Enhancement of fibroblast proliferation, collagen biosynthesis and production of growth factors as a result of combining sodium hyaluronate and aminoacids.

Fibroblasts play a key role in tissue healing by producing the majority of extracellular matrix components, favouring granulation tissue formation, and stimulating re-epithelialization. Hyaluronan is a component of ECM and its anti-inflammatory effects and properties in enhancing wound closure are well known. In this study, we examined the effects of Aminogam gel, a new pharmacological preparation suggested to improve wound healing, composed of hyaluronic acid, proline, lysine, glycine and leucine, on human fibroblasts. Results show that fibroblasts treated with hyaluronic acid plus aminoacid solution increased their proliferative activity, collagen I and III, and fibronectin synthesis. Moreover, HA plus aminoacid solution increased the expression of transforming growth factor beta, connective tissue growth factor, interleukin-6 and -8, assayed by RT-PCR. These results suggested that Aminogam gel, involved in several stages of wound healing, as fibroblast proliferation, granulation tissue formation, ECM component deposition, and production of cytokines, may be a useful device to favour and accelerate wound closure.

Survivin overexpression in head and neck squamous cell carcinomas as a new therapeutic target (Review).

Head and neck squamous cell carcinoma (HNSCC) is the sixth most commonly diagnosed cancer worldwide. It has poor clinical outcome due to intrinsic or acquired drug resistance. Deregulation of both apoptosis and autophagy contributes to chemotherapy resistance and disease progression. A new member of the inhibitors of apoptosis protein (IAP) family, namely survivin, is selectively overexpressed in tumors, including HNSCC, but not in normal tissues. Thus, it is considered a tumor biomarker. Here, we reviewed survivin expression and function in tumor progression focusing on its nodal role in the regulation of cell apoptosis and autophagy. Based on literature data, survivin targeting may be envisaged as a novel therapeutic strategy.

Halting pro-survival autophagy by TGFß inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients.

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Beta-catenin gene analysis in oral squamous cell carcinoma.

The molecular mechanisms involved in the development of oral squamous cell carcinomas (OSCC) are not yet well understood. Evidence of recent studies suggests that aberrant beta-catenin signalling may participate in the neoplastic transformation and that it is implicated in the development of several tumours. Beta-catenin is a component of the catenin family and plays a crucial role in cadherin mediated cell adhesion. However, it has recently been shown that beta-catenin is also involved in other functions such as intracellular signalling and the regulation of gene transcription. The aim of this study is to evaluate the presence of mutation in exon 3 of the beta-catenin gene in 20 OSCC cell lines. DNA was extracted using Qiagen Qiamp DNA minikit and a region encompassing the exon 3 of beta-catenin gene was amplified using a single PCR assay. The PCR products were analysed by SSCP and direct sequencing to detect any mutation of the gene. Most of the cell lines examined showed, by immunofluorescence, a beta-catenin delocalization. SSCP and sequence analysis of the PCR products did not show any mutation of the beta-catenin gene in any of the cell lines. In conclusion, although aberrant expressions or abnormal localization of beta-catenin have been detected in several OSCC cells, it appears that this finding has no relationship with beta-catenin gene mutations.

Inhibitory effect of retinoids on the generation of procoagulant activity by blood mononuclear phagocytes.

Retinoids are known to modulate several functions of mononuclear phagocytes. We have studied the effect of retinyl acetate (RAc) and retinoic acid (RA) on the production of procoagulant activity (PCA) by human peripheral blood mononuclear cells stimulated with endotoxin (1 microgram/ml, 4 or 20 h at 37 degrees C). Both compounds caused a dose-dependent reduction in the expression of cell-associated PCA (from 86 to less than 10% of control in the range of concentration comprised between 0.1 and 100 microM). This effect was also observed when the cells were exposed to retinoids for 10 min and washed before challenge with endotoxin, indicating that it is rapid and irreversible. In contrast, incubation of RAc or RA for 3 h at 37 degrees C with cells that have been already stimulated with endotoxin (20 h at 37 degrees C) remained without influence on cell PCA. The inhibitory action of retinoids was also observed when monocyte-enriched (greater than 85%) preparations or highly purified monocyte-derived macrophages (greater than 99%) were used instead of whole mononuclear cells. BW755C, an inhibitor of cyclo-oxygenase and lipoxygenase, reversed the inhibitory effect of retinoids, whereas acetylsalycilic acid, an inhibitor of cyclo-oxygenase, was inactive, suggesting the involvement of a lipoxygenase product. The inhibition of monocyte/macrophage PCA production and the subsequent reduction of cell potential for fibrin deposition might represent one of the mechanisms whereby retinoids exert their antiinflammatory and immunomodulatory activities.

