Component resolved diagnosis by recombinant allergens in patients with allergies to inhalants.

Molecular characterization of IgE reactivity of specific individual components of allergenic extracts is now possible due to the technology of recombinant allergens derived from studies of molecular biology of allergic pathology. The identification of the immunoreactivity to single allergenic components in allergic subjects allows to specifically define her/his allergic profile and obtain the so-termed Component Resolved Diagnosis (CRD). Molecular allergens can be classified into those that induce the respiratory allergic reactivity and those that identify the food-related allergic pathology. It is also essential to identify those molecular allergens whose immunoreactivity is able to connect the two clinical conditions: respiratory symptoms and food allergy symptoms. The present study was conducted on 50 patients with a clinical history of hypersensitivity to pollen and/or allergy and positivity to Skin Prick Test. The sera were analyzed in our laboratories and the panel of recombinant allergens was applied in the case of positivity of the specific IgE. Of the 50 patients enrolled, 31 were selected as positive to 4 main pan-allergen Bet v1, Par j2, Art v1 and Phl p1; among these, 14 subjects showed one allergen-specific IgE towards natural extracts of tested foods even in absence of clinical history. CRD allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in: a) resolving genuine vs cross-reactive sensitization in poly-sensitized patients, b) assessing, in selected cases, the risk of severe, systemic vs mild, local reactions in food allergy, and c) identifying patients and triggering allergens for specific immunotherapy (ITS). In light of our results, we believe that the transition from a diagnostic based on the use of allergenic extracts to another one based on the use of single allergenic molecules that is able to define the specific allergenic profile of each patient, seems to be able to revolutionize the allergy diagnosis.

Response of periodontium to mineral trioxide aggregate and Biodentine: a pilot histological study on humans.

BACKGROUND: The aim of this study was to investigate for the first time the histological response of human periodontium to mineral trioxide aggregate (MTA) and Biodentine. METHODS: Six patients scheduled for implant full-arch rehabilitation were randomly assigned to one of the two test groups: MTA or Biodentine treatment. For each patient, two teeth scheduled for strategic extraction were randomly assigned either to the test or to the control treatment. A lateral perforation was drilled on the root and either repaired with MTA/Biodentine or filled with gutta-percha(control). Three months later, the teeth were extracted along with the coronal third of the alveolar bone and a portion of gingival tissue, while performing implant placement, and processed for histological analysis. RESULTS: Biodentine resulted in less extrusion into the periodontal environment. All the materials showed good biocompatibility. A new mineralized cementum-like tissue incorporating periodontal fibres was visible in all cases treated with MTA. A small amount of new mineralized tissue was found in two Biodentine cases but not in control cases. Biodentine resulted in less damage to the periodontal ligament. CONCLUSIONS: Bioactivity and biocompatibility of MTA were confirmed in human models. Biodentine proved to be biocompatible, but it seems not to induce cementum regeneration.

Human cardiac multipotent adult stem cells in 3D matrix: new approach of tissue engineering in cardiac regeneration post-infarction.

Myocardial infarction is the leading cause of morbidity and mortality in developed countries. It causes a left ventricular dysfunction, mainly due to the loss of functional tissue, resulting in heart failure. New therapies are being developed, using a tissue engineering approach, with the ultimate goal of restoring cardiac function by regenerating and repairing the damaged myocardium. In the present study we investigated the behaviour of a specific population of c-kit positive human cardiac stem cells, called Multipotent Adult Stem Cells (MASCs), grown within three-dimensional collagen scaffolds (3D), to establish whether they could be used in post-infarction cardiac regeneration. We also evaluated the expression levels of the Granulocyte Macrophage-Colony Stimulating Factor Receptor (GM-CSFR) and endoglin, a component of the Transforming Growth Factor beta (TGF-ß) receptor complex. Finally, we also evaluated the expression of the α2ß1integrin. MASCs cultured within 3D collagen matrices are able to proliferate and migrate even in the absence of chemotactic agents and express high levels of factors involved in cell proliferation and migration, such as GM-CSFRα chain and integrins. They therefore represent a promising approach to tissue engineering aimed to restore cardiac function. Our results also suggest a role of GM-CSF in cell proliferation, while TGF-ß does not seem to be relevant.

