Assessment of host defence mechanisms induced by Candida species.

Some species of Candida are opportunistic pathogens that can cause disease in a host immunocompromised by underlying local or systemic pathological processes. C. albicans is the species most often associated with oral lesions, but other species of Candida, including C. glabrata, C. tropicalis and C. parapsilosis, have also been isolated in the saliva of subjects with and without candidiasis. In the present study we evaluated the host defence mechanisms induced by Candida albicans and other Candida species in monocytes and oral epithelial cells in order to establish the existence of a species-specific cellular response. Our results indicated that, during Candida species infection, the epithelial cells actively participate in the host defence by producing antimicrobial peptides and proinflammatory cytokines. Moreover, in infections caused by Candida tropicalis and Candida glabrata, the host defence may be strengthened by the release of perforin and granzyme by polymorphonuclear leukocytes recruited at the site of infection.

3-O-Methylfunicone, a metabolite produced by Penicillium pinophilum, modulates ERK1/2 activity, affecting cell motility of human mesothelioma cells.

OBJECTIVES: 3-O-methylfunicone (OMF), a secondary metabolite produced by Penicillium pinophilum, affects cell proliferation and motility in a variety of human solid tumours. The aim of this study was to demonstrate whether OMF has the ability to arrest cell division and motility, in a human mesothelioma cell line. Malignant mesothelioma is an aggressive cancer that does not respond to standard therapies the cells of which are considered to be highly resistant to apoptosis. MATERIAL AND METHODS: Cell motility and invasion were measured using a modified Boyden chamber. Gene expression was examined by RT-PCR, while ERK1/2 was investigated by Western blot analysis. All experiments were also performed on primary cultures of mesothelial cells. RESULTS: The present study shows that OMF inhibited motility of the NCI mesothelioma cell line by modulating ERK signalling activity, and affected alphaVbeta5 integrin and MMP-2 expression, inducing marked downregulation at both mRNA and protein levels. Substantial downregulation of VEGF gene expression was also demonstrated. These effects were not observed in normal mesothelial cell cultures. CONCLUSION: OMF may have potential as a naturally derived anti-tumour drug for treatment of mesothelioma.

Effect of temperature on the shift of Pseudomonas fluorescens from an environmental microorganism to a potential human pathogen.

Pseudomonas fluorescens is a Gram-negative bacterium generally considered of scarce clinical significance. However, in the last few years, the isolation of P. fluorescens as the causative agent of nosocomial infections has rapidly increased. P. fluorescens is a psychrophile microorganism which grows at an optimal temperature of 25-30 degrees Celcius. In spite of this constraint, it has recently been reported that the human physiological temperature does not appear to be a barrier for this microorganism. In this study we examined the ability of P. fluorescens, grown at 28 degrees C or at 37 degrees C, to adhere to cultured human A549 pulmonary cells and to form biofilm. The ability of P. fluorescens to induce expression of proinflammatory cytokines, beta-defensin 2 and the intercellular adhesion molecule-1 was also investigated. Our results clearly indicate that inflammatory mediators are induced when the microorganism is grown at a lower temperature, while biofilm is formed only at 37 degrees C. The results presented are consistent with previous reports indicating P. fluorescens as an opportunistic pathogen and underscore the urgent need for further studies to better characterize the virulence of this microorganism.

Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

Pemphigus serum and captopril induce heat shock protein 70 and inducible nitric oxide synthase overexpression, triggering apoptosis in human keratinocytes.

BACKGROUND: Captopril is an angiotensin-converting enzyme inhibitor with sulphydryl groups in its chemical structure. It is commonly used as an antihypertensive drug. The occurrence of pemphigus vulgaris has repeatedly been reported in patients receiving captopril. The capacity of captopril and pemphigus serum to induce acantholysis, in vivo or in vitro, has been demonstrated experimentally. OBJECTIVES: To show that captopril and pemphigus serum, acting by a biochemical and immunological mechanism, respectively, trigger apoptosis. METHODS: Human keratinocyte cells were treated with 15 mmol L-1 captopril or with pemphigus serum. DNA was extracted and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method was used to detect apoptosis. RESULTS: DNA fragmentation occurred after 72 h of treatment. Increased expression of p53, c-myc and inducible nitric oxide (NO) synthase (iNOS) mRNA were observed by polymerase chain reaction (PCR) in the treated cells compared with the untreated ones. The increase in iNOS gene expression was associated with overproduction of NO. Moreover, the addition of 1 mmol L-1N-monomethyl-L-arginine, a structural analogue of arginine, reduced nitrite levels by about 70% in cells treated with captopril or pemphigus serum. Western blot analysis revealed an overexpression of heat shock protein 70 (hsp70) in cells treated with captopril or pemphigus serum. Finally, total inhibition of the keratinocyte transglutaminase gene was shown by PCR analysis in the same samples, compared with control cells. CONCLUSIONS: These data demonstrate the involvement of apoptosis in keratinocytes treated with captopril or pemphigus serum, with induction of the iNOS gene and hsp70 in the cascade of events leading to programmed cell death.

