Neurotrophic regulation of epidermal dedifferentiation during wound healing and limb regeneration in the axolotl (Ambystoma mexicanum).

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.

Antibiotic-resistant Escherichia coli in wastewaters, surface waters, and oysters from an urban riverine system.

The antibiotic resistance (AR) patterns of 462 Escherichia coli isolates from wastewater, surface waters, and oysters were determined. Rates of AR and multiple-AR among isolates from surface water sites adjacent to wastewater treatment plant (WWTP) discharge sites were significantly higher (P < 0.05) than those among other isolates, whereas the rate of AR among isolates from oysters exposed to WWTP discharges was low (<10%).

Proinflammatory cytokine production by human keratinocytes stimulated with Propionibacterium acnes and P. acnes GroEL.

BACKGROUND: Keratinocytes form the first line of defence in the skin and alert the host to danger by the production of a number of cytokines and chemokines. However, the interaction of commensal microorganisms with keratinocytes has not been well studied. OBJECTIVES: To investigate the effect of viable and nonviable cells of Propionibacterium acnes in both exponential and stationary growth phases, and of P. acnes GroEL on cytokine production by human primary keratinocytes. METHODS: Actively proliferating or contact-inhibited keratinocytes were cocultured with viable or formaldehyde-killed P. acnes cells in either the exponential or stationary phase of growth. Culture supernatants were assayed by enzyme-linked immunosorbent assay for the cytokines interleukin (IL)-1alpha, tumour necrosis factor (TNF)-alpha and granulocyte/macrophage colony-stimulating factor (GM-CSF). Keratinocytes were also stimulated with different concentrations of P. acnes GroEL and supernatants assayed for cytokines. RESULTS: Viable P. acnes in the stationary phase of growth stimulated keratinocyte monolayers to produce significantly higher amounts of IL-1alpha, TNF-alpha and GM-CSF than unstimulated keratinocytes. Viable exponential-phase bacteria stimulated production of significantly higher amounts of TNF-alpha and GM-CSF but these levels were significantly lower than those for stimulation with stationary-phase bacteria. Nonviable P. acnes from either growth phase was not able to stimulate cytokine production. P. acnes GroEL at concentrations in the range 0.05-1.0 micro g mL(-1) was able to induce increased production of cytokines by keratinocytes in a dose-dependent manner. This was analogous to stimulation with Escherichia coli GroEL. CONCLUSIONS: Stimulation of cytokine production by P. acnes and P. acnes GroEL may be important in the pathogenesis of inflammatory acne vulgaris and may have wider implications for the immunomodulation of the human immune system by commensal skin microorganisms.

Modulation of the antigenic phenotype of human melanoma cells by differentiation-inducing and growth-suppressing agents.

Tumor cells often display alterations in their normal program of cellular differentiation. A promising approach for the treatment of cancer involves the induction of terminal differentiation and a loss of proliferative capacity in cancer cells. In human melanoma cells, the combination of mezerein (MEZ) and fibroblast interferon (IFN-beta), results in a rapid and irreversible suppression of cell growth with a concomitant increase in the synthesis of melanin. The induction of terminal differentiation is associated with alterations in the expression of several cellular genes, including fibronectin, ISG-15 and ISG-54, and changes in the expression of specific cell surface antigens, including intercellular adhesion molecule-1 (ICAM-1) and HLA Class I antigens. In the HO-1 human melanoma cell line, induction of terminal differentiation by MEZ plus IFN-beta results in an induction and/or increased expression of ICAM-1, HLA Class I antigens and HLA Class II antigens. IFN-beta and MEZ alone can modulate expression of these antigens to a lower extent than does the combination of compounds. Induction of terminal differentiation and the irreversible suppression of cell growth is not a prerequisite for antigenic modulation in HO-1 cells. This is indicated by the inability of immune interferon (IFN-gamma), a strong inducer of ICAM-1, HLA Class I antigens and HLA Class II antigens synthesis, or the combination of IFN-beta plus IFN-gamma which synergistically but reversibly suppresses HO-1 growth, to induce melanin synthesis or terminal differentiation in HO-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of recombinant fibroblast interferon and recombinant immune interferon on growth and the antigenic phenotype of multidrug-resistant human glioblastoma multiforme cells.

To study the effect of drug resistance on the response of stage IV astrocytomas to interferon, a human glioblastoma multiforme cell line, GBM-18, was transfected with an expression-vector plasmid containing a human multidrug resistance (MDR) gene (pHaMDR1/A), and clones surviving in colchicine were isolated. GBM-18 multidrug-resistant subclones displayed cross-resistance to other chemotherapeutic agents, including vincristine, doxorubicin, and dactinomycin. The multidrug-resistant phenotype was reversible when GBM-18 multidrug-resistant cells were cultured in colchicine and the calcium-channel blocker verapamil. The level of the MDR1 gene (also known as PGY1) message was increased in GBM-18 multidrug-resistant cells selected for increased resistance to colchicine, and this effect was not correlated with an amplification of the MDR1 gene. In both parental GBM-18 and GBM-18 multidrug-resistant cells, growth was suppressed to a greater degree when cultures were treated with the combination of fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma). Parental cells and multidrug-resistant subclones varied in their de novo and/or interferon-modulated expression of HLA class I and class II antigens, a high-molecular-weight melanoma-associated antigen, and intercellular adhesion molecule 1 (ICAM-1). Of the antigens tested, ICAM-1 and HLA class I antigens were the most sensitive to enhanced expression induced by IFN-beta and IFN-gamma when used alone or in combination. The results of the present study indicate that multidrug-resistant human glioblastoma multiforme cells retain their increased sensitivity to the antiproliferative activity of the combination of IFN-beta plus IFN-gamma, and differences in antigenic phenotype are apparent in independent multidrug-resistant glioblastoma multiforme clones.

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons.

Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN beta) and immune (IFN gamma) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN beta than to IFN gamma and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN beta plus IFN gamma was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN beta and IFN gamma differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN beta plus IFN gamma can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN beta or IFN gamma, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.

Production of growth factors by type 5 adenovirus transformed rat embryo cells.

Transformation of Sprague-Dawley rat embryo (RE) cells and a cloned Fischer rat embryo cell line (CREF) with wild-type (Ad5) or a temperature-sensitive DNA-minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In contrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]-EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]-EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF-receptor binding and induces anchorage-independent growth of normal cells.