Vitamin D receptor polymorphisms as tool for early screening of severe bone loss in women patients with rheumatoid arthritis.

OBJECTIVE: Rheumatoid Arthritis (RA) is an autoimmune inflammatory disease that leads to local and systemic arthritis and bone loss. Exploring genetic markers of candidate genes in osteoporosis and inflammatory cytokine genes could be a useful tool for the early identification of bone loss and fracture risk in RA patients. The target of this study is the evaluation and correlation between of Single Nucleotide Polymorphisms (SNPs) of Vitamin D Receptor (VDR) and possible effects on bone loss in RA. PATIENTS AND METHODS: 40 Caucasian patients with RA (26 of them with a severe bone loss) and 40 healthy donors as control samples were genotyped for the VDR SNPs (called BsmI, ApaI, TaqI and FokI). The detection method is based on Restriction Fragment Length Polymorphism (RFLP). RESULTS: Genotyping profile shown no difference between RA patients and controls. Only VDR-TaqI genotype (TT vs. tt) seem to influence the bone density in females, but not in males. The mean differences of Bone Mass Density (BMD) at the lumbar spine in RA women with the tt allele were 4.7% compared to 0.1% in women with the TT allele (p < 0.05). CONCLUSIONS: The results of these studies support an association between specific VDR alleles and bone loss in RA. The TaqI t and BsmI B alleles were associated with an accelerated bone loss in RA, but not with a focal bone loss. These effects of VDR genotypes and vitamin D supplementation are not unexpected, given that the central pathological feature in RA is bone and joint destruction. The VDR SNPs genotyping should be a useful tool to screen early women RA patients with the bone loss.

The plasminogen activator system in fibroblasts from systemic sclerosis.

Systemic sclerosis (SSc) is characterized by excessive fibrosis throughout the body. There are two major subsets of SSc, diffuse cutaneous Systemic sclerosis (dSSc) and limited cutaneous Systemic sclerosis (lSSc). Fibroblasts play a key role in SSc. The expression and function of the urokinase (uPA)-mediated plasminogen activation (PA) system, a well-characterized system of serine-proteases involved in several pathological processes, has been investigated in SSc fibroblasts. The expression of the components of the PA system, including uPA, its type-1 and type-2 inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), was examined by Western blot in fibroblasts from patients affected by limited and diffuse forms of SSc. uPA and PAI-1 secretion increased only in fibroblasts from lSSc lesions compared to normal fibroblasts. PAI-2 levels were decreased in fibroblasts from both SSc forms. Interestingly, fibroblasts from areas not adjacent to the lesions (not-affected) of the diffuse form showed reduced levels of PAI-1 and increased uPAR expression. Adhesion experiments showed reduced adherence to VN of fibroblasts from lSSc lesions and from non-affected areas of the diffuse form, as compared to normal controls. These results suggest a role for uPA and PAI-1 in the lSSc form, likely related to the activation of latent forms of cytokines and to the accumulation of ECM components, whereas a role for uPAR can be hypothesized in the evolvement of the diffuse form, based on its up-regulation in the non-affected areas.

Progressive topographical disorientation: a case of focal Alzheimer's disease.

We describe a follow-up study of a patient with a selective, progressive impairment of topographical orientation. The patient's topographical difficulties were evident only in unfamiliar surroundings at the beginning of the observation period but later on they were observed even at home. Serial neuropsychological tests demonstrated a progressive impairment of visuospatial abilities with sparing of the other cognitive domains; only at the last assessment, about six years after early disturbances and three years after the first evaluation, the patient developed the typical cognitive impairments of Alzheimer's disease (AD). This case represents a focal variant of AD not previously described and suggests that the neuronal pathways underlying spatial orientation may be selectively damaged by the degenerative process.

