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Cadmium (Cd) is a human carcinogen that likely acts via epigenetic mechanisms. However, the precise role of Cd in melanoma remains to be defined. The goals of this study are to: (i) examine the effect of Cd on the proliferation rate of cutaneous and uveal melanoma cells; (ii) identify the genes affected by Cd exposure; (iii) understand whether epigenetic changes are involved in the response to Cd. The cell growth capacity increased at 48 h after Cd treatment at doses ranging from 0.5 to 10 μM. The research on the key genes regulating proliferation has shown that aberrant methylation is responsible for silencing of p16(INK4A) and caspase 8 in uveal and cutaneous melanoma cells, respectively. The methylation and expression patterns of p14(ARF), death receptors 4/5, and E-cadherin remained unmodified after Cd treatment in all the cell lines analyzed. Ectopic expression of p16(INK4A) abolished the overgrowth of uveal melanoma cells in response to Cd and the overexpression of caspase 8 drastically increased the apoptotic rate of Cd-treated cutaneous melanoma cells. In conclusion, our data suggest that hypermethylation of p16(INK4A) and caspase 8 represents the most common event linked to Cd-induced stimulation of cell growth and inhibition of cell death pathway in melanoma.

Most melanomas occur on the skin, but a small percentage of these life-threatening cancers affect other parts of the body, such as the eye and mucous membranes, including the mouth. Given that most melanomas are caused by ultraviolet radiation (UV) exposure, close attention has been paid to the impact of oxidative stress on these tumors. The possibility that key epigenetic enzymes cannot act on a DNA altered by oxidative stress has opened new perspectives. Therefore, much attention has been paid to the alteration of DNA methylation by oxidative stress. We review the current evidence about (i) the role of oxidative stress in melanoma initiation and progression; (ii) the mechanisms by which ROS influence the DNA methylation pattern of transformed melanocytes; (iii) the transformative potential of oxidative stress-induced changes in global and/or local gene methylation and expression; (iv) the employment of this epimutation as a biomarker for melanoma diagnosis, prognosis, and drug resistance evaluation; (v) the impact of this new knowledge in clinical practice for melanoma treatment.

Cadmium is a highly toxic heavy metal, which has a destroying impact on organs. Exposure to cadmium causes severe health problems to human beings due to its ubiquitous environmental presence and features of the pathologies associated with pro-longed exposure. Cadmium is a well-established carcinogen, although the underlying mechanisms have not been fully under-stood yet. Recently, there has been considerable interest in the impact of this environmental pollutant on the epigenome. Be-cause of the role of epigenetic alterations in regulating gene expression, there is a potential for the integration of cadmium-induced epigenetic alterations as critical elements in the cancer risk assessment process. Here, after a brief review of the ma-jor diseases related to cadmium exposure, we focus our interest on the carcinogenic potential of this heavy metal. Among the several proposed pathogenetic mechanisms, particular attention is given to epigenetic alterations, including changes in DNA methylation, histone modifications and non-coding RNA expression. We review evidence for a link between cadmium-induced epigenetic changes and cell transformation, with special emphasis on melanoma. DNA methylation, with reduced expression of key genes that regulate cell proliferation and apoptosis, has emerged as a possible cadmium-induced epigenetic mechanism in melanoma. A wider comprehension of mechanisms related to this common environmental contaminant would allow a better cancer risk evaluation.

Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aberrant promoter methylation and resultant silencing of TRAIL decoy receptors were reported in a variety of cancers, but to date little is known about the relevance of this epigenetic modification in melanoma. In this study, we examined the methylation and the expression status of TRAIL receptor genes in cutaneous and uveal melanoma cell lines and specimens and their interaction with DNA methyltransferases (DNMTs) DNMT1, DNMT3a, and DNMT3b. DR4 and DR5 methylation was not frequent in cutaneous melanoma but on the contrary it was very frequent in uveal melanoma. No correlation between methylation status of DR4 and DR5 and gene expression was found. DcR1 and DcR2 were hypermethylated with very high frequency in both cutaneous and uveal melanoma. The concordance between methylation and loss of gene expression ranged from 91% to 97%. Here we showed that DNMT1 was crucial for DcR2 hypermethylation and that DNMT1 and DNMT3a coregulate the methylation status of DcR1. Our work also revealed the critical relevance of DcR1 and DcR2 expression in cell growth and apoptosis either in cutaneous or uveal melanoma. In conclusion, the results presented here claim for a relevant impact of aberrant methylation of decoy receptors in melanoma and allow to understand how the silencing of DcR1 and DcR2 is related to melanomagenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We previously showed that Staphylococcus aureus and Pseudomonas aeruginosa stimulate IL-8 expression in human conjunctival epithelial cells through different signal transduction pathways. As in some cell types both the bacteria may induce the release of prostaglandin E2 (PGE2) and PGE2 may affect the expression of IL-8, we aimed at investigating whether in human conjunctival cells infected with S. aureus or P. aeruginosa the activation of IL-8 transcription was mediated by PGE2 and which were the underlying molecular mechanisms. We found that S. aureus, but not P. aeruginosa, triggered IL-8 activation by increasing COX-2 expression and PGE2 levels in a time-dependent manner. Overexpression of nucleotide-binding oligomerization domain-2 (NOD2) resulted to be essential in the enhancement of IL-8 induced by S. aureus. It dramatically activated c-jun NH2-terminal kinase (JNK) pathway which in turn led to COX2 upregulation and ultimately to IL-8 transcription. The full understanding of the S. aureus-induced biochemical processes in human conjunctival epithelium will bring new insight to the knowledge of the molecular mechanisms involved in conjunctiva bacterial infections and develop novel treatment aiming at phlogosis modulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