Salivary up-regulation of human polymorphonuclear leucocyte chemotaxis and adhesion-molecule expression.

Chemotaxis and adhesion molecules were investigated in peripheral blood neutrophils incubated with saliva collected from healthy donors. A salivary concentration of 20% increased chemotactic responses and CD11a/CD18 and CD11b/CD18 expression, but had no effect on formyl-methionyl-leucyl-phenylalanine receptors.

Effect of cisplatin on the oxidative metabolism of polymorphonuclear leukocytes in cancer patients.

Peripheral blood polymorphonuclear leukocytes of patients with ovarian or uterine-cervix carcinoma were studied during cisplatin chemotherapy for phagocytic oxidative metabolism. In particular, superoxide production by these leukocytes after stimulation with opsonized Zymosan particles was determined. The basal value of oxidative metabolism of leukocytes from neoplastic patients was lower than in normals. Superoxide production decreased after each dose administration, but returned to normal at the end of the entire chemotherapeutic treatment. It is suggested that cisplatin treatment, despite a short-lived depression of oxidative metabolism in polymorphonuclear cells, improves this function, most probably by interfering with neoplastic growth.

Thymomodulin increases the depressed production of superoxide anion by alveolar macrophages in patients with chronic bronchitis.

Thymomodulin is an immunomodulating agent which is derived from calf thymus by partial acid lysis. It promotes T-cell maturation, enhances antibody synthesis and improves the phagocytic response of neutrophils. Clinical trials have revealed the effectiveness of this thymic derivative in the prevention of recurrent respiratory infections (RRI) in children and in adults; 11 patients (8 males and 3 females; age range 18-76 years) with chronic bronchitis dominated by recurrent respiratory infections were studied. They were treated orally for 6 months during the winter season with 120 mg/day of thymomodulin. All the subjects were asked to keep a diary recording the intensity of their symptoms, the number of working days lost (days of illness) and the use of antibiotic and/or mucolytic drugs. At the beginning and at the end of the trial each patient was subjected to a control with a flexible fibreoptic bronchoscope with bronchoalveolar lavage to evaluate the phagocytic response of alveolar macrophages. At the end of therapy a significant improvement of the clinical status, evaluated by the above-mentioned parameters, of the bronchial mucosa aspect and an increase in alveolar macrophage superoxide production was noticed (from 0.1 +/- 0.09 and 0.8 +/- 0.5 nmol to 1.6 +/- 0.8 and 4.1 +/- 2.2 nmol with PMA or zymosan particles respectively; p less than 0.001). During thymomodulin treatment no side-effects were recorded.

Inhibition of neutrophil functions by scrapie prion protein: description of some inhibitory properties.

The effect of scrapie prion protein (PrP) either in the native or in the denatured form was studied on in vitro responses of human neutrophils. Incubation of neutrophils with native PrP caused an inhibition of their aggregation induced by cytochalasin B. Moreover, the denatured form was in itself a strong aggregation inducer. When evaluating the effect on generation of neutrophil superoxide anion (O2) we found that neutrophils released O2 in response to the denatured from only but the native form was ineffective. Similarly, neutrophil discharge of beta-glucuronidase which represents the azurophilic granule marker was stimulated in a dose-dependent form by the denatured PrP 27-30 whereas the native form was almost completely devoid of any activity. These results indicate that several aspects of neutrophil function can be altered by the native form of prion protein PrP 27-30. This might be responsible for the impaired phagocytic cell activity explaining, at least in part, the absence of any inflammatory reaction during scrapie infection.

Bone marrow fibroblasts parallel multiple myeloma progression in patients and mice: in vitro and in vivo studies.