Ochratoxin A and zearalenone: a comparative study on genotoxic effects and cell death induced in bovine lymphocytes.

Ochratoxin A (OTA) and zearalenone (ZEA), two naturally occurring contaminants of animal feed, have been implicated in several mycotoxicoses in farm livestock but there is little information on their genotoxicity and toxicity in these species. Therefore, we investigated on the cytogenetic and cytotoxic effects of both OTA and ZEA in in vitro cultures of bovine lymphocytes. We determined chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as the mitotic index (MI) and cell viability following OTA and ZEA treatment. This report is the first to provide evidence of a statistically significant increase of structural CAs and of SCEs/cell associated with a reduction of the MI in all OTA- and ZEA-treated bovine lymphocyte cultures and a clear reproducible reducing effect of OTA on cell viability mediated by enhanced apoptosis. OTA-induced programmed cell death was not limited to bovine lymphocytes, as comparable data were demonstrated in the human leukemic T-cell line Jurkat.

Behavior of SaOS-2 cells cultured on different titanium surfaces.

Surface properties may affect the clinical outcome of titanium dental implants. The aim of the present study was to investigate the effects of 3 different titanium surfaces-smooth (S), sandblasted (SB), and titanium plasma-sprayed (TPS)-on proliferation, differentiation, and apoptosis of human osteoblast-like cells, SaOS-2. Cell proliferation was significantly (p < 0.05) higher on the S surface, and synthesis of extracellular matrix proteins was more abundant on TPS and SB than on S surfaces. Analysis of integrin receptors showed a higher expression of alpha2, alpha5, alphaVbeta3, and ss1 on TPS as compared with SB and S surfaces. An increase in alkaline phosphatase activity was detected only on SB and TPS surfaces. Analysis of cell apoptosis did not demonstrate any significant difference among the 3 different surfaces. The results indicate that titanium surface topography affects proliferation and differentiation of osteoblast-like SaOS-2 cells, suggesting that surface properties might be important for bone response around dental implants in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the osteoblastic differentiation of the human osteosarcoma cell line SaOS-2.

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

Inhibition of human breast cancer cell growth by blockade of the mevalonate-protein prenylation pathway is not prevented by overexpression of cyclin D1.

Overexpression of the cyclin D1 (CCND1) gene, encoding a downstream effector of mitogenic signals that plays a central role in G1 phase progression, is often found in cancerous cells. In sporadic breast cancer (BC), this is one of the most frequent and early genetic lesions identified so far, found in more than 50% of the tumors. Inhibitors of the mevalonate/protein prenylation pathway belong to a new family of cancer therapeutic agents that act by blocking intracellular mitogenic signal transduction pathways, thereby preventing expansion of pre-cancerous foci and inhibiting growth of transformed cells. It is not known at present whether constitutively high intracellular levels of cyclin D1 might interfere with the cytostatic actions of mevalonate/protein prenylation inhibitors. This possibility was investigated here by assessing the cell cycle effects of Simvastatin, a non-toxic upstream inhibitor of the mevalonate pathway, on human BC MCF-7 cells expressing either normal or enhanced levels of cyclin D1 from of a stably transfected, tet-inducible expression vector. Results show that constitutive overexpression of this protein, such as that found in sporadic BCs, does not influence the growth inhibitory effects of Simvastatin in vitro. In addition, D1-overexpressing embryo fibroblasts were also found to be responsive to the cell cycle effects of mevalonate/protein prenylation pathway blockade, further suggesting that high intracellular levels of cyclin D1 do not prevent the cytostatic actions of compounds targeting this metabolic pathway.

The antiestrogen ICI 182,780 inhibits proliferation of human breast cancer cells by interfering with multiple, sequential estrogen-regulated processes required for cell cycle completion.