High levels of serum interleukin-8 (IL-8) have been detected in chronic hepatitis B (CHB) patients during episodes of hepatitis flares. We investigated whether hepatitis B virus (HBV) may directly induce IL-8 production and whether IL-8 may antagonize interferon-alpha (IFN-α) antiviral activity against HBV. We showed that CHB patients had significantly higher IL-8 levels both in serum and in liver tissue than controls. In HBV-replicating HepG2 cells, IL-8 transcription was significantly activated. AP-1, C/EBP and NF-kB transcription factors were concurrently necessary for maximum IL-8 induction. Moreover, HBx viral protein was recruited onto the IL-8 promoter and this was paralleled by IL8-bound histone hyperacetylation and by active recruitment of transcriptional coactivators. Inhibition of IL-8 increases the antiviral activity of IFN-α against HBV. Our results indicate that HBV activates IL-8 gene expression by targeting the epigenetic regulation of the IL-8 promoter and that IL-8 may contribute to reduce HBV sensitivity to IFN-α., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To study the correlation of gender, contact lens (CL) wearing, chronic drinking and chronic smoking with wound healing cytokine levels and corneal recovery after photorefractive keratectomy (PRK)., Materials and Methods: One hundred and twenty-eight age-matched patients (180 eyes) undergoing PRK were enrolled. PDGF, EGF, VEGF, HGF and TGF-β(1) protein levels were measured in tears by enzyme-linked immunosorbent assay either preoperatively or 2, 7 and 15 days after PRK. Patients were seen between one day and five days postoperatively for the evaluation of epithelial healing. Delayed re-epithelialization was defined as healing after day 5. All patients were followed for haze formation for a minimum of three months., Results: All cytokines increased significantly during the first two postoperative days (p < 0.001). PDGF, EGF, HGF decreased to the preoperative levels by day 7, whereas TGF-β1 and VEGF remained elevated over the entire period of observation of 15 days, although to a lesser extent than the second day after surgery, in CL-wearers and smokers, respectively (p < 0.01). The Pearson correlation analysis showed that: (i) CL-wearing positively correlated with TGF-β1 amounts, while chronic smoking positively correlated with VEGF production; (ii) CL-wearing and TGF-β1 amount were found to be associated with early haze formation, whereas chronic smoking and VEGF level with delayed re-epithelialization. No association was found between gender or alcohol consumption and cytokine levels or wound healing., Conclusions: Our findings highlight for the first time the important role that cigarette smoking and CL wearing may have in altering the tear cytokine network and impairing corneal epithelial wound repair after surgical injury.

We have recently reported that in astrocytoma cells the expression of interleukin-8 (IL-8) is upregulated by prostaglandin E2 (PGE2). Unfortunately, the exact mechanism by which this happens has not been clarified yet. Here, we have investigated whether IL-8 activation by PGE2 involves changes in DNA methylation and/or histone modifications in 46 astrocytoma specimens, two astrocytoma cell lines and normal astrocytic cells. The DNA methylation status of the IL-8 promoter was analyzed by bisulphite sequencing and by methylation DNA immunoprecipitation analysis. The involvement of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), as well as histone acetylation levels, was assayed by chromatin immunoprecipitation. IL-8 expression at promoter, mRNA and protein level was explored by transfection, real-time PCR and enzyme immunoassay experiments in cells untreated or treated with PGE2, 5-aza-2'-deoxycytidine (5-aza-dC) and HDAC inhibitors, alone or in combination. EMSA was performed with crude cell extracts or recombinant protein. We observed that PGE2 induced IL-8 activation through: (1) demethylation of the single CpG site 5 located at position -83 within the binding region for CEBP-β in the IL-8 promoter; (2) C/EBP-β and p300 co-activator recruitment; (3) H3 acetylation enhancement and (4) reduction in DNMT1, DNMT3a, HDAC2 and HDAC3 association to CpG site 5 region. Treatment with 5-aza-dC or HDAC inhibitors of class I HDACs strengthened either basal or PGE2-mediated IL-8 expression. These findings have elucidated an orchestrated mechanism triggered by PGE2 whereby concurrent association of site-specific demethylation and histone H3 hyperacetylation resulted in derepression of IL-8 gene expression in human astrocytoma.

Background and Aims: To investigate the single and joint effects of chronic cigarette smoking and alcohol consumption on oxidative stress in age-related macular degeneration (ARMD)., Methods: Superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (CAT) activities; malondialdehyde (MDA) levels; and DNA damage were measured in patients with early ARMD (n=211) and late ARMD (n=205), and control persons (n=262)., Results: When compared with healthy controls, early- and late-ARMD patients showed significant decreases in the activities of SOD and GSHPx, but not CAT, along with marked enhancements of MDA levels and tail parameters (p<0.01). No notable differences were observed in the early- vs the late-ARMD group for each of the above mentioned dependent variables. Multiple regression analysis revealed that in healthy subjects chronic smoking had the strongest impact on SOD and GSHPx activities, MDA levels, and amount of DNA damage, whereas in ARMD patients, the combination of smoking and drinking habits was the greatest predictor of oxidative stress., Conclusions: The combination of chronic cigarette smoking and alcohol consumption appears to be an aggravating factor that contribute to serious oxidative imbalance and DNA damage in ARMD. Thus, combined smoking/drinking by persons with this pathological condition should be considered harmful. Identification of factors exacerbating ARMD-associated oxidative stress can facilitate development and adoption of effective preventative measures for this disease.

Background and Aims: The aim of the present study was to determine whether age and gender affect the imbalance between oxidant production and antioxidant levels in age-related macular degeneration (ARMD) patients., Methods: Total superoxide dismutase (T-SOD), total glutathione peroxidase (T-GSHPx), and catalase (CAT) activities, as well as malondialdehyde (MDA), protein carbonyl (PC), 8-Hydroxy-29-deoxyguanosine (8-OHdG) and total oxidation status (TOS) levels, were measured in the following groups subdivided by age and gender: 156 early-ARMD patients; 80 wet-late ARMD patients; 72 dry-late ARMD patients; and 207 healthy controls., Results: Among all study participants, women aged 50-54 had higher T-SOD and T-GSHPx activities and lower MDA, PC, TOS and 8-OHdG levels than age-matched men (p<0.05), whereas older women were not significantly different from agematched older men. Significantly increased oxidative damage was associated with ARMD patients >60 years of age in both sexes compared with controls (p<0.01 for 60-64 and 65-69-year-old ARMD subgroups; p<0.001 for 70-74 and 75-80-year-old ARMD subgroups). Multiple regression analysis demonstrates that age significantly affects antioxidant status and oxidative damage in ARMD patients compared with controls (controls, p<0.05; ARMD patients, p<0.001). A direct correlation with antioxidant enzyme activities and an inverse correlation with oxidative DNA, protein and lipid damage were also observed in premenopausal women (controls, p<0.05; ARMD patients, plt;0.001)., Conclusions: Aging and postmenopausal status may be aggravating factors contributing to redox imbalance and oxidative damage in ARMD patients.

Background: The upregulation of microsomal prostaglandin E synthase-1 (mPGES-1) and the overexpression of interleukin-8 (IL-8) have been separately linked to glioma malignancy., Objective: To evaluate (1) the correlation between the mRNA levels of IL-8, mPGES-1, and the main transcription factors (TFs) activating the IL-8 promoter in human brain tumors of different grades; (2) the role of prostaglandin E2 (PGE2) on IL-8 activation and the expression of these TFs in tumor-derived cells; and (3) the biological impact of PGE2 treatment and mPGES-1 silencing on IL-8 synthesis and tumorigenesis., Methods: Quantitative real-time polymerase chain reaction, transfection experiments, and cell proliferation and apoptosis assays were performed., Results: Regardless of histological grade, a significant positive association between IL-8 expression and mPGES-1, CCAAT/enhancer-binding protein-β (C/EBP-β) and C/EBP Homologous Protein (CHOP) mRNA levels was found only in astrogliomas (P < .001). The correlation was not significant in the other brain tumors. PGE2-treated astroglioma cells showed a marked upregulation of IL-8, C/EBP-β, and CHOP, as well as increased proliferation and decreased apoptosis compared with untreated cells. mPGES-1-silenced astroglioma cells displayed decreased IL-8 synthesis, accompanied by reduced cell growth and an increased rate of apoptosis. The other brain tumor cells were unaffected either by PGE2 treatment or by mPGES-1 knockout., Conclusion: (1) PGE2 is responsible for IL-8 overexpression, independently of the malignancy grade, in astrogliomas only. (2) C/EBP-β and CHOP may be involved in mediating PGE2-induced IL-8 activation in these tumors. (3) mPGES-1 inhibition may have potential as a form of adjuvant therapy for astrogliomas.

