High frequency of immature dendritic cells and altered in situ production of interleukin-4 and tumor necrosis factor-alpha in lung cancer

Introduction Antigen-presenting cells, like dendritic cells (DCs) and macrophages, play a significant role in the induction of an immune response and an imbalance in the proportion of macrophages, immature and mature DCs within the tumor could affect significantly the immune response to cancer. DCs and macrophages can differentiate from monocytes, depending on the milieu, where cytokines, like interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) induce DC differentiation and tumor necrosis factor (TNF)-alpha induce DC maturation. Thus, the aim of this work was to analyze by immunohistochemistry the presence of DCs (S100+ or CD1a+), macrophages (CD68+), IL-4 and TNF-alpha within the microenvironment of primary lung carcinomas. Results Higher frequencies of both immature DCs and macrophages were detected in the tumor-affected lung, when compared to the non-affected lung. Also, TNF-alpha-positive cells were more frequent, while IL-4-positive cells were less frequent in neoplastic tissues. This decreased frequency of mature DCs within the tumor was further confirmed by the lower frequency of CD14-CD80+ cells in cell suspensions obtained from the same lung tissues analyzed by flow cytometry. Conclusion These data are discussed and interpreted as the result of an environment that does not oppose monocyte differentiation into DCs, but that could impair DC maturation, thus affecting the induction of effective immune responses against the tumor.

Mesenchymal Stem Cells and Bovine Bone Mineral in Sinus Lift Procedures-An Experimental Study in Sheep

Introduction: New reconstructive and less invasive methods have been searched to optimize bone formation and osseointegration of dental implants in maxillary sinus augmentation. Purpose: The aim of the presented ovine split-mouth study was to compare bovine bone mineral (BBM) alone and in combination with mesenchymal stem cells (MSCs) regarding their potential in sinus augmentation. Material and Methods: Bilateral sinus floor augmentations were performed in six adult sheep. BBM and MSCs were placed into the test side and only BBM in the contra-lateral control side of each sheep. Animals were sacrificed after 8 and 16 weeks. Augmentation sites were analyzed by computed tomography, histology, and histomorphometry. Results: The initial volumes of both sides were similar and did not change significantly with time. A tight connection between the particles of BBM and the new bone was observed histologically. Bone formation was significantly (p = 0.027) faster by 49% in the test sides. Conclusion: The combination of BBM and MSCs accelerated new bone formation in this model of maxillary sinus augmentation. This could allow early placement of implants.

Mesenchymal stem cells and inorganic bovine bone mineral in sinus augmentation: comparison with augmentation by autologous bone in adult sheep

Our aim was to compare the osteogenic potential of mononuclear cells harvested from the iliac crest combined with bovine bone mineral (BBM) (experimental group) with that of autogenous cancellous bone alone (control group). We studied bilateral augmentations of the sinus floor in 6 adult sheep. BBM and mononuclear cells (MNC) were mixed and placed into one side and autogenous bone in the other side. Animals were killed after 8 and 16 weeks. Sites of augmentation were analysed radiographically and histologically. The mean (SD) augmentation volume was 3.0 (1.0) cm(3) and 2.7 (0.3) cm(3) after 8 and 16 weeks in the test group, and 2.8 (0.3) cm(3) (8 weeks) and 2.8 (1.2) cm(3) (16 weeks) in the control group, respectively. After 8 weeks, histomorphometric analysis showed 24 (3)% BBM, and 19 (11)% of newly formed bone in the test group. The control group had 20 (13%) of newly formed bone. Specimens after 16 weeks showed 29 (12%) of newly formed bone and 19 (3%) BBM in the test group. The amount of newly formed bone in the control group was 16 (6%). The results show that mononuclear cells, including mesenchymal stem cells, in combination with BBM as the biomaterial, have the potential to form bone. (C) 2009 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Biological effects of insulin on murine melanoma cells and fish erythrophoroma cells: A comparative study

