Osteoblastlike Cell Adhesion on Titanium Surfaces Modified by Plasma Nitriding

Purpose: The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Materials and Methods: Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nit riding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. Results: There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Conclusions: Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:237-244

Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy

The objective of the present study was to investigate the correlation between macrophage activity and apoptosis in the polar forms of leprosy because the immunopathological phenomena involved in these forms are still poorly understood For this purpose, 29 skin biopsy samples obtained from patients with the polar forms of leprosy were analyzed. Macrophage activity and apoptosis were evaluated by immunohistochemistry using lysozyme, CD68, iNOS and caspase 3 as markers The nonparametric Mann-Whitney test and Spearman`s linear correlation test were used for statistical analysis The results suggest that the apoptosis rate is under the direct influence of macrophage activity in lesions of patients with the tuberculoid form In contrast, in lepromatous lesions other factors seem to induce programmed cell death, possibly TGF-beta. Further studies are necessary to identify additional factors involved in the immunopathogenesis of leprosy. (C) 2010 Elsevier Ltd. All rights reserved

Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?

Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were pet-formed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.

Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells

Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, Including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom After treating endothelial cells with venom toxins, we observed that the venom Interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L interned:a venom on endothelial cells is not mediated by venom internalization (C) 2010 Elsevier Ltd. All rights reserved

Recognition by toll-like receptor 2 induces antigen-presenting cell activation and Th1 programming during infection by Neospora caninum

Neospora caninum is an apicomplexan parasite responsible for major economic losses due to abortions in cattle. Toll-like receptors (TLRs) sense specific microbial products and direct downstream signaling pathways in immune cells, linking innate, and adaptive immunity. Here, we analyze the role of TLR2 on innate and adaptive immune responses during N. caninum infection. Inflammatory peritoneal macrophages and bone marrow-derived dendritic cells exposed to N. caninum-soluble antigens presented an upregulated expression of TLR2. Increased receptor expression was correlated to TLR2/MyD88-dependent antigen-presenting cell maturation and pro-inflammatory cytokine production after stimulation by antigens. Impaired innate responses observed after infection of mice genetically deficient for TLR2((-/-)) was followed by downregulation of adaptive T helper 1 (Th1) immunity, represented by diminished parasite-specific CD4(+) and CD8(+) T-cell proliferation, IFN-gamma:interleukin (IL)-10 ratio, and IgG subclass synthesis. In parallel, TLR2(-/-) mice presented higher parasite burden than wild-type (WT) mice at acute and chronic stages of infection. These results show that initial recognition of N. caninum by TLR2 participates in the generation of effector immune responses against N. caninum and imply that the receptor may be a target for future prophylactic strategies against neosporosis. Immunology and Cell Biology (2010) 88, 825-833; doi:10.1038/icb.2010.52; published online 20 April 2010

Anandamide prior to sensitization increases cell-mediated immunity in mice

The endocannabinoid system has become a topic of great interest in pharmacology due to its remarkable distribution in mammal organisms and capacity to play a modulatory role on several physiological systems, including modulation of immunity. Many studies have shown that administration of cannabinoids causes inhibitory effects on immune cells, including decreased proliferation and antigen-presenting cell (APC) costimulatory activity. In contrast, other groups have shown that some cannabinoids might present stimulatory actions on macrophage activity and T cell activation. Therefore, we aimed to investigate whether a treatment in vivo with a low dose of anandamide (0.1 mg/kg) immediately prior to sensitization would have an immunosuppressive or immunostimulatory effect on cell-mediated immunity (Th1 response) in mice. We report here that anandamide, prior to sensitization, was able to increase the Th1 response to ovalbumin in vivo and ex vivo. Anandamide increased delayed type hypersensitivity (DTH), splenocyte proliferation, and IFN-gamma production in a co-culture of adherent and non-adherent splenocytes. Moreover, anandamide prior to sensitization increased both the expression of DC co-stimulatory molecules (CD80/CD86) and IL-12/IL23 (p40) production ex vivo. We have also assessed direct effects of anandamide in the IFN-gamma/IL-4 balance of ConA-stimulated splenocytes in vitro. Anandamide at nanomolar concentrations increased the production of IFN-gamma, while such production decreased at micromolar range. Thus, anandamide induced both the increment of DC activation and IFN-gamma production, which are likely the mechanisms involved in the increase of Th1 response reported here. (C) 2010 Elsevier B.V. All rights reserved.

