Cigarette smoke is associated with up-regulation of inducible NOS and COX-2 protein expression and activity in granulosa cells of women undergoing in vitro fertilization.

Cigarette smoke exposure represents a well-established ovotoxic exogenous stress, but the molecular mechanisms underlying of this effect are still unclear. Cigarette smoke upregulates inflammatory genes in the female reproductive organs, therefore an abnormal inflammation response may contribute to the impairment of female fertility. In this study we investigated for the first time the effect of cigarette smoke exposure on NOS and COX expression and activity and on their transcription factors (CREB and NF-kB) in human GCs and on the release of NO and PGE2 in the FF in smoking and non-smoking patients undergoing IVF treatment. In addition, correlation analysis between AMH serum levels, an index of ovarian reserve, and smoking exposure or iNOS and COX-2 protein expression levels were performed using a Pearson correlation method. Cigarette smoke exposure resulted in a significant increase of iNOS and COX-2 protein expression together with an increase of iNOS activity and PGE2 levels. pNF-kB and pCREB protein expression were upregulated in the GCs of smokers compared to non-smokers. The habit of smoking was negatively correlated with serum AMH levels, and positively correlated with iNOS and COX-2 protein expression levels. The data presented in the current study revealed a novel molecular mechanism underlying the toxic effects of cigarette smoke on fertility. Additional pathways mediating the effects of cigarette smoke exposure in human GCs cannot be excluded and should be investigated in future studies.

Liposomes embedded with differentiating factors as a new strategy for enhancing DPSC osteogenic commitment.

Human dental pulp stem cell (DPSC) differentiation toward the osteoblastic phenotype is enhanced when culture media are supplemented with differentiating factors, i.e. ascorbic acid, ß-glycerophosphate and dexamethasone. Liposomes, spherical vesicles formed by a phospholipid bilayer, are frequently used as carriers for drugs, growth factors and hydrophobic molecules. The aim of this work was to speed up DPSC commitment to the osteogenic lineage by embedding differentiating factors within liposomes. Firstly, liposomes were prepared by rehydrating a phospholipidic thin film and characterised in terms of dimensions. Secondly, liposome-exposed DPSCs were characterised by their immunophenotypic profile. Levels of CD90 were significantly decreased in the presence of liposomes filled with ascorbic acid, ß-glycerophosphate and dexamethasone (Lipo-Mix) with respect to normal differentiation medium (DM), while CD73 and CD29 expression were enhanced, suggesting osteogenic commitment. Additionally, an appreciable extracellular matrix deposition is detected. Thirdly, the Lipo-Mix formulation better increases alkaline phosphatase activity and levels of Collagen I secretion with respect to DM. In parallel, the new liposome formulation is capable of decreasing the release of H2O2 and of triggering a precocious antioxidant cell response, redressing the redox balance required upon mesenchymal stem cell commitment to osteogenesis. It can be therefore hypothesised that Lipo-Mix could represent a suitable tool for clinical regenerative purposes in the field of tissue engineering by speeding up DPSC osteogenic commitment, mineralised matrix deposition and remodelling.

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.

HEMA-induced cytotoxicity: oxidative stress, genotoxicity and apoptosis.

Dental resin composites consist of organic polymers with inorganic fillers used as bonding resins and direct filling materials in dentine adhesives and as sealing agents for inlays, crowns and orthodontic brackets. Despite various modifications in the formulation, the chemical composition of composite resins includes inorganic filler particles and additives, which are incorporated into a mixture of an organic resin matrix. Among them, 2-hydroxyethylmethacrylate (HEMA) is one of the most frequently used. Several studies have attempted to clarify the mechanisms underlying HEMA cytotoxicity. Most of them support the hypothesis that this compound, once released in the oral environment, increases reactive oxygen species (ROS) production and oxidative DNA damage through double-strand breaks evidenced by in vitro presence of micronuclei. As a consequence, the glutathione detoxifying intracellular pool forms adducts with HEMA through its cysteine motif and inflammation begins to occur: transcription of early genes of inflammation such as tumour necrosis factor α or inducible cyclooxygenase up to the secretion of prostaglandins 2. These phenomena are counteracted by N-acetylcysteine (NAC), a nonenzymatic antioxidant, but not by vitamin E or other antioxidant. Consequently, NAC prevents HEMA-induced apoptosis acting as a direct ROS scavenger. This minireview collects the most significant papers on HEMA and tries to make an overview of its cytotoxicity on different cell types and experimental models.

Comparison between the accumulation capacity of four lichen species transplanted to a urban site.

