Molecular mechanisms driving Streptococcus mitis entry into human gingival fibroblasts in presence of chitlac-nAg and saliva.

The molecular mechanisms leading to Streptococcus mitis capability of entering oral cells were investigated in a co-culture of S. mitis and Human Gingival Fibroblasts (HGFs) in the presence of saliva. An innovative colloidal solution based on silver nanoparticles (Chitlac-nAg), a promising device for daily oral care, was added to the experimental system in order to study the effects of silver on the bacterial overgrowth and ability to enter non-phagocytic eukaryotic cells. The entry of bacteria into the eukaryotic cells is mediated by a signalling pathway involving FAK, integrin ß1, and the two cytoskeleton proteins vinculin and F-actin, and down-regulated by the presence of saliva both at 3 and 48 h of culture, whereas Chitlac-n Ag exposure seems to influence, by incrementing it, the number of bacteria entering the fibroblasts only at 48 h. The formation of fibrillary extrusion from HGFs and the co-localization of bacteria and silver nanoparticles within the fibroblast vacuoles were also recorded. After longer experimental times (72 and 96 h), the number of S. mitis chains inside gingival cells is reduced, mainly in presence of saliva. The results suggest an escape of bacteria from fibroblasts to restore the microbial balance of the oral cavity.

MRJF4, a novel histone deacetylase inhibitor, induces p21 mediated autophagy in PC3 prostate cancer cells.

Autophagy is a cellular defense mechanism which occurs through degradation and recycling of cytoplasmic constituents and represents a caspase—independent alternative to cell death by apoptosis. It is generally accepted that the suppression of autophagy in many cancer cells is directly correlated to malignancy; hence, the control of autophagy genes could represent a target for cancer therapy. The inhibition of cell proliferation through autophagy activation could be an important mechanism for many anti—tumor drugs. Here we report the effects of a novel histone deacetylase inhibitor MRJF4 (racemic mixture) and of its two enantiomers [(+)—MRJF4 and (—)—MRJF4] on the morphological and molecular mechanisms causing death and migration of PC3 prostatic cancer cells. In particular, we investigated the occurrence of the autophagic process, both at morphological and molecular levels (LC3 expression), and its relationship with p21, a key molecule which regulates cell cycle and autophagy cell death. Moreover, pERK/Nf—kB driven intracellular signaling, the expression of MMP9 protein — a key component of cell migration — invasion, and metastasis were assayed. Our results showed that the anti—proliferative effects of MRJF4 due to autophagy occurrence, documented by LC3 increase and ultrastructural modifications, and the reduction of invasiveness seem to be mediated by the down—regulation of pERK/NF—kB signaling pathway, along with p21 up—regulation.

A dual role for ß1 integrin in an in vitro Streptococcus mitis/human gingival fibroblasts co-culture model in response to TEGDMA.

AIM: To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. METHODOLOGY: ß1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase ß (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). RESULTS: When HGFs are co-cultured with S. mitis, ß1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P < 0.05 HGFs versus HGFs co-cultured with S. mitis). A higher level of MMP3 in HGFs treated with TEGDMA was recorded (P < 0.05 HGFs versus HGFs exposed to TEGDMA). COX-2 inflammatory factor mRNA level appeared higher in HGFs exposed to 1 mmol L(-1) TEGDMA (P < 0.01 HGFs versus HGFs exposed to TEGDMA), whereas TNF-α gene expression was higher in HGFs co-cultured with S. mitis (P < 0.05 HGFs versus HGFs co-cultured with S. mitis). CONCLUSIONS: ß1 integrin triggered the signalling pathway, transduced by p-PKCα and involving ERK 1 and 2 and MMPs. This pathway resulted in an unbalanced equilibrium in tissue remodelling process, along with inflammatory response when HGFs are exposed to bacteria or biomaterial alone. On the contrary, the TEGDMA/S. mitis combination restored the balance between extracellular matrix deposition and degradation and prevented an inflammatory response.

NF-kB mediated down-regulation of collagen synthesis upon HEMA (2-hydroxyethyl methacrylate) treatment of primary human gingival fibroblast/Streptococcus mutans co-cultured cells.

