findings raise the possibility that there is a significant contribution from autocrine growth factors to endothelial wound repair. To evaluate the potential role of insulin-like growth factor I (IGF-I) in the wound repair process, we used a radioimmunoassay to measure IGF-I secretion. Wounded cultures exhibited a 187 +/- 58% increase in IGF-I production when compared with nonwounded cultures (P < 0.05). To determine the extent to which endogenous IGF-I mediates the proliferative response of endothelial cell monolayers to wounding, wounded cultures were incubated with inactivating concentrations of IGF-I antibody. When IGF-I antibody was present in the culture media, only 26 +/- 3% of the nuclei at the wound edge were labeled with [3H]thymidine (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Jasmonic acid stimulates shoot and bulb formation of garlic in vitro

Abstract: Although much information is available concerning the involvement of jasmonates in the regulation of plant development, few reports are devoted to their effects in tissue culture. In the present study, the influence of jasmonic acid (JA) on shoot and bulb formation in tissue culture of garlic (Allium sativum cv. Ptuj) was studied. Isolated basal plates were placed onto Gamborg's B5 medium. JA significantly enhanced the shoot and bulb development in concentrations from 1–10 μM. When the combination of 10 μM JA and 5 μM 2-iP was used in the initiation media, the average number of shoots was 30 after 6 weeks of culture. The bulbs formed on approximately 50% of shoots. Results described in this article show that JA might play an important role in the formation of storage organs in plants, in this case on garlic bulbs.

Bioactive material template for in vitro synthesis of bone tissue

Abstract: Novel non-crystalline, porous bioactive glass and ceramic materials that permit the in vitro formation of bone tissue when exposed to a tissue culture medium and inoculated with cells are disclosed. The present invention also discloses methods of treating bioactive glass materials to control pH so that when the glass is exposed to a tissue culture medium and then inoculated with cells, bone tissue growth occurs in vitro. The glass material disclosed is preferably formed from SiO2, CaO, Na2O and P2O5 and the porous, non-crystalline structure is most preferably created by melting the constituents, cooling and pulverizing the resulting glass, and then forming and hot pressing the powder. The glass of the present invention may be formed to produce templates that are useful for various indications, as well as granules that may be formed into a paste.

In vitro Organogenesis and Somatic Embryogenesis: Physiological and Biochemical Aspects

Abstract: The study of plant morphogenesis is one area of research with which tissue culture long has been associated, and one in which the use of the in vitro technology has made significant contributions to both fundamental knowledge and application (Thorpe, 1990). In particular, our understanding of the processes of xylogenesis (cytodifferentiation), somatic organogenesis and somatic embryogenesis has been clearly enhanced through use of tissue cultures. These processes enable cells and tissues, which are quiescent or committed to some function or pathway of development to be channelled into some different aspect of organized de novo development.