The capacity to accumulate trace elements from the atmosphere of the lichens Hypogymnia physodes, Parmelia sulcata, Pseudevernia furfuracea and Usnea gr. hirta transplanted to an urban site of N Italy was compared. Twenty-nine elements (Al, As, Br, Ca, Cd, Ce, Cl, Co, Cr, Cs, Cu, Fe, Hg, I, K, La, Mg, Mn, Ni, Pb, Rb, Sb, Sc, Se, Sm, Th, Ti, V, Zn) were analyzed by Instrumental Neutron Activation Analysis (INAA) and Electro-Thermal Atomic Absorption Spectroscopy (ET-AAS). The ratio between the concentrations of each element in exposed samples to that of control samples (exposed-to-control ratio and EC ratio) was used to investigate the accumulation rates of lichen thalli. The results showed that in general elements did not exhibit well defined trends, but rather showed fluctuations, and indicated that H. physodes, P. furfuracea and U. gr. hirta have a similar accumulation capacity, while that of P. sulcata is lower.

Neuromelanin and iron in human locus coeruleus and substantia nigra during aging: consequences for neuronal vulnerability.

In this study a comparative analysis of iron molecules during aging was performed in locus coeruleus (LC) and substantia nigra (SN), known targets of Parkinson's Disease (PD) and related disorders. LC and SN neurons, especially the SN pars compacta, degenerate in PD and other forms of parkinsonism. Iron and its major molecular forms, such as ferritin and neuromelanin (NM), were measured in LC and SN of normal subjects at various ages. Iron levels were lower, H-ferritin/iron ratio was higher and the iron content in NM was lower in LC than in SN. Iron deposits were abundant in SN tissue, very scarse in LC tissue and completely absent in pigmented neurons of both SN and LC. In both regions H- and L-ferritins were present only in glia. This suggests that in LC neurons iron mobilization and toxicity is lower than that in SN and is efficiently buffered by NM. Ferritins accomplish the same buffering function in glial cells.

Determination of baseline element composition of lichens using samples from high elevations.

The results of a survey aimed at providing baseline element composition of lichens from unpolluted or very low polluted areas are reported. Lichen samples collected at high elevation areas in Himalayas (Nepal), Mt. Kenya (Kenya) and the Alps (Italy) were analyzed for their trace element content, mostly by INAA, allowing for a multi-element analysis. The elemental composition of lichen samples was essentially influenced by natural occurrence, mainly airborne soil dust. However, also anthropogenic input determined by long-distance atmospheric transport of pollutants was involved for some highly volatile elements such as Br, Cd and Sb. To avoid the influence of soil contamination and air pollution, and obtain baseline concentrations to be used as reference (the "natural" elemental composition of lichens), the average of the lowest concentrations is suggested.

The neuromelanin of human substantia nigra: structure, synthesis and molecular behaviour.

The pigmented neurons of the substantia nigra (SN) are typically lost in Parkinson's disease: however the possible relationship between neuronal vulnerability and the presence of neuromelanin (NM) has not been elucidated. Early histological studies revealed the presence of increasing amounts of NM in the SN with aging in higher mammals, showed that NM granules are surrounded by membrane, and comparatively evaluated the pigmentation of SN in different animal species. Histochemical studies showed the association of NM with lipofuscins. However, systematic investigations of NM structure, synthesis and molecular interactions have been undertaken only during the last decade. In these latter studies, NM was identified as a genuine melanin with a strong chelating ability for iron and affinity for compounds such as lipids, pesticides, and MPP+. The affinity of NM for a variety of inorganic and organic toxins is consistent with a postulated protective function for NM. Moreover, the neuronal accumulation of NM during aging, and the link between its synthesis and high cytosolic concentration of catechols suggests a protective role. However, its putative neuroprotective effects could be quenched in conditions of toxin overload.

Determination of trace elements and evaluation of their enrichment factors in Himalayan lichens.

Within the framework of the Ev-K2-CNR research program of the Italian National Research Council a specific task project has been initiated for the identification of trace elements in lichens in remote high altitude areas. This is to evaluate the possibility of using lichens as biomonitors of trace elements of atmospheric pollution. In this preliminary study, more than 20 elements have been determined in some species of lichens collected at different altitudes (from 1300 to 5000 m) in the region of the Sagarmatha National Park (Nepal). Samples of superficial soils were also collected and analyzed to calculate the trace element enrichment factors (EF) using scandium as a normalizing element. Most of the trace elements were determined by instrumental neutron activation analysis (INAA), while lead and cadmium measurements were carried out with electrothermal atomic absorption spectroscopy (ET-AAS). The EF values as well as the elemental concentrations are reported and discussed.

The neuromelanin of human substantia nigra and its interaction with metals.