The role of cancer-associated fibroblasts (CAFs) has not been previously studied in multiple myeloma (MM). Here, cytofluorimetric analysis revealed higher proportions of bone marrow (BM) CAFs in patients with active MM (both at diagnosis and relapse) compared with patients in remission or those with monoclonal gammopathy of undetermined significance or deficiency anemia (controls). CAFs from MM patients produced increased levels of transforming growth factor-ß, interleukin-6, stromal cell-derived factor-1α, insulin-like growth factor-1, vascular endothelial growth factor and fibroblast growth factor-2 and displayed an activated and heterogeneous phenotype, which supported their origin from resident fibroblasts, endothelial cells and hematopoietic stem and progenitor cells via the endothelial-mesenchymal transition as well as mesenchymal stem cells via the mesenchymal transition, as both of these processes are induced by MM cells and CAFs. Active MM CAFs fostered chemotaxis, adhesion, proliferation and apoptosis resistance in MM cells through cytokine signals and cell-to-cell contact, which were inhibited by blocking CXCR4, several integrins and fibronectin. MM cells also induced the CAFs proliferation. In syngeneic 5T33MM and xenograft mouse models, MM cells induced the expansion of CAFs, which, in turn, promoted MM initiation and progression as well as angiogenesis. In BM biopsies from patients and mice, nests of CAFs were found in close contact with MM cells, suggesting a supportive niche. Therefore, the targeting of CAFs in MM patients may be envisaged as a novel therapeutic strategy.

Sulfide influence on polymorphonuclear functions: a possible role for Ca2+ involvement.

Polymorphonuclear cells (PMN) of gingival sulcus play an important role in host defense against periodontal tissue-invading bacteria, but their phagocytic activity is conditioned by several virulence factors released by oral pathogens. In this report we have studied the influence of sulfide, a toxic bacterial metabolite, on the main PMN functions: chemotaxis, degranulation and oxidative burst. PMN exposed to sodium sulfide (up to 2 mM) used as a source of H2S showed a depression of the calcium-dependent cytoskeleton activities such as chemotaxis and azurophilic granule release induced by FMLP. No effect was observed on the calcium-independent specific granule release obtained by PMA. These data were in agreement with the sulfide inhibition of cytosolic free Ca2+ concentration [Ca2+]i increase normally induced by ionomycin. On the other hand, hydrogen sulfide was able to prime PMN for a stronger oxidative response both to calcium-dependent or calcium-independent stimulation. This finding may account for a more efficient oxidative killing under reoxygenation of the anaerobic infectious areas.

[Effects of cis-platin on the contractile system of polymorphonuclear leukocytes]. / Effetti del cis-platino sul sistema contrattile di leucociti polimorfonucleati.

The effects of cis-Platinum, a new antitumor drug, on guinea pig polymorphonuclear functions related to subplasmalemmal fibrillar structures, were investigated. The compound markedly decreased chemotaxis. Zymosan particles engulfment and degranulation. The depression of these leucocyte activities adds to the already known drug induced myelotoxicity .

Modifications of macrophage chemotaxis in rats bearing the transplantable tumour reticulosarcoma 'IRE'.

Macrophage chemotactic activity was studied as a marker of the anti-inflammatory effect accompanying tumour growth. An inhibiting effect of the tumour on the macrophage mobility was observed by comparing the macrophages collected from normal rats and those from animals bearing the IRE reticulosarcoma. To explain these results, it is postulated that a MIF, or a similar inhibiting factor is released by the tumour. Both the the nature and function of such a factor(s) remain to be clarified.

Salivary effects on polymorphonuclear leukocyte functions.

Respiratory burst, enzymatic degranulation and bacterial killing were investigated on peripheral blood polymorphonuclear leukocytes (polymorphonuclear leukocytes) incubated with a pool of salivary fluids elicited from healthy donors. Low saliva concentrations primed polymorphonuclear leukocytes for enhancement of O2 consumption, O2- and beta-glucuronidase release and Staphylococcus aureus killing. Whole saliva, on the contrary, depressed all tested phagocytic activities.

Different priming on human neutrophil respiratory burst by lipopolysaccharides from phase I and phase II Coxiella burnetii.