Insulin is the hormone that plays an essential role in metabolism and mitosis of normal and tumor cells, exerting its pleiotropic effects through binding to specific membrane receptors and promoting the phosphorylation of tyrosine residues of the receptor itself and of other components of the signaling pathway. The aim of this study was to investigate the effects of insulin on melanogenesis and cell growth in three different cell lines: the goldfish GEM-81 erythrophoroma cells (undifferentiated and differentiated with 1.5% dimethylsulfoxide-DMSO), and the murine B16F10 and Cloudman S91 melanoma cells. Undifferentiated GEM-81 and B16F10 cells responded to insulin with a small increase of cell proliferation, whereas S91 cells responded with a decrease of growth. In the two mammalian cell lines, and in DMSO-differentiated GEM-81 cells, the hormone strongly inhibited melanogenesis, by decreasing tyrosinase activity. In undifferentiated GEM-81 cells, insulin had no effect on tyrosinase activity. An increase in the tyrosine phosphorylation status of pp 185 (insulin receptor substrate 1 and 2-IRS-1/2) phosphorylation degree was observed in S91 mouse melanoma and in differentiated GEM-81 erythrophoroma cells, suggesting that this specific protein was maintained during transformation process and participates in insulin signaling. Our results imply an ancient and diverse history of the insulin signaling system in vertebrate pigment cells. (C) 2008 Elsevier Inc. All rights reserved.

Upregulation of E2F1 in cerebellar neuroprogenitor cells and cell cycle arrest during postnatal brain development

In the developing cerebellum, proliferation of granular neuroprogenitor (GNP) cells lasts until the early postnatal stages when terminal maturation of the cerebellar cortex occurs. GNPs are considered cell targets for neoplastic transformation, and disturbances in cerebellar GNP cell proliferation may contribute to the development of pediatric medulloblastoma. At the molecular level, proliferation of GNPs is regulated through an orchestrated action of the SHH, NOTCH, and WNT pathways, but the underlying mechanisms still need to be dissected. Here, we report that expression of the E2F1 transcription factor in rat GNPs is inversely correlated with cell proliferation rate during postnatal development, as opposed to its traditional SHH-dependent induction of cell cycle. Proliferation of GNPs peaked at postnatal day 3 (P3), with a subsequent continuing decrease in proliferation rates occurring until P12. Such gradual decline in proliferating neuroprogenitors paralleled the extent of cerebellum maturation confirmed by histological analysis with cresyl violet staining and temporal expression profiling of SHH, NOTCH2, and WNT4 genes. A time course analysis of E2F1 expression in GNPs revealed significantly increased levels at P12, correlating with decreased cell proliferation. Expression of the cell cycle inhibitor p18 (Ink4c) , a target of E2F1, was also significantly higher at P12. Conversely, increased E2F1 expression did not correlate with either SMAC/DIABLO and BCL2 expression profiles or apoptosis of cerebellar cells. Altogether, these results suggest that E2F1 may also be involved in the inhibition of GNP proliferation during rat postnatal development despite its conventional mitogenic effects.

Amino acids and diabetes: implications for endocrine, metabolic and immune function

Aberrant alterations in glucose and lipid concentrations and their pathways of metabolism are a hallmark of diabetes. However, much less is known about alterations in concentrations of amino acids and their pathways of metabolism in diabetes. In this review we have attempted to highlight, integrate and discuss common alterations in amino acid metabolism in a wide variety of cells and tissues and relate these changes to alterations in endocrine, physiologic and immune function in diabetes.

TLR2 Is a Negative Regulator of Th17 Cells and Tissue Pathology in a Pulmonary Model of Fungal Infection

To study the role of TLR2 in a experimental model of chronic pulmonary infection, TLR2-deficient and wild-type mice were intratracheally infected with Paracoccidioides brasiliensis, a primary fungal pathogen. Compared with control, TLR2(-/-) mice developed a less severe pulmonary infection and decreased NO synthesis. Equivalent results were detected with in vitro-infected macrophages. Unexpectedly, despite the differences in fungal loads both mouse strains showed equivalent survival times and severe pulmonary inflammatory reactions. Studies on lung-infiltrating leukocytes of TLR2(-/-) mice demonstrated an increased presence of polymorphonuclear neutrophils that control fungal loads but were associated with diminished numbers of activated CD4(+) and CD8(+) T lymphocytes. TLR2 deficiency leads to minor differences in the levels of pulmonary type 1 and type 2 cytokines, but results in increased production of KC, a CXC chemokine involved in neutrophils chemotaxis, as well as TGF-beta, IL-6, IL-23, and IL-17 skewing T cell immunity to a Th17 pattern. In addition, the preferential Th17 immunity of TLR2(-/-) mice was associated with impaired expansion of regulatory CD4(+)CD25(+)FoxP3(+) T cells. This is the first study to show that TLR2 activation controls innate and adaptive immunity to P. brasiliensis infection. TLR2 deficiency results in increased Th17 immunity associated with diminished expansion of regulatory T cells and increased lung pathology due to unrestrained inflammatory reactions. The Journal of Immunology, 2009, 183: 1279-1290.