Antiestrogens are widely used for breast cancer treatment, where they act primarily by inhibiting the mitogenic action of estrogens on tumor cells. The effects of the pure antiestrogen ICI 182,780 on estrogen-regulated cell cycle phase-specific events were investigated here in synchronously cycling human breast cancer (HBC) cells. In early G(1)-arrested MCF-7 or ZR-75.1 cells, 17beta-estradiol (E2) induces rapid activation of the cyclin/Cdk/pRb pathway, as demonstrated by D-type G(1) cyclins accumulation during the first few hours of hormonal stimulation, followed by sequential accumulation of E, A and B1 cyclins and progressive pRb phosphorylation, as cells progress through the cell cycle. When added to quiescent cells together with E2, ICI 182,780 prevents all of the above hormonal effects. Interestingly, in mid-G(1) cells (2-8 h into estrogen stimulation) the antiestrogen causes rapid reversal of hormone-induced D-type cyclins accumulation and pRb phosphorylation, and still fully inhibits G(1)-S transition rate, while in late-G(1) cells it does not prevent S phase entry but still inhibits significantly DNA synthesis rate, S-phase cyclins accumulation and pRb hyperphosphorylation. These results indicate that pure antiestrogens prevent multiple estrogen-induced cell cycle-regulatory events, each timed to allow efficient G(1) completion, G(1)-S transition, DNA synthesis and cell cycle completion.

Urokinase-type plasminogen activator up-regulates the expression of its cellular receptor.

The expression of the receptor for the urokinase-type plasminogen activator (uPAR) can be regulated by several hormones, cytokines, tumor promoters, etc. Recently, it has been reported that uPAR is capable of transducing signals, even though it is lacking a transmembrane domain and a cytoplasmatic tail. We now report that uPAR cell surface expression can be positively regulated by its ligand, uPA, in thyroid cells. The effect of uPA is independent of its proteolytic activity, since inactivated uPA or its aminoterminal fragment have the same effects of the active enzyme. The increase of uPAR on the cell surface correlates with an increase of specific uPAR mRNA. Finally, uPA up-regulates uPAR expression also in other cell lines of different type and origin, thus suggesting that the regulatory role of uPA on uPAR expression is not restricted to thyroid cells, but it occurs in different tissues, both normal and tumoral.

HLA class I antigen downregulation by interleukin (IL)-10 is predominantly governed by NK-kappaB in the short term and by TAP1+2 in the long term.

The present study was designed to determine the molecular mechanisms by which interleukin (IL)-10 prevents the HLA class I antigen expression at the cell surface. In this context, the potential role of transporter associated with antigen presentation 1+2 (TAP1+2) molecules and NF-kappaB transcription factors was addressed. The IL-10 effect was investigated in a human lymphoblastoid cell system defective for TAP1+2 genes (T2 cell line) and in the related TAP1+2 transfectants (T3 cell line). In this experimental system, after 48 h of incubation in the presence of IL-10, the HLA class I antigen downmodulation was observed in the T3 but not in the T2 cell line, suggesting a potential role of TAP1+2 molecules. In the same experimental conditions, the NF-kappaB activity was unaffected. Instead, after 3 h of exposure to IL-10, the HLA downmodulation was observed in both cell lines, the NF-kappaB factors activity being strongly reduced. In addition, the transfection of the inhibitor of NF-kappaB, IkappaBalpha, prevented the IL-10 effect on HLA class I antigen expression in the T3 cell line. This phenomenon was observed after 3 h but not 48 h of IL-10 incubation. These evidences indicate a time dependent involvement of TAP1+2 antigens and of NF-kappabeta activity in the IL-10-induced major histocompatibility complex (MHC) class I downmodulation.

Iodide excess induces apoptosis in thyroid cells through a p53-independent mechanism involving oxidative stress.