PURPOSE: In vitro studies have evidenced the cytotoxic effect of HEMA (2-hydroxyethyl methacrylate), the most common component of dental resin-based restorative material, which is released within the oral cavity, on eukaryotic cells such as gingival fibroblast and epithelial cells. However, since the presence of microorganisms within the oral cavity cannot be excluded and little is known about the interactions occurring between eukaryotic cells and the human oral microbiota, our attention has been addressed to investigate the effect of 3 mM HEMA on the molecular mechanisms driving the response of human gingival fibroblasts (HGFs) co-cultured with Streptococcus mutans. METHODOLOGY: HGF/S. mutans co-culture has been set up in our lab, and upon HEMA treatment, S.mutans and HGF cells' viability and adhesion along with type I collagen gene and pro-collagen I, Bax, Bcl2, nuclear factor kB (NF-kB), IkBα, pIkBα protein expression by PCR, Western blotting and ELISA assays have been investigated. RESULTS: HEMA treatment determines a significant decrease of type I collagen protein production, even in the presence of S. mutans, in parallel to a decrease of cell viability and adhesion, which seem to be regulated by NF-kB activation. In fact, when SN50, NF-kB-specific pharmacological inhibitor, is added to the culture, cell proliferation along with collagen synthesis is restored. CONCLUSION: The modulation exerted by S. mutans on the cytotoxic effect of HEMA suggests that within the oral cavity, the eukaryotic/prokaryotic cell interactions, maintaining the balance of the environment, allow HEMA to perform its adhesive and bonding function and that the use of a co-culture system, which simulates the oral cavity organization, improves the knowledge concerning the biocompatibility of this dental material.

pPKCα-mediated effect on in vitro Aß production in response to gamma secretase inhibitor LY411575 in rat CTXTNA2 astrocytes.

Alzheimer's Disease implies memory and cognitive impairment due to beta amyloid accumulation, presence of reactive microglia and astrocytes, loss of synapses, neural network dysfunctions and modifications of neuronal signalling. A key role in such events is played by astrocytes, which actively secrete high levels of beta amyloid protein originating from sequential cleavage of APP by alpha, beta and gamma secretases. Since inhibition of such process could represent an important strategy against the occurrence of Alzheimer's Disease, in this paper the role played by pPKC alpha in the in vitro beta amyloid production in response to gamma secretase inhibitor in rat cortical astrocytes is reported. pPKC alpha increased expression seems to be related to decreased beta amyloid production in parallel to increased astrocytes viability and decreased iNOS expression in the presence of 10 microM LY411575. Thus gamma secretase inhibitor, activating pPKC alpha intracellular pathway could be suggested to prevent or reduce downstream toxic events, representing a useful strategy to counteract Alzheimer's disease.

NAD(P)H oxidase and pro-inflammatory response during maximal exercise: role of C242T polymorphism of the P22PHOX subunit.

Intense exercise induces a pro-inflammatory status through a mechanism involving the NAD(P)H oxidase system. We focused our attention on p22phox, a subunit of the NAD(P)H oxidase, and on its allelic polymorphism C242T, which is known to affect the functional activity of the enzyme. We investigated whether the p22phox C242T variants exhibit systemic effects in healthy subjects by analyzing the proinflammatory and cardiocirculatory responses to physical exercise in endurance athletes. The group of study consisted of 97 long distance runners, 37 +/- 4.4 yrs of age, with similar training history. The subjects underwent a maximal stress test during which both inflammatory and cardiopulmonary parameters were monitored. Our results demonstrate that T allele deeply influences the neutrophil activation in response to intense exercise, since T carriers were characterized by significantly lower release of myeloperoxidase (MPO), a classical leukocyte derived pro-inflammatory cytokine. In addition, the presence of T allele was associated with a higher cardiopulmonary efficiency as evidenced by a significantly lower Heart Rate (HR) at the peak of exercise and, when a dominant model was assumed, by a higher maximal oxygen uptake (VO2 max). On the other hand, no effects of 242T mutation on the plasmatic total antioxidant capacity (TAC) and on the cortisol responses to the physical exercise were detected. In conclusion, our data support a systemic role for p22phox C242T polymorphism that, modifying the intensity of the inflammatory response, can influence the cardiovascular adaptations elicited by aerobic training. These results contribute to support the hypothesis of a systemic effect for the C242T polymorphism and of its possible functional rebound in healthy subjects.

Histochemical and biochemical analysis of phospholipase C isoforms in normal human gastric mucosa cells.

The expression and activity of PIP2-specific phospholipase C (PLC) in healthy human gastric mucosa cells were investigated by means of Western blotting, immunohistochemistry and in vitro activity assays. The results provide direct evidence for an almost exclusive expression of the PLC beta family and at the same time supply a cellular cartography of each represented isoform of this family. In this context, the putative roles of each isoform in the signaling events regulating the gastric mucosa metabolic machinery are discussed. These data provide a unique map of the specific expression and cellular distribution of the most represented PLC isoforms in healthy human gastric mucosa cells, which may constitute a reference point in future studies aimed at highlighting possible cytochemical and biochemical hallmarks of metaplastic or malignant transformation.