CD4(+) CD25(+) Foxp3(+) Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4(+) CD25(+) Foxp3(+) Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123(+)), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-alpha) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and longlasting protective immunity to malaria.

Reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells and diminished FOXP3 expression in patients with Common Variable Immunodeficiency: A link to autoimmunity?

Common Variable Immunodeficiency (CVID) is a primary immunodeficiency disease characterized by defective immunoglobulin production and often associated with autoimmunity. We used flow cytometry to analyze CD4(+)CD25(HIGH)FOXP3(+) T regulatory (Treg) cells and ask whether perturbations in their frequency in peripheral blood could underlie the high incidence of autoimmune disorders in CVID patients. In this study, we report for the first time that CVID patients with autoimmune disease have a significantly reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells in their peripheral blood accompanied by a decreased intensity of FOXP3 expression. Notably, although CVID patients in whom autoimmunity was not diagnosed had a reduced frequency of CD4(+)CD25(HIGH)FOXP3(+) cells, FOXP3 expression levels did not differ from those in healthy controls. In conclusion, these data suggest compromised homeostasis of CD4(+)CD25(HIGH)FOXP3(+) cells in a subset of CVID patients with autoimmunity, and may implicate Treg cells in pathological mechanisms of CVID. (C) 2009 Elsevier Inc. All rights reserved.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

Cellular and tissue effects induced by photogemA (R) and red LED in photodynamic therapy

In order to consider the photodynamic therapy (PDT) as a clinical treatment for candidosis, it is necessary to know its cytotoxic effect on normal cells and tissues. Therefore, this study evaluated the toxicity of PDT with PhotogemA (R) associated with red light-emitting diode (LED) on L929 and MDPC-23 cell cultures and healthy rat palatal mucosa. In the in vitro experiment, the cells (30000 cells/cm(2)) were seeded in 24-well plates for 48 h, incubated with PhotogemA (R) (50, 100, or 150 mg/l) and either irradiated or not with a red LED source (630 +/- 3 nm; 75 or 100 J/cm(2); 22 mW/cm(2)). Cell metabolism was evaluated by the MTT assay (ANOVA and Dunnet`s post hoc tests; p < 0.05) and cell morphology was examined by scanning electron microscopy. In the in vivo evaluation, PhotogemA (R) (500 mg/l) was applied to the palatal mucosa of Wistar rats during 30 min and exposed to red LED (630 nm) during 20 min (306 J/cm(2)). The palatal mucosa was photographed for macroscopic analysis at 0, 1, 3, and 7 days posttreatment and subjected to histological analysis after sacrifice of the rats. For both cell lines, there was a statistically significant decrease of the mitochondrial activity (90-97%) for all PhotogemA (R) concentrations associated with red LED regardless of the energy density. However, in the in vivo evaluation, the PDT-treated groups presented intact mucosa with normal characteristics both macroscopically and histologically. From these results, it may be concluded that the association of PhotogemA (R) and red LED caused severe toxic effects on normal cell cultures, characterized by the reduction of mitochondrial activity and morphological alterations, but did not cause damage to the rat palatal mucosa in vivo.

Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing Mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial, DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was upregulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.

Unravelling the Chemical Morphology of a Mesoporous Titanium Dioxide Interface by Confocal Raman Microscopy: New Clues for Improving the Efficiency of Dye Solar Cells and Photocatalysts

The presence of anatase and rutile domains on nanocrystalline films of P25 TiO(2), as well as the distinct coordination modes of carboxylates on those phases, were revealed by confocal Raman microscopy, a technique that showed to be suitable for imaging the chemical morphology down to submicrometric size.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.