Human neutrophils produce small amounts of superoxide anion when stimulated with the chemotactic peptide FMLP; preincubating neutrophils with low concentrations of lipopolysaccharides (LPS) markedly increases this response, an effect referred to as priming. In this work LPS from Coxiella burnetii either phase I (virulent) or phase II (avirulent) were examined for their ability to induce priming. Results clearly show that only LPS from phase II microorganism was able to increase the release from neutrophils upon subsequent stimulation with FMLP. This effect was abolished by preincubation of LPS with polymyxin B. This finding may account for the ability of Coxiella burnetii phase I to escape intracellular phagocyte killing during persistent infections.

Sulfide enhancement of PMN apoptosis.

Hydrogen sulfide is a toxic metabolite released by several bacterial agents under anaerobic conditions. In the present paper, we investigated the effects of sulfide on polymorphonuclear cell (PMN) apoptosis, a mechanism suggested for limiting the toxic potential of neutrophils in inflammatory sites. We showed that 1 mM sulfide (concentration not conditioning PMN viability) is able to enhance the apoptotic fate of human granulocytes by increasing: i) the number of cells containing pyknotic nuclei, ii) the internucleosomal cleavage, and, iii) the intensity of tubulin immunofluorescence staining. The sulfide effect is partially prevented by ionomycin and this finding is consistent with the hypothesis of the inhibiting role played by high levels of cytosolic calcium in PMN apoptosis modulating.

Retinoids inhibit the respiratory burst and degranulation of stimulated human polymorphonuclear leukocytes.

Retinoids exhibit a wide spectrum of activities, including antiinflammatory properties. We have investigated the effect of retinoic acid (RA) and retinyl acetate (RAc) on the production of reactive oxygen metabolites and the release of lysosomal enzymes by human polymorphonuclear leukocytes (PMN). Incubation of PMN with RAc or RA (1-100 microM) caused a dose-dependent inhibition (upto 90%) in O2- production and chemiluminescence induced by phorbol myristate acetate (PMA), N-formyl-methionyl-leucyl-phenylanaline (fMLP), opsonized zymosan or ionophore A23187. Both retinoids (1-100 microM) also inhibited, in a dose-dependent way, degranulation induced by fMLP (upto 85% at the highest concentration of RA). These inhibitory effects appear irreversible, since they persist after the drugs are removed and the cells washed before stimulation. Inhibitors of cyclo-oxygenase activity such as acetylsalicyclic acid and indomethacin did not influence the effects of RAc. In contrast, BW755, an inhibitor of both cyclooxygenase and lipoxygenase, reversed the inhibitory action of RAc, suggesting that the effect of retinoids occurs possibly through the mediation of lipoxygenase products. The modulation of PMN oxidative metabolism and degranulation might help explain the antiinflammatory properties of retinoids.

In vitro cisplatin effects on phagocyte functions.

The effects of cis-diamminedichloroplatinum (II) (cisplatin), a drug used in cancer chemotherapy, on the oxidative metabolism, endocytosis, chemotaxis and exocytosis of guinea-pig polymorphonuclear leukocytes were studied. All these functions were negatively influenced, but the same effect (50% inhibition) was observed at different drug concentrations (3 X 10(-5) M for chemotaxis, 10(-4) M for O2 consumption by FMLP and beta-glucuronidase release, 10(-3) M for O2 consumption by PMA and for zymosan engulfment). The effects of the drug can be explained by its ability to bind to membrane proteins, essentially to -SH groups.

Cytochrome b and FAD content in polymorphonuclear leucocytes in a family with X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD), an immunodeficiency syndrome characterized by extreme susceptibility to bacterial infections, is due to a defect of the respiratory burst in human phagocytes. NADPH oxidase, the enzyme that catalyzes the reduction of oxygen and the release of oxidative radicals, was studied in polymorphonuclear leucocytes (PMNs) in a family affected by an x-linked inheritance form at high penetrance of the disease. The contents of cytochrome b, suggested as the terminal component of the oxidase electron transport chain, and FAD, the hypothetical proximal component of the chain, were determined in patients and in carriers. Cytochrome b showed the typical behaviour of x-linked CGD: total absence in patients, intermediate values in carriers. FAD content evaluated on plasma membranes was less decreased than cytochrome b. Carriers also showed a decrease of this flavoprotein. Cytochrome b and FAD contents were compared to NBT test and superoxide production: a clear correlation was observed for the cytochrome b, but FAD plasma membrane evaluation could also be an interesting tool for the metabolic characterization of the disease in patients and in carriers.