Epigenetic deregulation contributes to diseases including cancer, neurodegeneration, osteodystrophy, cardiovascular defects, and obesity. For this reason, several inhibitors for histone deacetylases (HDACs) are being validated as novel anti-cancer drugs in clinical studies and display important anti-proliferative activities. While most inhibitors act on both class I, II, and IV HDACs, evidence is accumulating that class I is directly involved in regulation of cell growth and death, whereas class II members regulate differentiation processes, such as muscle and neuronal differentiation. Here, we show that the novel class II-selective inhibitor MC1568 interferes with the RAR- and peroxisome proliferator-activated receptor γ (PPARγ)-mediated differentiation-inducing signaling pathways. In F9 cells, this inhibitor specifically blocks endodermal differentiation despite not affecting retinoic acid-induced maturation of promyelocytic NB4 cells. In 3T3-L1 cells, MC1568 attenuates PPARγ-induced adipogenesis, while the class I-selective MS275 blocked adipogenesis completely thus revealing a different mode of action and/or target profile of the two classes of HDACs. Using in vivo reporting PPRE-Luc mice, we find that MC1568 impairs PPARγ signaling mostly in the heart and adipose tissues. These results illustrate how HDAC functions can be dissected by selective inhibitors.

DSS1 and p53 are required for homologous recombination, but, although p53 inactivation has a key function in skin tumorigenesis, there is no clear evidence supporting a function of DSS1. We screened the entire DSS1 coding sequence and p53 exons 5-8 in a series of 60 cases of skin squamous cell carcinoma (SCC). Mutational analysis of p53 revealed tumor-associated mutations in 28 (46.7%) of the cases. No tumor-associated DSS1 mutations were detected; however, the germline DSS1 c.143G>A synonymous polymorphism had a significantly higher frequency in patients with SCC (16.67%) versus healthy subjects (5%). DSS1 expression was evaluated by quantitative real-time RT-PCR and immunohistochemistry. With respect to c.143G>G genotype, SCCs and adjacent normal tissues carrying the c.143G>A polymorphism showed significantly lower DSS1 RNA and protein levels and a prevalent cytoplasmic rather than nuclear localization of DSS1 protein. The assay for mRNA stability revealed that the c.143G>A polymorphism affects DSS1 expression efficiency, but not mRNA decay. We clearly showed that the c.143G>A variant is associated with reduced DSS1 expression at both the RNA and protein levels and with altered traffic of the DSS1 protein from the cytoplasm to the nucleus. These alterations could impair DSS1 function in DNA repair and may be implicated in skin cancer.

Introduction: End stage renal disease (ESRD) is associated with a high incidence of cardiovascular disease and cancer. Patients undergoing hemodialysis show a reduced number and an impaired function of endothelial progenitor cells (EPCs), which in physiological conditions contribute to repair the vascular damage. In patients with ESRD, massive oxidative genome damage has been demonstrated but the role of HD in causing it is still a controversial issue. The aim of our study was to analyze the effects of a single HD session on the number of cells marked with CD34 (including sub-type cells known to be EPCs); we then evaluated the genomic damage in these cells using COMET assay., Patients and Methods: We quantified CD34(+) cells in blood samples in 30 patients in hemodiafiltration treatment for 3.5 to 4 hours 3 times/week and in 30 healthy volunteers. In HD patients, blood samples were drawn at different time intervals: start of dialysis (T(0)), at the end of the treatment (T(end)) and 24 hours afterwards in the interdialytic day (T(inter)). Staining and analysis was performed using the ISHAGE (International Society of Hematotherapy and Graft Engineering) guidelines. EPCs count was conducted using a multiparameter flow cytometric lyse no-wash method. Genomic damage was evaluated by Comet assay., Results: The number of CD34(+) cells in the HD patients at the beginning of the dialysis session (T(0)) was significantly lower than in healthy controls. HD patients showed a significant increase in CD34 number at the end of the session (T(end)) with respect to T(0). In the interdialytic period (T(int)), the number of CD34(+) cells was significantly reduced with respect to T(end). COMET assay performed on CD34(+) cells showed a higher basal level of genomic damage in HD patients than in controls; it increased in a statistically significant manner after the hemodialysis session, while in the interdialytic period it came back to T(0) level., Conclusions: Uremic status is characterized by lower levels of circulating EPCs, which increase after a single session of HD together with genomic damage to the CD34(+) cells.

Bisphosphonates (BPs) are the current standard of care for bone lesions in patients with multiple myeloma (MM) but they are associated with a number of side effects such as osteonecrosis of the jaw. The exact mechanisms of osteonecrosis are not elucidated, and its physiopathology is based on several hypotheses such as a decrease in bone remodeling or an inhibitory effect on angiogenesis. The aim of our study was to investigate the mechanism involved in the pathogenesis of osteonecrosis. We examined the apoptosis of circulating endothelial progenitor cells in MM subjects before and after BP treatment and in osteonecrosis patients using a flow-cytometric analysis. Our data showed an increase in endothelial cell apoptosis in MM patients after BP administration and in osteonecrosis subjects. Our study seems in agreement with the hypothesis that BPs can inhibit angiogenesis interfering with endothelial cell proliferation and survival, leading to loss of blood vessels and avascular necrosis., (Copyright © 2010 S. Karger AG, Basel.)