Early vitronectin receptor downregulation in a melanoma cell line during all-trans retinoic acid-induced apoptosis.

BACKGROUND: Recent evidence assigns the vitronectin receptors (VnRs) an important role in regulating tumour cell invasion and dissemination. In vivo and in vitro studies document that all trans-retinoid acids (ATRAs) inhibit growth-inducing apoptosis in melanomas. OBJECTIVES: We have analysed the effects of ATRA treatment on melanoma cell adhesion and motility. METHODS: Human M14 melanoma cells were treated with 10 micromol L-1 ATRA for different times and stained with rhodamine-phalloidin to analyse the effect of treatment on cytoskeleton organization. Cell adhesion and cell migration assays were performed to analyse the role of VnRs in the ATRA-induced early stages of apoptosis. VnR expression was evaluated by Western blot, immunoprecipitation and immunocytochemistry assays. RESULTS: First, using an annexin V assay, we found that apoptosis was triggered by 48 h with 10 micromol L-1 ATRA exposure. At this time point, decrease in the F-actin polymerization as well as inhibition of cell adhesive ability to vitronectin (Vn) was exerted by ATRA treatment. In the presence of serum, exposure to 10 micromol L-1 ATRA for 48 h produced a dramatic inhibition of the cell adhesion ability that was comparable with that exerted by untreated cells preincubated with anti-alpha(v)beta(3) or anti-alpha(v)beta(5) VnR monoclonal antibodies. Functionally, the treatment of melanoma cells with 10 micromol L-1 ATRA for 48 h causes an inhibition of directional cell migration towards Vn-coated filters. Therefore, we analysed the effect of ATRA on the VnR expression. Both alpha(v)beta(3) and alpha(v)beta(5) VnR levels were reduced upon exposure to 10 micromol L-1 ATRA for 48 h as shown by Western blot, immunoprecipitation and immunocytochemistry assays. CONCLUSIONS: Altogether, our data indicate that treatment of M14 melanoma cells with ATRA downregulates VnR expression and that this reduction is closely correlated with the ATRA-dependent inhibition of actin-fibre organization, cell adhesion and migration. Although the mechanism by which ATRA regulates the expression of VnR in M14 melanoma cells needs further elucidation, this system may represent a model for understanding the molecular basis of ATRA therapy in melanoma.

Immunomodulatory effects of peptide T on human keratinocyte cells.

BACKGROUND: Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. PT is from the V2 region of HIV-1 gp120, an exterior envelope glycoprotein that is a target for host immune responses. The anti-inflammatory mechanisms of PT are not well understood. OBJECTIVES: We studied the immunomodulatory effects of PT on the human keratinocyte cells. METHODS: Cultured human keratinocytes were treated with PT, proteins extracted and analysed by Western blotting and reverse transcriptase polymerase chain reaction. RESULTS: Our findings show reduced expression of intercellular adhesion molecule 1 and an increase in transforming growth factor (TGF)-beta and heat shock protein (HSP)-70 in human keratinocyte cells treated with PT. The HSP-70 increase is modulated by TGF-beta. In fact, we demonstrated that anti-TGF-beta antibodies reduce HSP-70 overexpression. In addition, we show a modulation of alphav integrins after 4 hours of treatment with PT. These receptors favour keratinocyte migration and epidermal regeneration. It has been reported that overexpression of HSP results in dramatic changes to intermediate filaments. These proteins act on keratin intermediate filaments and determine their retraction. The consequence is cell-cell contact detachment and inhibition of cellular hyperproliferation. CONCLUSIONS: Our results support the beneficial effect of PT found in vivo, suggesting, moreover, the primary role of keratinocytes upon which PT acts directly by stimulating the anti-inflammatory function and favouring the regeneration of tissue.

Uptake of Malassezia furfur by human dermal fibroblasts: effect of ketoconazole and cytoskeleton inhibitors.