Neuromelanin (NM) is a peculiar biochemical component of several neurons in the Substantia Nigra (SN), the target area of the degenerative process in Parkinson Disease (PD). SN NM has peculiarities as to its composition and an impressive capacity of chelating metals, iron in particular, but not exclusively. Gaining insights into the structural and functional characteristics of NM should help understanding the reasons of selective vulnerability of nigral neurons in many parkinsonian conditions. From the present data a protective role of NM can be postulated until the buffering capability toward heavy metals are exhausted. The overloading of NM with iron and other metals in neurons may trigger inflammatory and degenerative processes aggravating the underlying pathological condition.

Trace elements determination in lichens and in the airborne particular matter for the evaluation of the atmospheric pollution in a region of northern Italy.

Lichens as biomonitors and neutron activation analysis as analytical technique have been employed to study the distribution of trace elements (TE) in a mountain region of north Italy (Biella) characterized by settlements of wool industry. Samples of airborne particulate matter collected onto filters, different species of lichens and samples of soils have been analyzed for the calculation of the enrichment factors (EFs) of more than 25 TE. By comparison of the corresponding EFs, the most suitable lichen species (Parmelia caperata) was selected as specific TE biomonitor of the area investigated. Samples of this lichen were collected and analyzed for the evaluation of the TE distribution in four different locations. The results obtained from the analysis of the lichens gave information about the predominant direction of pollutants transportation, while those related to the concentrations found in the air particulate allowed the evaluation of the degree of the local TE atmospheric pollution.

Preconcentration and preseparation procedure for platinum determination at trace levels by neutron activation analysis.

A procedure that simultaneously allows both preconcentration and preseparation of platinum for its determination by neutron activation analysis at trace levels (<0.1 microg x g(-1)) in environmental related matrixes is developed. The method is mainly based on a two-step chemical procedure: (i) a preirradiation concentration/separation based on a column chromatography separation where the platinum is previously retained and subsequently separated from Au, Ca, Na, Br, and P; (ii) a postirradiation separation/purification of the 199Au at 158.4 keV used for the platinum determination via [198Pt (n,gamma)-->199Pt (beta-)-->199Au]. The method eliminates the following radionuclides with the gamma lines in the region of 158 keV: 47Sc (daughter of Ca) at 159.3 keV; 199Au (produced by double neutron capture onto the natural 197Au) at 158.4 keV; 123m Te at 158.8 keV. In addition, the background activity deriving from 24Na, 32p, and 82Br is drastically reduced. The method was tested by the analysis of certified rock material and then applied for platinum determination in airborne particulate matter.

Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes.

Information on the molecular distribution and ageing trend of brain iron in post-mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H-ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of approximately 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L-Ferritin also showed an increasing trend during life although the concentrations were approximately 50% less than that of H-ferritin at each age point. Neuromelanin was not detectable during the first year, increased to approximately 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high-spin trivalent iron is probably arranged in a ferritin-like iron--oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H- and L-ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.

Some analytical problems encountered for trace elements determination in the airborne particulate matter of urban and rural areas.

The determination of trace elements (TE) in airborne particulate matter collected onto filters requires an accurate evaluation of the entire analytical procedure. Since many elements have to be determined at a few milligrams of air dust at nanogram levels, possible sources of error can arise from uncontrolled parameters such as blank of the filters, sample homogeneity, pre-analytical treatment, primary and comparator standards. These potential critical points are presented here and discussed on the basis of the experience developed in our laboratory in previous studies where several TE monitoring campaigns have been performed for air particulate matter collected in urban as well as in rural residential areas of north Italy. Instrumental neutron activation analysis (INAA) and electrothermal atomic absorption spectroscopy (ET-AAS) have been used for the investigation of more than 30 trace elements.

Trace elements total content and particle sizes distribution in the air particulate matter of a rural-residential area in north Italy investigated by instrumental neutron activation analysis.

The concentrations (ng/m3) of more than 30 trace elements have been determined in the total air particulate of a rural-residential area in north Italy. By collecting the aerosols with multistage impactors the distribution of the trace elements in the different size-fractionated particles has been also investigated. The fine 'inhalable' fraction with particles of less than 10 microns in equivalent aerodynamic diameter (PM10) as well as the subsequent finest 'respirable' fractions with particles of 0-1.1 microns (alveolar), 1.1-4.6 microns (bronchial) and 4.6-9 microns (tracheo-pharynx) have been analyzed and evaluated. Apart from Pb, Cd and, in some cases, Ni and Cu which have been determined by ETAAS (electrothermal atomic absorption spectroscopy), all measurements have been carried out by instrumental neutron activation analysis (INAA).

Content of trace elements in the respirable fractions of the air particulate of urban and rural areas monitored by neutron activation analysis.

The concentrations (ng/m3) of more than 30 trace elements have been determined in the total air particulate matter and in the size-segregated fractions collected in urban, industrialized, and rural residential areas in northern Italy by means of a multistage inertial impactor with the PM10 inlet. All measurements have been carried out by instrumental neutron activation analysis, except for Pb and Cd, which have been determined by electrothermal atomic absorption spectroscopy. Analytical quality assurance procedures have been developed with special regard to blanks, reagents, and sampling. Total concentrations and the granulometric distribution found in the different locations are reported and compared.