Background: Diabetes and periodontal diseases are often associated. Both have highly inflammatory components, but the role played by distinct phlogistic mediators in their pathogenesis is not fully understood and remains controversial. The purpose of this study is to evaluate whether type 2 diabetes alters the expression of inflammatory mediators in sites with chronic periodontitis (CP) or peri-implantitis (P-IM)., Methods: The expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and -8, and monocyte chemotactic protein (MCP)-1 plus key CC chemokine receptors (CCR1 through 5) and CXC chemokine receptors (CXCR1 through 3) was quantified by real-time polymerase chain reaction (PCR) in gingival or peri-implant biopsies from 135 patients with well-controlled or poorly controlled diabetes and periodontal disease, 65 patients with periodontal disease but otherwise healthy, and 90 systematically and periodontally healthy subjects. Western blots were performed., Results: Relative to controls, in patients without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 expression was exclusively higher in sites with P-IM (P <0.01), whereas IL-6 and -8 were overexpressed in sites with CP and, even more, in sites with P-IM (P <0.01). In patients with poor glycemic control, TNF-alpha, CCR5, and CXCR3 mRNAs were increased in sites with CP (P <0.01). A statistically significant higher IL-6 and -8 expression from patients without diabetes and patients with well-controlled diabetes was observed compared to patients with poorly controlled diabetes. Regardless of metabolic/glycemic status, MCP-1 and CCR2 and 4 were markedly higher in both of the oral pathologies examined (P <0.01). At the protein levels, Western blot experiments confirmed the real-time PCR results., Conclusions: These findings showed that: 1) in subjects without diabetes and patients with well-controlled diabetes, TNF-alpha, CCR5, and CXCR3 may constitute distinctive biomarkers of P-IM; 2) poor glycemic control abolished the differences between CP and P-IM regarding the expression of these mediators; and 3) type 2 diabetes affected the expression of TNF-alpha, IL-6 and -8, CCR5, and CXCR3.

Bone marrow-derived, CD34+ progenitor cells have been shown to promote the repair of damaged tissues, offering promise for the treatment of hereditary and acquired human diseases. These cells in fact differentiate into endothelia, hematopoietic cells and possibly neurons, fibroblasts and muscle. CD34+ and AC133+ progenitor cells may participate in neovascularization by differentiating into endothelial cells. Circulating bone marrow-derived endothelial cells home to sites of neovascularization and stimulate healing of injured tissues but also promote restenosis, tumor growth and inflammatory disease. These cells may thus participate in tissue regeneration or pathogenesis of several diseases. Although the molecular mechanisms that promote the homing and recruitment of bone marrow-derived progenitor cells to remodeling tissues remain unclear the evidence that these cells promote tissue repair is strong.

The purpose of this study was to identify the Pseudomonas aeruginosa-activated signaling pathway leading to interleukin (IL)-8 gene expression and protein synthesis by human conjunctival epithelium. IL-8 protein and mRNA were determined by enzyme-linked immunosorbent assay and reverse transcription-PCR, respectively. Activation of MAPKs and NF-kappaB was analyzed by Western blotting using phosphospecific antibodies. We used transfection with wild-type or mutated IL-8 promoters and cotransfection with transcription factor overexpressing plasmids or small interfering RNAs. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) were performed for in vitro and in vivo protein-DNA binding studies, respectively. P. aeruginosa increased IL-8 expression at the transcriptional level by phosphorylating CCAAT/enhancer-binding protein beta (C/EBPbeta) via p38MAPK and activating NF-kappaB. The simultaneous involvement of RelA and C/EBPbeta and the integrity of the corresponding consensus sites were required, whereas c-Jun was involved only in basal IL-8 expression. Re-ChIP experiments showed that RelA and C/EBPbeta act together at the IL-8 promoter level upon P. aeruginosa infection. Taken together, our results suggest that P. aeruginosa induces IL-8 promoter expression and protein production in conjunctival epithelial cells by activating RelA and C/EBPbeta and by promoting the cooperative binding of these transcription factors to the IL-8 promoter that in turn activates transcription.

Background: Endosseous dental titanium implants have revolutionized restorative dentistry and have made a significant impact on improved patient care. The aim of this study was to compare and evaluate the influence of the placement technique on periodontal health., Methods: A baseline examination was performed in patients with submerged and non-submerged titanium implants, including an evaluation of plaque index (PI), gingival index (GI), periodontal probing depth (PD), clinical attachment level (CAL), and bone level, as well as histamine and arachidonic acid metabolite concentrations, in the peri-implant crevicular fluid. Examinations were repeated after 12, 24, and 36 months., Results: Bone loss was significantly higher in the submerged group relative to the non-submerged group at 3 years (P <0.01), with a slight increase at 24 months. All clinical parameters were significantly higher in the submerged group relative to the non-submerged group at 24 and 36 months (P <0.05 for PI; P <0.01 for GI, PD, and CAL). The mean levels of histamine and other inflammatory mediators were significantly higher, whereas 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid concentrations were significantly reduced in the submerged group, with a high correlation with periodontal indices at 24 and 36 months (P <0.001)., Conclusion: This longitudinal study suggested that submerged implants present a number of risks for periodontal complications compared to non-submerged implants, which can be evidenced by inflammatory mediator variations in the peri-implant crevicular fluid.

Aim and Background: In breast cancer, as in almost all neoplastic diseases, the prognosis is strictly related to the invasive capacity, local and distant, that characterizes the growth of all tumors. Since the mechanisms that regulate replication of the neoplastic cells, with consequent capacity to metastasize, are not completely known, identification of new markers represents the gold standard of research in the stratification of patients with such a pathology. Osteopontin, a specific phosphoglycoprotein isolated from extracellular bone matrix and actively involved in mechanisms of bone reabsorption, appears to play a key role in osteoclastogenesis at the level of the skeleton in some pathologic situations. It has been found that patients with metastatic bone lesions from breast or prostate cancer present, with respect to subjects without repetitive bone lesions, elevated serum levels of the protein, indicating that osteopontin could play an important role in the development and progression of the neoplastic disease at the bone level., Methods and Study Design: The authors studied 26 patients with breast cancer, evaluating as a marker also serum osteopontin levels., Results and Conclusions: The results, although obtained on a small number of patients, showed that osteopontin evaluation in breast cancer patients can be a particularly interesting method of research in staging of the disease as well as in the prognosis, thereby attributing a role of a biotumoral marker also in the follow-up of the therapy.

Background: Three different homozygous loss-of-function mutations of the Forkhead box E1 (FOXE1) gene have been associated with syndromic cleft palate. Here, we screened the entire promoter region to identify the variations in significant consensus motifs affecting FOXE1 transcription., Method: Genomic DNAs of 35 cleft palate patients, 10 of whom with CHARGE association, 80 unrelated healthy people and 80 unaffected first-degree relatives were analysed by automatic sequencing. The Transcription Element Search System program was employed to identify transcription factor binding sites. The protein-DNA complexes were observed using DNA band-shift assays and oligonucleotide competition analyses. Real-time PCR was used to estimate FOXE1 expression at mRNA level., Results: In 11 non-syndromic cleft palate patients, a novel non-coding polymorphism (C-->G) in the 5'-untranslated region of FOXE1 was found. The variation fell into a putative consensus sequence for the transcription factor MYF-5 and completely impaired the ability of MYF-5 to bind to its motif, as shown by EMSA experiments. As a consequence, a significantly reduced FOXE1 mRNA expression was observed., Conclusions: In 45% of non-syndromic cleft palate patients, a novel homozygous polymorphism that prevented the binding of MYF-5 to FOXE1 promoter and affected the FOXE1 expression was found. As recent data show the role of MYF-5 in the muscle-dependent craniofacial skeletal development and in the fusion of primary palate and secondary palate, the results reported here strongly suggest a more significant involvement of this factor in the cleft palate onset.