Inefficient phospholipase C activation and reduced Lck expression characterize the signaling defect of umbilical cord T lymphocytes.

Adult and neonatal immunocompetent cells exhibit important functional distinctions, including differences in cytokine production and susceptibility to tolerance induction. We have investigated the molecular features that characterize the immune response of cord blood-derived T lymphocytes compared with that of adult T lymphocytes. Our findings demonstrate that phospholipase C (PLC) isozymes, which play a pivotal role in the control of protein kinase C activation and Ca2+ mobilization, are differently expressed in cord and adult T lymphocytes. PLCbeta1 and delta1 are expressed at higher levels in cord T cells, while PLCbeta2 and gamma1 expression is higher in adult T lymphocytes. PLCdelta2 and gamma2 appear to be equally expressed in both cell types. In addition, a functional defect in PLC activation via CD3 ligation or pervanadate treatment, stimuli that activate tyrosine kinases, was observed in cord blood T cells, whereas treatment with aluminum tetrafluoride (AlF4-), a G protein activator, demonstrated a similar degree of PLC activation in cord and adult T cells. The impaired PLC activation of cord blood-derived T cells was associated with a a very low expression of the Src kinase, Lck, along with a reduced level of ZAP70. No mitogenic response to CD3 ligation was observed in cord T cells. However, no signaling defect was apparent downstream of PLC activation, as demonstrated by the mitogenic response of cord T cells to the pharmacologic activation of protein kinase C and Ca2+ by treatment with PMA and ionomycin. Thus, neonatal cord blood-derived T cells show a signaling immaturity associated with inadequate PLCgamma activation and decreased Lck expression.

Novel evidence of expression and activity of ecto-phospholipase C gamma1 in human T lymphocytes.

Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCgamma1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCgamma1-positive (PLCgamma1(+)) cells (approximately 37%) were CD8(+) and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and activity, which were restored by treatment with an antiproliferative agent like natural interferon beta. Moreover, the different kinetics of formation of its hydrolytic products, inositol 1 phosphate and inositol 1:2 cyclic phosphate (Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a selective involvement of the two inositol phosphates in the mechanisms regulating the metabolism of particular T-lymphocyte subsets.

Interferon mediated phosphatidylinositol uptake and processing in nuclei isolated from Burkitt lymphoma cells.

The kinetic analysis of exogenous [3H]phosphatidylinositol (PI) uptake and processing by nuclei isolated from Daudi lymphoma cells upon interferon alpha treatment has been performed. Results have disclosed that, with respect to controls, interferon induces an evident stimulation of label incorporation into nuclei. The incorporated [3H] PI has been found for phosphorylation and hydrolytic cleavage, indicating that the intranuclear transduction system activated by interferon at plasma membrane level, might involve the PI cycle as a possible route of intracellular signalling.

Evidence for nuclear phosphoinositidase C activity in the antiproliferative signals produced by interferon in Burkitt lymphoma cells.

The influence of interferon alpha on nuclear phosphoinositidase C (PIC) in Daudi cells has been analysed. Results showed an early increase of PIC activity detectable within 90 min of interferon treatment concomitant with an increase of diacylglcerol (DAG) levels. Since the interferon-induced DAG production is not modified by the addition of propranolol, a compound known to inhibit production of DAG from phosphatidylcholine hydrolysis, it is suggested that the interferon antiproliferative signal is transduced into the nucleus via the inositol lipid pathway. A parallel analysis performed on intact cells showed a rapid inhibition of PIC activity accompanied by an increase of DAG level thus suggesting that interferon-generated signals at plasma-membrane level use pathways different from that of inositol lipids. A selected clone of Daudi cells resistant to interferon action provided a control for specificity of results.

Dental pulp lipids from Bos taurus during odontogenesis.

During the fetal development of the dental pulp, the various lipid classes show no substantial differences in their relative ratios but differences occur between deciduous and permanent teeth. By chromatography, the amount of free cholesterol was found decreased in deciduous and permanent teeth as compared to fetal teeth. Esterified cholesterol increased in permanent teeth and triglyceride levels were high only in developing permanent teeth. Phosphatidylcholine, sphingomyelin and phosphatidylserine were present in higher concentration in permanent unerupted teeth, while phosphatidylethanolamine was at first constant but then decreased during development in the permanent unerupted teeth. These data suggest that lipid changes are related to the assembly of plasma membranes and to the establishment of the innervation during ontogeny and postnatal development of dental pulp.