We showed the ability of human dermal fibroblasts to take up Malassezia furfur and the effect of ketoconazole and cytoskeleton inhibitors, including cytochalasin D and colchicine, on invasivity. Engulfment was evaluated by May Grunwald Giemsa stain and confirmed by acridine orange staining and electron microscopy. Both revealed the different steps of engulfment, including a fusion event between lysosomes and phagosomes containing M. furfur. Subinhibitory concentrations of ketoconazole (5 microg/ml) reduced the invasive capacity compared to controls (52.0+/-6.3 vs 10.0+/-1.2). M. furfur induced changes in the cytoskeleton of human dermal fibroblasts, with signs of disaggregation of actin fibres. We also studied the effect of the cytoskeleton inhibitors, cytochalasin D (1 microg/ml) and colchicine (1 microg/ml), on engulfment. Cytochalasin D, an inhibitor of actin polymers, inhibited the uptake of M. furfur by human dermal fibroblasts. Colchicine, a microtubule inhibitor, reduced the uptake of M. furfur less markedly. This suggests that the process of engulfment is F-actin-dependent, but the integrity of microtubules is also important in "non-professional" phagocytic cells such as dermal fibroblasts.

Malassezia furfur invasiveness in a keratinocyte cell line (HaCat): effects on cytoskeleton and on adhesion molecule and cytokine expression.

The lipophilic yeast Malassezia furfur is a member of the cutaneous microbiota, also associated with several chronic diseases such as pityriasis versicolor, folliculitis, seborrhoeic dermatitis, and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. In this study we determined the immunomodulatory and invasive capacity of M. furfur in a human keratinocyte cell culture, HaCat. At a yeast cell to HaCat ratio of 30:1, M. furfur penetration was only 30% with poor phagolysosome fusion and with cytoskeleton modification. Transglutaminase I gene expression was also inhibited, supporting the hypothesis that M. furfur causes an initial break in the barrier function of the epidermis. Moreover, we demonstrated that M. furfur modulates proinflammatory and immunomodulatory cytokine synthesis by downregulating IL-1alpha and by inhibiting IL-6 and TNF-alpha and by upregulating IL-10 and TGF-beta1. The suppressed inflammatory response induced by M. furfur may play a role in chronic disease.

Histopathological features and modulation of type IV collagen expression induced by Pseudomonas aeruginosa lipopolysaccharide (LPS) and porins on mouse skin.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) is responsible for serious infections in the immunocompromised host. Many skin lesions induced by P. aeruginosa have been described. Few investigations have been performed on the local action of P. aeruginosa components. OBJECTIVES: To shed light on the "in vivo" activity of lipopolysaccharide (LPS) and porins extracted from P. aeruginosa, by verifying their effects after inoculation in mouse skin through the observation of histological changes and immunohistochemical expression of collagen IV. RESULTS: Both substances were able to induce a similar inflammatory process and a characteristic reversible change in collagen IV distribution. Interestingly, a fibroblast increase was observed at 24 h in the skin treated with porins, while it appeared later in the skin treated with LPS. Besides these changes, porins particularly increased collagen edema, together with disgregation of hypodermal structures. Moreover "in vitro", porins were able to stimulate fibloblasts 3T3 to convert 72 kDa type IV collagenase into the activated 62 kDa form and to release the 92 kDa collagenase. CONCLUSION: LPS and porins, released by gram-negative bacteria during cell growth and lysis, interact with the host at target cells, such as keratinocytes, fibroblasts and immunocompetent cells, thus contributing significantly to the pathogenesis of P aeruginosa skin infections.

A half life: women caregivers of older disabled relatives.

This paper explores the impact of caring on caregivers' life style and health. Interviews were carried out with women caregivers over 50 years old. Transcripts from the interview are analyzed through a detailed discourse analysis within an ethnomethodological framework showing how the caring tasks are constructed in the interaction as a women's duty, inscribed in a gendered moral order. A difference is noticeable between family caregivers who describe the caring tasks as the cause of intense stress, physical and psychological problems and voluntary or paid caregivers who see caring as a source of satisfaction and well being.

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Expression of teratocarcinoma-derived growth factor-1 (TDGF-1) in testis germ cell tumors and its effects on growth and differentiation of embryonal carcinoma cell line NTERA2/D1.

The teratocarcinoma-derived growth factor-1 (TDGF-1) gene codes for a 188-aminoacid glycoprotein that shares structural homology with the epidermal growth factor (EGF) family of growth factors. TDGF-1 is highly expressed in the undifferentiated embryonal carcinoma stem cell line NTERA2 clone D1 (NT2/D1) and its expression is downregulated in response to differentiating agents such as retinoic acid (RA) and hexamethylen-bisacetamide (HMBA). To assess the role of TDGF-1 in the onset and/or progression of human germ cell tumors, we analysed TDGF-1 expression by Northern blot and immunostaining in a panel of 59 human germ cell tumors of different histological origins. We show that TDGF-1 expression is markedly elevated in a subset of human testicular germ cell tumors as compared to normal testes. TDGF-1 overexpression occurs in about 100% of tumors with non-seminomatous phenotype, such as embryonal carcinomas and malignant undifferentiated teratocarcinomas. To address the questions of how TDGF-1 (previously called CRIPTO) may affect the growth and/or the differentiation of embryonal carcinoma cells, we have characterized the effects of exogenous recombinant TDGF-1 protein on the proliferation rate and differentiation 'potential of NT2/D1. Exogenous TDGF-1 protein stimulated DNA synthesis and cell proliferation in both undifferentiated and differentiated NT2/D1 cells. However, TDGF-1 protein treatment was unable to block differentiation induced by both RA and HMBA. These results suggest that TDGF-1 growth factor may represent an autocrine growth factor that may be involved in the process of development of testicular neoplasms.

Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic.

The placental-derived growth factor (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial growth factor. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic 21-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this factor functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial growth factor and basic fibroblast growth factor under the same conditions and at the same concentration. PIGF-1 was shown to induce cell growth and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial growth factor and basic fibroblast growth factor on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.

Two periods of sensitivity to mutagens in induced Mel cells with different outcomes.

Mouse erythroleukemia (Mel) cells are particularly sensitive to mutagenic agents between 18 and 24 h from the start of induction (Foresti, M.L. Gaudio, G. Geraci and P. Manduca (1986) Inhibition of dimethyl sulfoxide induced erythropoietic differentiation of murine erythroleukemia cells in culture. Cancer Res., 46, 6260-6263). We show here the occurrence of another period of sensitivity during the initial 5 h after the addition of the inducer dimethyl sulfoxide (DMSO) to the culture medium. The sensitivity to the mutagenic action of a sublethal 3-s pulse of UV light (13.5 J/m2) was monitored on the progeny of the irradiated cells at day 5 after the start of induction. The effects were analysed on functions strictly linked to the final expression of the differentiated phenotype: hemoglobin concentration, percent cells producing hemoglobin (%B+), activity of delta-amino levulinic acid dehydrase (ALA-DH) and presence of globins. Each function appeared differently and selectively affected in the progeny of the cells depending on the exact time of irradiation during the period of sensitivity Specifically, cells irradiated at hour 3 after induction show both hemoglobin concentration and ALA-DH activity values increased by a factor 3 over controls. Cells irradiated at hour 5 show an almost complete halt in cell induction and the other tested functions show minimal values. Cells are nearly insensitive to irradiation at later times, until hour 20, after which a second period of sensitivity with peak value at hour 22 occurs at which time hemoglobin concentration in the progeny of irradiated cells is increased by a factor 3 over controls, ALA-DH activity is increased by a factor 15 while percent B+ value is at its minimum. The differential effects of UV irradiation on Mel cell functions in the first and in the second period of sensitivity to mutagens confirm the hypothesis that the consequences of a mutational event are strictly dependent on the functional state of the cell. The 1-5 h period of sensitivity in which Mel cells fix the effects of the mutagen in their genome corresponds to increased thymidine incorporation not correlated with cell duplication.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

Inhibition of erythroid differentiation in MEL cells by UV irradiation. Cell cycle and DNA repair activity.

Irradiation with a 3-s pulse of 254 nm UV light has been used to study sensitivity to mutagenic agents of mouse erythroleukemia (MEL) cell cultures in correlation with the cell cycle. A dose of UV irradiation was chosen that had no consequences for cell viability and growth. For this reason phenotypic effects were monitored on the progeny of all cells of the irradiated cultures by scoring those unable to undergo erythroid differentiation upon induction with dimethyl sulfoxide. The very short period of irradiation made it possible to show that MEL cells, synchronized by two sequential blocks of deoxythymidine and one of hydroxyurea (HU), are sensitive to UV irradiation only in a very short period of time at about 60 min after release from HU block. Determinations of deoxythymidine incorporation into DNA show that this time period corresponds only marginally to the initial part of the S phase during which irradiation has no consequences for cell properties. Cells are not sensitive to UV irradiation in G1 and in G2/M unless, immediately after irradiation and for the following 2 h, cultures are treated with 1 mM HU to interfere with DNA repair. Alkaline sucrose gradient analyses show at all tested times that irradiation leads to fragmentation of cell DNA. The data suggest that an immediate increase of deoxythymidine incorporation into DNA following irradiation is not necessary for the efficient repair of damaged DNA. In fact, the percent of cells expressing the erythroid phenotype is normal in the progeny of cells irradiated in G2/M, when TdR incorporation is at a minimum. Repair activities appear then to be mechanistically divided into two phases, (1) recognition labeling of the altered sites and (2) reconstitution of the DNA sequences. The first activity appears to be operative at all phases of the cycle, the second activity is little or not operative in G2/M, possibly delayed to the following G1 period.