For a long period of time, the transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP) has been thought to inhibit transcriptional activity for its ability to interact with CCAAT enhancer-binding protein family factors, thus preventing their binding to DNA. We have previously shown that in human T lymphocytes the CHOP phosphorylation induced by prostaglandin E(2) (PGE(2))-increased interleukin-8 (IL-8) gene expression. Given the CHOP positive role in the regulation of transcription, here we have investigated the molecular mechanism(s) by which CHOP increases IL-8 gene activity under PGE(2) stimulus. Transfection experiments with mutants showed both that the CHOP transactivation domain is essential for IL-8 transcription and that the IL-8/activator protein 1 (AP-1) promoter mutated in NF-kappaB and NF-IL-6, but not in the AP-1 site, harbors essential CHOP-responsive elements. CHOP silencing confirmed its role in the IL-8 transcriptional regulation and protein production, whereas c-Jun small interfering RNA experiments showed that the PGE(2)-induced activation of IL-8 promoter is mainly c-Jun-independent. Moreover, PGE(2) induced CHOP-DNA complexes only when the entire IL-8/AP-1 promoter or the wild type sequences encompassing the AP-1 upstream region were employed. Mutations introduced in these sequences prevented the DNA-CHOP complex formation. The IL-8/AP-1 mutant promoter lacking the sequence immediately upstream the AP-1 site is PGE(2)-unresponsive. Finally, chromatin immunoprecipitation data confirmed in vivo that PGE(2) induces CHOP binding to the IL-8 promoter. Taken together, our results suggest that the increased expression of CHOP in response to PGE(2) exerts a positive transcriptional regulation of the IL-8 promoter mediated by direct binding to a novel consensus site.

Endothelium was initially considered an inert lining of the blood vessels. Recently, it was suggested that damaged cells are continuously replaced by novel cells, hematopoietic stem cells (HSCs), which are directly mobilized by the bone marrow and then transformed into endothelial progenitor cells (EPCs). Initial triggers of vessel remodeling are physical forces such as blood pressure and fluid shear stress. We investigated whether or not a stress stimulus on vessels applied by a cold pressor test (CPT) would stimulate the mobilization of progenitor cells. Twenty-two healthy subjects, 20 patients with essential hypertension, and 18 with chronic kidney disease (CKD) underwent CPT by dipping their hands in icy water for 4 min. Immediately before and after 4 and 60 min, we quantified HSCs and EPCs identified by flow cytometry. We measured also adhesion soluble molecules (sICAM-1, sVCAM-1, and sE-selectin) as markers of endothelial activation. In healthy and hypertensive subjects, but not in CKD subjects, the number of HSCs was elevated as a direct response to CPT stress. Levels of EPCs and adhesion soluble molecules increased significantly, but to a different extent in every group. In CKD patients, the number of EPCs did not return to basal levels either after 60 min. Levels of adhesion soluble molecules directly correlated with the number of progenitor cells in hypertensive and healthy subjects. CPT caused an increase in adhesion soluble molecules. Discrepancies in the numbers of HSCs and EPCs in CKD patients could suggest a specific impairment in blood vessel remodeling correlated with recognized endothelial dysfunction.

Purpose: To identify signal transduction pathways involved in interleukin (IL)-8 expression by human conjunctival cells challenged with Staphylococcus aureus., Methods: Conjunctival cells were cultured in the presence of live or heat-killed S. aureus. IL-8 protein and mRNA were determined by ELISA and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs) and NF-kappaB was analyzed by Western blot analysis with phosphospecific antibodies. Conjunctival cells were transfected with wild-type (wt) or mutated IL-8 promoters (IL-8-97, lacking the AP-1 site; IL-8-97 mutant C/EBP; IL-8-97 mutant NF-kappaB; IL-8/AP-1 double mutant for C/EBP and NF-kappaB) or c-Jun-NH(2)-terminal kinase (JNK)-responsive GAL-c-Jun. In further experiments, cells were cotransfected with wt IL-8 promoter and expression plasmids for p38MAPK-responsive C/EBP homologous protein (CHOP) or wt or dominant negative transactivation domain mutant (TAM-67) c-Jun. A protein-DNA binding study was performed by electrophoretic mobility shift assay (EMSA), to identify the transcription factors bound to the IL-8 promoter., Results: S. aureus induced significant IL-8 expression and synthesis in human conjunctival epithelial cells by activating c-Jun phosphorylation and transactivation potential via JNK. The IL-8 promoter activation was NF-kappaB- and p38MAPK-independent. Transfection and EMSA experiments suggested that only AP-1 transcription factors were necessary for optimal IL-8 expression., Conclusions: Human conjunctival epithelial cells possess the ability to respond to Gram-positive S. aureus and to activate the innate immune response by the IL-8 gene expression. These results are the first to delineate the transcription factors involved in S. aureus-induced IL-8 release by conjunctival epithelium.

Objective: The aim of the present study was to analyze the concentrations of endothelial precursor cells (EPCs) during the 3 trimesters of normal pregnancy and to compare the EPC counts in women with normal pregnancy, gestational diabetes, and gestational hypertension., Study Design: The study was conducted on 21 pregnant women with single pregnancies (age range, 22 to 35 years). EPCs were quantified by flow cytometry. The subjects were divided into 3 groups, each consisting of 7 subjects: patients with gestational diabetes; patients with gestational hypertension; patients with normal pregnancy., Results: A progressive increase was found in the concentrations of EPCs during pregnancy in healthy women. In the third trimester of pregnancy, the number of CD34+ cells was significantly lower in patients with gestational diabetes than in hypertensive patients and controls; no significant differences were found between the levels of circulating CD34+ cells in hypertensive patients and those in controls. There were no significant differences between the diabetic and hypertensive patients for the percentage of cells expressing CD133 and VEGFR2, whereas in both groups the percentage of CD133+/VEGFR2+ elements was significantly higher than in the healthy control subjects., Conclusion: Our findings confirm that EPCs isolated from the maternal circulation increase gradually throughout the gestational trimesters. These cells were derived from the endothelial cells lineage, as demonstrated by CD133+/VEGFR2+ cell assay. Moreover, the concentration of EPCs in pregnant women with gestational diabetes and hypertension differs from that in subjects with a normal pregnancy, CD34+ cells being reduced but CD133+/VEGFR2+ cell concentrations being increased. These results not only substantiate recent insights into the mechanisms regulating maternal vascular modifications during pregnancy but also throw light upon the activation of different patterns in the mobilization of endothelial progenitor cells during pathologic states in which endothelial disorders occur.