Thyroid toxicity of iodide excess has been demonstrated in animals fed with an iodide-rich diet; in vitro iodide is cytotoxic, inhibits cell growth, and induces morphological changes in thyroid cells of some species. In this study, we investigated the effect of iodide excess in an immortalized thyroid cell line (TAD-2) in primary cultures of human thyroid cells and in cells of nonthyroid origin. Iodide displayed a dose-dependent cytotoxicity in both TAD-2 and primary thyroid cells, although at different concentrations, whereas it had no effect on cells of nonthyroid origin. Thyroid cells treated with iodide excess underwent apoptosis, as evidenced by morphological changes, plasma membrane phosphatidylserine exposure, and DNA fragmentation. Apoptosis was unaffected by protein synthesis inhibition, whereas inhibition of peroxidase enzymatic activity by propylthiouracil completely blocked iodide cytotoxicity. During KI treatment, reactive oxygen species were produced, and lipid peroxide levels increased markedly. Inhibition of endogenous p53 activity did not affect the sensitivity of TAD-2 cells to iodide, and Western blot analysis demonstrated that p53, Bcl-2, Bcl-XL, and Bax protein expression did not change when cells were treated with iodide. These data indicate that excess molecular iodide, generated by oxidation of ionic iodine by endogenous peroxidases, induces apoptosis in thyroid cells through a mechanism involving generation of free radicals. This type of apoptosis is p53 independent, does not require protein synthesis, and is not induced by modulation of Bcl-2, Bcl-XL, or Bax protein expression.

Urokinase-type plasminogen-activator and normal thyroid cell adhesion to the extracellular matrix.

The urokinase-type plasminogen activator receptor (uPA-R) focuses the proteolytic activity of its ligand, the urokinase-type plasminogen activator (uPA), on the cell surface, and can also act as an adhesion receptor for vitronectin (VTN). uPA increases uPA-R affinity for VTN and is also able to cleave its receptor. We have previously shown that uPA-R is involved in the adhesion of normal thyroid cells to VTN. In the present report, we have investigated the effect of uPA on normal thyroid cell adhesion to some extracellular matrix (ECM) components. We show that a short-term treatment with uPA does not change normal thyroid cell adhesion to fibronectin (FNT), collagen (CGN), laminin (LMN) and VTN. The prolongation of uPA treatment increases cell adhesion to VTN, and, less efficiently, to other ECM components. Since the short term uPA treatment causes a partial cleavage of uPA-R, that does not increase with time, the observed increase in cell adhesivity cannot be related to the cleavage of uPA-R. We show that the adhesion improvement after the long term uPA treatment is instead due to a strong increase of the cell-surface expression of the integrin beta3 and a moderate increase of the integrin alpha(v). Both alpha(v) beta3 and alpha(v) beta1 are integrinic receptors for VTN.

Expression of integrins of the beta1 family in thyroid cells from patients with Graves' disease in vivo and in vitro.

The expression of the beta1 family of integrins was determined in thyroid follicular cells from patients with Graves' disease (GD). Integrin expression was quantitated by flow fluorocytometry of single cell suspensions with antibodies against the common beta1 chain and the alpha1-alpha6 subunits. Results indicated that also in thyroid glands of GD, as previously observed in nodular goiters, two follicular cell populations with different patterns of beta1 integrin expression coexist (VLAalpha3beta1 and VLAalpha1,3,5,6beta1). The VLAalpha1,3,5,6beta1 thyrocyte population in GD was more abundant than in nodular goiters, ranging from 40 to 70% of the total follicular cells and the overall expression of the beta1 integrins was a two-fold higher. In thyrocytes from patients with GD cultured in vitro, alpha3 and alpha2 expression was regulated by cell-to-cell contact as previously described in normal thyroid cells, while the expression of alpha1, alpha5 and alpha6 was quickly lost during the culture. Our data suggest that the integrin profile of the VLAalpha1,3,5,6beta1 thyrocyte population in GD is induced by micro-environmental conditions rather than being the expression of a constitutive phenotype.