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

A Role for the Substance P/NK-1 Receptor Complex in Cell Proliferation in Oral Squamous Cell Carcinoma

Objective: To investigate the presence and distribution of substance P (SP) and neurokinin I receptor (NK-IR) in oral squamous cell carcinoma (OSCC) and their relationship with proliferation. Patients and Methods: Ninety OSCCs from 73 patients were immunohistochemically analyzed using monoclonal antibodies against SP, NK-IR and Ki-67 in a case and control study. Results: Seventy-one percent (n=49) of cases expressed SP on tumour cell membrane, 81.3% (n=69) in cytoplasm, 39.4% (n=28) in nucleus, 81.6% (n=71) in infiltrating lymphocytes, and 58.1% (n=43) in peritumoural or intratumoural blood vessels; 14% (n=12) of cases expressed NK-1R on tumour cell membrane, 50% (n=43) in cytoplasm, 48.3% (n=42) in infiltrating lymphocytes and 22.5% (n=18) in tumour blood vessels. All cases expressed Ki-67, which was expressed in >25% of tumour cells in 79.8% of cases (n=63). Direct significant associations were observed in SP expression between different tissue levels (p<0.01), between SP and NK-IR tumour cell membrane expression (p<0.01), and between joint,SP and NK-IR expression in tumour cell cytoplasm and a higher expression of Ki-67 (p<0.05). Conclusion: The ubiquitous presence of SP strongly suggests a role for SP/NK-1R complex in tumour development and progression and possibly for NK-IR antagonists, such as L-773060, in the management of patients with oral cancer.

G2 phase cell cycle arrest in human skin following UV irradiation

The contribution of the short wavelength ultraviolet (UV) component of sunlight to the aetiology of skin cancer has been widely acknowledged, although its direct contribution to tumour initiation or progression is still poorly understood. The loss of normal cell cycle controls, particularly checkpoint controls, are a common feature of cancer. UV radiation causes both GI and G2 phase checkpoint arrest in vitro cultured cells. In this study we have investigated the cell cycle responses to suberythemal doses of UV on skin. We have utilized short-term whole organ skin cultures, and multi parameter immunohistochemical and biochemical analysis to demonstrate that basal and suprabasal layer melanocytes and keratinocytes undergo a G2 phase cell cycle arrest for up to 48 h following irradiation. The arrest is associated with increased p16 expression but no apparent p53 involvement. This type of organ culture provides a very useful model system, combining the ease of in vitro manipulation with the ability to perform detailed molecular analysis in a normal tissue environment.

Interpretation of the reversible inhibition of adenosine deaminase by small cosolutes in terms of molecular crowding

Published results on the inhibitory effects of small cosolutes on adenosine deamination by adenosine deaminase [Kurz. L. C.. Weitkamp, E., and Frieden, C. (1987) Biochemistry 26, 3027-3032; Dzingeleski, G., and Wolfenden, R. (1993) Biochemistry 32, 9143 -9147] have been reexamined. Results for sucrose, dioxane, methanol, and ethanol are shown to be qualitatively consistent with thermodynamic interpretation in terms of molecular crowding effects arising from the occurrence of a minor increase in enzyme volume and/or asymmetry during the kinetic reaction-a conformational transition that could be either preexisting or ligand induced. Direct evidence for the existence of the putative isomeric transition is provided by active enzyme gel chromatography on Sephadex G-100, which demonstrates a negative dependence of enzyme elution volume upon substrate concentration and is therefore consistent with substrate-mediated conformational changes that favor a larger (or more asymmetric) isomeric state of the enzyme. There are thus experimental grounds for adopting the present description of the inhibitory effects of unrelated cosolutes on the kinetics of adenosine deamination by adenosine deaminase in terms of thermodynamic nonideality.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

P-gingivalis-specific T-cell lines produce Th1 and Th2 cytokines

Cytokines produced by T-cells in periodontal lesions may determine the nature of the adaptive immune response. Since different antigen-7 presenting cells (APC) may direct the Th1/Th2 response, P. gingivalis-specific T-cell lines were established by different APC subpopulations, and their cytokine profiles were determined. Peripheral blood mononuclear cells induced similar percentages of IL-4+ and IFN-gamma+ T-cells and lower percentages of IL-10+ T-cells, Epstein-Barr virus-trans formed B-cells (LCL) induced higher percentages of IL-4+ cells than IFN-gamma+ cells, with lower percentages of IL-10+ cells. Peripheral blood mononuclear cells induced a higher percent of IFN-gamma+ CD8 cells than LCL (p = 0.004). Purified B-cells, monocytes, and dendritic cells induced similar percentages of IL-4+ and IFN-gamma+ cells, although again, the percentage of IL-10+ cells was lower. The results of the present study have demonstrated that, as measured by FACS analysis of intracytoplasmic cytokines, P. gingivalis-specific T-cells produce both Th1 and Th2 cytokines, regardless of the APC population.