During haemodialysis (HD) the endothelium is the first organ to sense and to be impaired by mechanical and immunological stimuli. We hypothesized that a single HD session induces mobilization of endothelial progenitor cells (EPCs) and that cardiovascular risk factors may influence this process. We quantified EPCs at different maturational stages (CD34+, CD133+/VEGFR2+) in blood samples from 30 patients, during HD and on the interdialytic day, and in 10 healthy volunteers. Samples were drawn at the start of HD, 1, 2 and 3 h after, at the end of HD and at 24 h on the interdialytic day. Patients were divided into two groups based on a recent risk scoring system (SCORE project): low-risk (LR) and high-risk groups (HR). HD patients showed a significantly reduced basal number of EPCs with respect to healthy volunteers. In contrast, we observed increasing EPCs during HD whereas they diminished on the interdialytic day. The EPC number was directly correlated with HD time progression. The EPC number during HD was increased in the HR group with respect to the LR group. We had a direct correlation between risk score and number of EPCs. Cardiovascular risk factors influenced the mobilization of stem cells from the bone marrow. This feature could be the direct consequence of an augmented request of stem cells to respond to the most important endothelial impairment but could also show a defective capacity of EPCs to home in and repair the sites of vascular injury., (Copyright 2007 S. Karger AG, Basel.)

Background: Some previous investigations underscored the role of histamine in periodontal disease, especially in diabetic patients, but the behavior of this inflammatory mediator in the early phases of periodontal involvement remains unclear. The aim of the present study was to correlate the presence of histamine in saliva with clinical parameters in healthy, periodontitis-affected, and diabetic subjects to ascertain whether this amine may serve as a predictive index of periodontal risk., Methods: For this purpose, subjects were selected as follows: 1) with newly diagnosed type 2 diabetes mellitus; 2) with neither diabetes nor periodontitis; 3) with no diabetes but with chronic, untreated periodontal disease. Histamine salivary levels were measured at the initial time (T0) and after 6, 12, and 24 months using high-performance liquid chromatography. The main periodontal indexes were recorded at the same time intervals., Results: At T0, a very typical shape of the histamine chromatogram was found for all patients of the three groups; at this time, the salivary histamine levels of diabetic patients were increased and comparable to those of healthy patients with periodontal disease, whereas healthy subjects with no periodontitis showed reduced histamine levels. Further controls at 6, 12, and 24 months showed a statistically significant correlation between the increase of salivary histamine and the worsening of the periodontal indexes in diabetic and non-diabetic subjects., Conclusion: These results suggest that salivary histamine may serve as a predictive index in the prevention of periodontal disease.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) and liver X receptor-alpha (LXRalpha) are nuclear ligand-activated transcription factors, which regulate lipid metabolism and inflammation. Murine J774.2 macrophages were stimulated with Escherichia coli lipopolysaccharide (concentration, 10 microg/mL) with or without the PPARgamma ligand, 15-deoxy-Delta prostaglandin J2 (15d-PGJ2), or the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317 (concentration range, 0.01-10 micromol/L), alone or in combination. Nitric oxide (NO) metabolites and tumor necrosis factor alpha production, inducible NO synthase expression, and mitochondrial respiration were measured. When added to the cells as single agents, 15d-PGJ2, 22(R)-hydroxycholesterol, or T0901317 reduced the lipopolysaccharide-induced NO and tumor necrosis factor alpha production and the inducible NO synthase expression, and partially maintained mitochondrial respiration in a concentration-dependent manner. When added to the cells in combination at suboptimal concentrations, 15d-PGJ2 with 22(R)-hydroxycholesterol, or 15d-PGJ2 with T0901317, exerted anti-inflammatory effects similar to much higher concentrations (10,000-fold to 100,000-fold) of each ligand alone. The anti-inflammatory effects of these ligands, alone or in combination, were associated with reduction of nuclear factor-kappaB activation and with enhancement of PPARgamma DNA binding. LXRalpha expression was upregulated in response to 15d-PGJ2 and to the LXRalpha ligands when added alone or in combination. Immunoprecipitation experiments revealed that PPARgamma interacted with LXRalpha. Our data demonstrate that the PPARgamma ligand, 15d-PGJ2, and the LXRalpha ligands, 22(R)-hydroxycholesterol and T0901317, although binding to different nuclear receptors (i.e., PPARgamma and LXRalpha, respectively), affect mediator production through common cell signaling events and exert a synergistic potentiation in a combined treatment at suboptimal concentrations. Thus, our data suggest that PPARgamma and LXRalpha may interact in controlling the inflammatory response in macrophages.

Denaturing high-performance liquid chromatography (DHPLC) has established itself as one of the most powerful tools for DNA variation screening. FOXE1, a highly GC-rich gene involved in syndromic cleft palate, is under investigation in thyroid dysgenesis, nonsyndromic cleft palate and squamous cell carcinoma. A technique for fast and simultaneous detection of sequence variants in the entire coding region of the FOXEl gene based on multiplex PCR/DHPLC is presented here. Given its characteristics of high sensitivity and rapidity, the testing strategy developed by us appears to be a reliable approach for FOXE1 analysis in the screening of a large population at risk.

Using PCR-SSCP screening and direct sequencing we analyzed a series of 28 colorectal carcinomas for mutations in p53 (exons 5-8) and Ki-ras (codons 12, 13 and 61), and for micro-satellite instability (MSI) at BAT25 and BAT26, supplementing data with the analysis of the IARC colorectal cancer p53 mutation database. Mutations were correlated with the site of tumor origin (proximal or distal to the splenic flexure). We identified 19 mutations in p53, 9 in Ki-ras, and 4 MSI-positive cases in a total of 20 tumors. Only 6/20 cases (30%) carried mutations in both p53 and Ki-ras. Mutations in p53 were detected at similar frequencies in proximal and distal tumors, while IARC data pointed to a strong association of p53 mutations with distal cancers. Ki-ras mutations were more frequent in proximal tumors, and MSI occurred at similar frequencies in proximal and distal tumors and was associated with mutations in p53 or Ki-ras. The p53 mutations detected in the series analyzed, as well as those retrieved from the IARC database, were predominantly transitions, with no preferential sequence localization or nucleotide position. Ki-ras mutations were predominantly transversions in position 2 at codon 12. MSI-H occurred at similar frequencies in proximal and distal tumors and was associated with either p53 or Ki-ras mutations. Overall these data suggest that distinct mutagenic factors target p53 and Ki-ras in colorectal epithelium irrespective of MSI-H status.