Cell cycle block at G1-S or G2-M phase correlates with differentiation of Caco-2 cells: effect of constitutive insulin-like growth factor II expression.

BACKGROUND & AIMS: We have previously shown that autocrine insulin-like growth factor (IGF)-II synthesis through IGF-I receptor stimulates proliferation and inhibits differentiation of Caco-2 cells. To demonstrate whether differentiation of Caco-2 cells is dependent on cell growth status, we analyzed the effect of cell cycle arrest on differentiation of wild-type and IGF-II-overexpressing cells. METHODS: Cells were treated with drugs that inhibit the progression either to S phase (l-b-D-arabinofuranosylcytosine or M phase (nocodazole). Cell differentiation was analyzed by assessing apolipoprotein A-1 and sucrase-isomaltase expression. Cell proliferation and DNA content were assessed by thymidine incorporation and fluorescence-activated cell sorter analysis, respectively. Cell cycle regulatory molecules were analyzed by assessing p21 and retinoplasma protein (pRb) expression and pRb phosphorylation. RESULTS: Cell cycle block at G1-S phase was associated with increased expression of differentiation markers in both parental and IGF-II-transfected cells. On the contrary, cell cycle arrest at G2-M phase correlated with the expression of differentiation markers in parental but not in IGF-II-transfected cells. Constitutive IGF-II-expressing cells actively incorporated thymidine and showed an increase in the proportion of cells with >4N DNA ploidy in the presence of nocodazole. Nocodazole treatment of constitutive IGF-II-expressing cells stimulated p21 expression in the presence of hyperphosphorylated pRb. CONCLUSIONS: The data show that cell cycle arrest increases differentiation of Caco-2 cells. IGF-II-mediated proliferation may prevent cell differentiation through effects on control cell checkpoint proteins.

Enhanced glutathione levels and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression.

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappaB activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD.

Expression of GM-CSF receptor and "in vitro" effects of GM-CSF on human fibroblasts.

In the present study the effects of Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on fibroblast growth and activity have been studied. In this regard the AA have evaluated in primary cultures of human gengival normal fibroblasts (PG1 cells): a)-the expression of GM-CSF receptor (GM-CSFR) (alfa unit) on the cell surface; b)-the in vitro effects of different doses of GM-CSF on the GM-CSFR expression and on the proliferation and activity of fibroblasts. PG1 cells have been stimulated in vitro with different concentrations of GM-CSF (10, 50, 80, 100 and 150 ng/ml) using promonocytic cell line U937 as positive control for GM-CSFR expression. GM-CSFR was investigated by flow cytometry, with mouse monoclonal antibody (mAb) against the alfa chain of the human GM-CSFR and fluorescein-conjugated goat antimouse immunoglobulin G (IgG). At high GM-CSF concentration (80 ng/ml) the AA observed: 1)-A marked increase of GM-CSFR expression evaluated as fluorescence intensity (about three fold in respect to the controls); 2)-Maximal increase of PG1 cells proliferation. Moreover immunofluorescence on fibroblasts obtained from culture plates showed increased actin stress fibers and fibronectin production with low stimulation by GM-CSF, while higher concentration of this cytokine determined increased proliferation of cells, but a decreased formation of actine fibers and vinculin plaques. These results demonstrate: 1)-The presence of GM-CSFR on the surface of fibroblasts; 2)-The proliferation and the synthesis activity of these cells (in vitro) are modulated by different concentration of GM-CSF. We hypothesize that GM-CSF until 80 ng/ml can upregulate the expression of the receptor. Therefore, on the basis of previous findings of high serum levels of GM-CSF in course of scleroderma, a disease characterized by fibroblast hyperactivity, a possible role of this cytokine in the pathogenic process of this disease can be hypothesized.