Cell kinetic patterns in early gastric cancer.

Early gastric cancers (EGC) may be subdivided into 2 groups by means of the use of mitotic index, apoptotic index and cell density: EGCs with high cell turnover and low cell density, which show high cell dissociation and, more frequently, lymph node invasion; EGCs with low cell turnover and high cell density. The same parameters discriminate among intestinal type tumors, when separately considered from diffuse ones. No correlation is noted of these 2 groups with transforming growth factor-alpha, epidermal growth factor receptor and p53 expression, gross type, entity of neoangiogenesis, and submucosal invasion.

Growth patterns of diffuse non-Hodgkin's lymphomas estimated from mitotic and apoptotic indices.

Growth rates of neoplasms could be calculated only on the basis of mitotic and apoptotic indices (MI and AI, respectively), assessed on tissue sections, if the duration of mitosis and apoptosis (Tm and Ta, respectively) in vivo were known. For humans, this is practically never the case. What use then can be made of MI and AI to arrive at a relative, crude estimate of the state of growth? As a model system to study this problem, we chose diffusely growing stage I + II non-Hodgkin's lymphomas (dNHL, n = 94). Cluster analysis revealed the existence of 3 highly distinct groups of dNHL (clusters I, II and III) in the MI vs. AI per case plot, with a roughly linear relation between both parameters. Most nosologic entities defined by the REAL classification comprise cases that were represented in more than one cluster. We adopted the simple formula GI (growth index) = XMI - AI, where X (= Ta/Tm) remains to be evaluated. Based on the assumption that spontaneous regressions of dNHL are rare but do occur, we estimated that X = 2 or, possibly, 3 are best fits for the pooled dNHLs studied. With the assumption of X = 2, (i) 2MI - AI gave relatively lower values for dNHL than proliferative indices such as %Ki-67+ cells; (ii) values for 2MI/AI per cluster showed a pattern inverse to that for %bcl-2+ cells; and (iii) a plot of 2MI - AI vs. 2MI/AI per case allowed the recognition, especially among NHLs with a low cell turnover, of cases where accumulation of presumably longer-lived cells is an important factor in determining growth.

Cell kinetics, morphology, and molecular IgVH gene rearrangements in Hodgkin's disease.

The present study dealt with the question of whether any cellular kinetic patterns correlate with clonal rearrangement of the IgVH gene as revealed by polymerase chain reaction on DNA extracted from lymph nodes with classical Hodgkin's disease (HD) and/or from single CD30+ cells (Hodgkin [H] and Reed-Sternberg [RS] cells). In 15/4 cases with H-RS cells of B or Null phenotype, signs of such monoclonality could be detected (group I) but not in the others (group II). CD30+/H-RS cells in group I differed slightly but significantly from those in group II in that they a) exhibited a larger fraction of cells attaining the anaphase/telophase stage of mitosis, and b) produced relatively more mononucleated cells (H) at the expense of multinucleated (RS) cells. In addition, reactive lymphoid cell (CD30-) infiltrates were considerably less dense in group I that in group II. These findings suggest that the cytokinesis of H-RS cells in group I was moderately more efficient than in group II. However, signs of monoclonality were not associated with the normalization of the mitotic process, which also proved to be disturbed in group I.

Cellular kinetic and phenotypic heterogeneity in and among Burkitt's and Burkitt-like lymphomas.

This study asks whether the known genotypic heterogeneity within and between endemic or sporadic Burkitt's lymphomas (eBLs and sBLs, n = 10 each), and Burkitt-like lymphomas (BLLs, n-12), is reflected in divergent cytokinetics and related immunophenotypes. There was strong evidence that eBL and BLL grow markedly faster than sBL, as shown by differences in mitotic and apoptotic indices. Furthermore, in BLL, the median percentage of neoplastic cells immunoreactive for the bcl-2 protein was much higher than that observed in eBL and sBL. The reverse was true for the median fraction of cells containing c-myc protein. In eBL and sBL, the median fraction of bcl-6 protein-positive cells reached values above 50 per cent, while cells of 8/12 BLLs did not contain detectable amounts of this protein. This observation indicates that in this respect, eBL and sBL resemble normal germinal centres of lymphatic tissue much more than do BLL. Evidence for infection of neoplastic cells by the Epstein-Barr virus (EBV) was observed in 9/10 cases of eBL and in 3/10 of sBL, but not in BLL. EBV-positive lymphomas were associated with distinctly lower apoptotic indices and smaller median percentages of bcl-6-positive cells than EBV-negative tumours.