Background: The risk of developing cardiovascular diseases is higher in patients on haemodialysis than in the general population. These patients may develop arrhythmias that depend on the extra- and intracellular concentrations of potassium. ECG findings, particularly the QT interval and its dispersion (QT(d)) and the QT(c) (QT interval corrected for heart rate according to Bazett's formula) and its dispersion (QT(cd)), may be direct indicators of the risk of developing arrhythmia., Methods: Our cohort comprised 28 patients who were dialysed for 3.5-4 h three times per week, first with haemodiafiltration with a constant potassium concentration (HDF) in the dialysis bath then with haemodiafiltration with variable concentrations of potassium (HDF(k)). ECGs were done at different time intervals: at the start of dialysis (T(0)), at 15 (T(15)), 45 (T(45)), 90 (T(90)) and 120 min (T(120)) after the beginning of the session, and at the end of treatment (T(end)). ECG-derived data (QT, QT(d), QT(c) and QT(cd)) were measured. At the same time points, plasma electrolytes, intra-erythrocytic potassium and the electrical membrane potential at rest (REMP) of the erythrocytic membrane were measured., Results: Plasma potassium concentration diminished more gradually in HDF(k) than in HDF, the difference being statistically significant at T(15) and T(45) (P<0.05), and T(90) (P<0.01). The intra-erythrocytic potassium concentration remained constant throughout the observation period. In both HDF and HDF(k), REMP was lower at all points after T(0) (P<0.05), but the reduction was greater and more significant in HDF than in HDF(k) at T(15) and T(120) (P<0.05). ECG revealed a statistically significant diminution in HDF(k) vs HDF in the measures of dispersion of QT and QT(c) at T(15), T(90), T(120) and T(end) (P<0.01) and of QT(cd) at T(45) (P<0.05). The mean of QT(d), adjusted for plasma potassium, increased over time in HDF with large alternate mean increase and decrease peaks and error intervals. In HDF(k), instead, there was a progressive and constant diminution with minor error intervals. QT(cd) adjusted for plasma potassium had the same trend. A marked difference was found between the final values in standard HDF and those in HDF(k)., Conclusions: HDF and HDF(k) have significantly different effects on QT(c). ECG data demonstrate that the risk of arrhythmia could be lower, with a variable removal of potassium during haemodialysis. With HDF but not HDF(k), hyperpolarization of the cell membrane is detected, and this could have a destabilizing effect on different types of cardiac cell, giving rise to retrograde circuits.

The effect of prostaglandin E(2) (PGE(2)) in regulating the synthesis of the pro-inflammatory chemokine interleukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE(2) induced IL-8 mRNA transcription through prostaglandin E(2) receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE(2)-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-kappaB-independent. Our data suggest that PGE(2) acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE(2) regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.

To ascertain the effect of acetate-free biofiltration (AFB), performed with polyacrilonitrile filters, on serum concentrations of osteoprotegerin (OPG) and other bone-acting cytokines (interleukin (IL) IL-1, IL-6, tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta)) in end-stage renal disease (ESRD) patients, we evaluated these parameters during an AFB session and 24 hr after it ended. In second time we verified the existence of eventual correlations among serum levels of all these cytokines at different times. We investigated 48 subjects: 24 healthy volunteers (controls) (12 females, 12 males, mean age 55 +/- 9 yrs) and 24 ESRD patients (12 females, 12 males, mean age 58 +/- 6.7 yrs, mean dialytic age 2.7 +/- 1.6 yrs, residual glomerular filtration rate (GFR) 2.3 +/- 0.6 ml/min). All dialyzed patients received regular AFB with polyacrilonitrile filters for 4 hr thrice-weekly. Statistical analysis showed significant increase in basal serum OPG, IL-6 and TNF-alpha concentrations in dialyzed patients compared to controls, while it did not show significant variations for the other cytokines. During the dialytic session, OPG and TGF-beta concentrations did not show significant variations, while serum TNF-alpha, IL-6 and IL-1 levels significantly decreased from the 1st hour of AFB. None of the cytokines showed significant differences between basal and interdialytic values. We did not find correlations between OPG, IL-1, IL-6, TNF-alpha and TGF-beta concentrations during hemodialytic sessions and during the interdialytic interval. It is our opinion that the lack of correlation between serum concentrations, observed in our study, could not exclude the presence of local interferences between OPG and the other cytokines.

Background/aims: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells., Methods: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration., Results: In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH., Conclusion: The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation., (Copyright (c) 2005 S. Karger AG, Basel.)

The histamine content of tears of healthy sex- and age-matched subjects and patients affected by allergic or nonallergic inflammatory ocular diseases was determined through a new competitive reverse-phase high-performance liquid chromatography (HPLC) method. Tear samples from 50 healthy subjects, 30 patients affected by seasonal allergic conjunctivitis, 12 patients with bacterial conjunctivitis associated with Haemophilus influenzae and 8 patients with bacterial conjunctivitis associated with Streptococcus pneumoniae were analyzed for histamine concentration by O-phthaldialdehyde precolumn derivatization-based HPLC. In physiological conditions, the tear histamine content was low (2.26 ng/ml) and did not vary in relation to age and sex. Histamine levels were significantly higher in all the patients studied, to a greater extent in those affected by allergic (23.61 ng/ml) or Haemophilus influenzae-associated (21.53 ng/ml) conjunctivitis., (Copyright 2004 S. Karger AG, Basel)

Based on its ability to inhibit the tyrosine kinase activity of ABL, as well as the c-kit and the Platelet Derived Growth Factor Receptor tyrosine kinases, the spectrum of diseases that may respond to STI571 is increasing. A recently recognized subgroup of myeloproliferative disorders/myelodysplastic syndromes (MPD/MDS) has a t(5;12)(q33;p13) with the activation of the gene for PDGFBR which encodes a receptor tyrosine kinase. Here, we present the case of a patient, with MPD/MDS, and eosinophilia, carrying a translocation t(5;12)(q33;p13) who achieved a complete remission following treatment with STI571, 400 mg daily. At the time of writing he still remains in complete remission with an excellent performance status. There is clearly a need for further studies of STI 571in MPD/MDS with chromosomal translocations involving PDGFBR to confirm this promising initial result.

The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators.

TNF-alpha is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-alpha production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-kappa B in TNF-alpha production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as I kappa B alpha, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-alpha production, were delayed by 30-60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-kappa B (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-alpha production, although none of the inhibitors used alone was able to completely prevent TNF-alpha release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-kappa B, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-alpha production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-alpha release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.

The retina is characterized by glycolysis under aerobic conditions, mediated by lactate dehydrogenase isoenzyme-5 (LDH-5) as well as by the soluble isoenzyme of malate dehydrogenase. Bovine retina LDH and MDH isoenzymes and their activities were studied after polyamine treatment. Our results showed that LDH-5 isoenzyme presented the highest activity in untreated as well as in putrescine-treated retina. Decreased activity was present when the retina was treated with spermidine or spermine. It was demonstrated that retinic LDH-5 had a high affinity for lactate which enabled the isoenzyme to be more effective than the other LDH isoenzymes in the conversion of NADH to NAD. Therefore, the putrescine enhancing LDH-5 activity appeared to be capable of stimulating NAD-mediated rhodopsin regeneration. Putrescine induced a marked increase of both MDH isoenzymes--soluble (s-MDH) and mitochondrial (m-MDH), while spermine and spermidine mostly affected the soluble form of the enzyme. Putrescine induced a three-fold increase in s-MDH and m-MDH activities, while spermine and spermidine induced a four to five-fold increase in s-MDH. These results document the differential effects of polyamine treatment on LDH and MDH isoenzyme activities.

Both prostaglandins (PGs) and vitamin E are known to deeply affect immune responses. It is shown here that they both influence T cell-mediated immunity through reciprocal interference on the expression of cyclic-AMP responsive element binding (CREB) family proteins. CREB1 protein of human T lymphocytes was significantly modulated by a brief treatment of 5 to 10 min with PGE2. On the contrary, vitamin E appeared to be ineffective on the CREB1 behavior, while it abolished the PGE2-induced modulation of this protein. The CREB2 protein expression was also affected by PGE2 treatment, but a longer period of incubation (>20 min) was needed to observe these changes. Vitamin E showed a strong enhancing effect on CREB2 that was partially reversed by the subsequent treatment with PGE2. Our results support the idea that there is reciprocal interference between PGE2 and vitamin E on PGE2-induced signals in T lymphocytes. These data are in agreement with the reports concerning different cell systems and experimental conditions.

Background and Methods: In the present study, we tested the hypothesis that peroxynitrite and subsequent activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) play a role in the pathogenesis of multiple organ failure induced by peritoneal injection of zymosan in the rat. Animals were randomly divided into six groups (ten rats for each group). The first group was treated with ip administration of saline solution (0.9% NaCl) and served as the sham group. The second group was treated with ip administration of zymosan (500 mg/kg suspended in saline solution). In the third and fourth groups, rats received ip administration of 3-aminobenzamide (10 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. In the fifth and sixth groups, rats received ip administration of nicotinamide (50 mg/kg) 1 and 6 hrs after zymosan or saline administration, respectively. After zymosan or saline injection, animals were monitored for 72 hrs to evaluate systemic toxicity (conjunctivitis, ruffled fur, diarrhea, and lethargy), loss of body weight, and mortality., Results: A severe inflammatory response, characterized by peritoneal exudation, high plasma and peritoneal levels of nitrate/nitrite (the breakdown products of nitric oxide), and leukocyte infiltration into peritoneal exudate, was induced by zymosan administration. This inflammatory process coincided with the damage of lung, small intestine, and liver as assessed by histologic examination and by an increase of myeloperoxidase activity, which is indicative of neutrophil infiltration. Zymosan-treated rats showed signs of systemic illness, significant loss of body weight, and high mortality rates. Peritoneal administration of zymosan in the rat also induced a significant increase in the plasma levels of peroxynitrite as measured by the oxidation of the fluorescent dihydrorhodamine 123. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific "footprint" of peroxynitrite, in the lung of zymosan-shocked rats. In vivo treatment with ip administration of 3-aminobenzamide (10 mg/kg, 1 and 6 hrs after zymosan injection) or nicotinamide (50 mg/kg, 1 and 6 hrs after zymosan injection) significantly decreased mortality, inhibited the development of peritonitis, and reduced peroxynitrite formation. In addition, PARS inhibitors were effective in preventing the development of organ failure because tissue injury and neutrophil infiltration, by myeloperoxidase evaluation, were reduced in the lung, small intestine, and liver., Conclusions: In conclusion, the major findings of our study are that peroxynitrite and the consequent PARS activation exert a role in the development of multiple organ failure and that PARS inhibition is an effective anti-inflammatory therapeutic tool.

This study examined the role of social support and relationship quality on the well-being of pregnant adolescents. Sixty-seven Black and 43 White single pregnant teens participated in the study. The reciprocal exchange of support between parents and teens was correlated with increased mastery and life satisfaction and decreased depression and anxiety. However, the reciprocal exchange of support with friends did not correlate with well-being. A high quality relationship with a significant other was associated with increased self-esteem among pregnant teens dating the father of their child. This study extends the adolescent pregnancy literature by considering the reciprocal exchange of support and relationship quality with the partner., (Copyright 1999 The Association for Professionals in Services for Adolescents.)

PGE2 treatment of mononuclear cells from patients with different types of neoplasias was unable to decrease either the number of plaque-forming cells or the expression of CD71 and CD25 in PWM-driven cultures. In contrast, in previous studies, PGE2 inhibited these parameters in cultured mononuclear cells from normal volunteers. Surgical treatment of cancer patients did not modify the lymphocyte sensitivity to PGE2 after 1 week, but at 2 and 6 months after therapeutical treatment, the inhibition values of the parameters studied were almost similar or very similar to those of normal lymphocytes. The reduction of PGE2 sensitivity in cancer patients was related to the increase of PGE2 levels and, probably, to a PGE2 receptor saturation. A restoration of PGE2-induced inhibition some months after therapy could be due to the decrease in PGE2 levels and to receptor unsaturation.

Interactive coordination was observed in laboratory play interactions of pairs of 29 clinically depressed and 14 nondepressed mothers and their 13-29-month-old children (M = 18.9 months). Nondepressed mothers and their children displayed more interactive coordination than depressed-mother dyads (p < .001). Depressed mothers were less likely to repair interrupted interactions, and their toddlers were less likely to maintain interactions than nondepressed controls. Toddlers matched their nondepressed but not their depressed mothers negative behavior rates. Results suggested that early interventions focus on training mothers to attend to maintain, and repair mother-child interactions to more closely approximate normal levels of interactive coordination.

Construct validity of the newly developed Preschool Assessment of Attachment (PAA) was examined in a sample of depressed and nondepressed mothers and their preschoolers, focusing on attachment related differences in children's general caregiving environments, maternal psychosocial functioning, and child behavior during interactions with mother. Mothers of secure children were more emotionally and verbally responsive to their children than were mothers of insecure children, and secure children were emotionally more positive to their mothers than were insecure children. Mothers of secure children also reported higher levels of social supports than did mothers of insecure children. Finally, dyads with children who lacked unitary, coherent attachment strategies (i.e., anxious depressed, defended/coercive, and insecure other) showed the worst functioning in all domains relative to all other attachment groups. Similar but slightly less robust findings were obtained with socioeconomic variables statistically controlled. These results lend support to the PAA as a valid system for the conceptualization and measurement of quality of attachment among preschoolers. Future research applications with the PAA are discussed.