Characterization of a monoclonal antibody recognizing mast cells

Immunohistochemical screening for monoclonal antibodies prepared by immunization of mice with a rat osteoblastic cell population led to identification of one antibody that reacted against a small population of cells present in the soft connective tissue compartment of 21 days fetal rat calvaria. The morphology of the cells and the immunohistochemical staining characteristics (a distinct intracellular granular pattern) suggested that the antibody might be reacting specifically against mast cells. We used combined histochemistry and immunohistochemistry to further characterize this antibody, designated RCJ102. Cryosections containing calvaria bone, soft connective tissues and skin were prepared from the top of the head of 21 days fetal rats, and from adult rats cryosections of lung, muscle, adipose tissue and small intestine were prepared. Some sections were labelled by indirect immunofluorescence with RCJ102; corresponding sections were labelled histochemically with toluidine blue. There was a direct correspondence between mast cells identified histochemically and cells labelling with RCJ102 in all tissues except intestine, in which the mast cell detectable by histochemistry were not labelled by RCJ102. These results suggest that the RCJ102 antibody will be a valuable new reagent for further elucidation of the heterogeneity described between connective tissue and intestinal mucosal mast cells.

Anti-genotoxic effect of aqueous extracts of sun mushroom (Agaricus blazei Murill lineage 99/26) in mammalian cells in vitro

The sun mushroom is the popular name for the Agaricus blazei Murill fungus, a mushroom native to south-eastern Brazil, which has been frequently used in popular medicine mainly in the form of tea to treat various ailments (stress, diabetes, etc.). In the present study, the genotoxic and/or anti-genotoxic effects ofA. blazei on mammalian cells in culture was assessed by checking the increase or reduction of micronucleus (MN) frequency and comets. The sun mushroom (lineage 99/26) was used as aqueous extracts prepared (2.5%) at three different temperatures (60, 25 and 4°C). The in vitro micronucleus (MN) test in binucleated cells and comet assay were used in V79 cells cultivated in HAM-F10+DMEM medium (1:1), supplemented with 10% of fetal bovine serum. The experiments were divided into four treatment types: 1. Negative control; 2. Positive control with MMS; 3. Treatments with the three forms of extracts (60, 25 and 4°C); and 4. Treatments with the extracts in different associations (simultaneous, pre-treatment, post-treatment and simultaneous after pre-incubation for 1 h) with MMS. None of the A. blazei extracts show genotoxic activity. In the comet assay no protecting effect was found. The results obtained in the MN test showed that the three forms of extracts used had protective activity, suggesting that the compound or active ingredients of A. blazei are always present in these extracts. The greater protective efficiency of the simultaneous treatment and simultaneous treatment with pre-incubation mixture with MMS suggests that the extracts have an antimutagenic action of the desmutagenic type. © 2002 Elsevier Science Ltd. All rights reserved.

Lack of genotoxicity of formocresol, paramonochlorophenol, and calcium hydroxide on mammalian cells by comet assay

Formocresol, paramonochlorophenol, and calcium hydroxide are widely used in dentistry because of their antibacterial activities in root canal disinfection. However, the results of genotoxicity studies using these materials are inconsistent in literature. The goal of this study was to examine the genotoxic potential of formocresol, paramonochlorophenol, and calcium hydroxide using mouse lymphoma cells and human fibroblasts cells in vitro by the comet assay. Data were assessed by Kruskal-Wallis nonparametric test. The results showed that all compounds tested did not cause DNA damage for the tail moment or tail intensity parameters. These findings suggest that formocresol, paramonochlorophenol, and calcium hydroxide do not promote DNA damage in mammalian cells and that the comet assay is a suitable tool to investigate genotoxicity.

γ-Radiation induces apoptosis via sarcoplasmatic reticulum in guinea pig ileum smooth muscle cells

We investigated the effects of γ-radiation on cells isolated from the longitudinal smooth muscle layer of the guinea pig ileum, a relatively radioresistant tissue. Single doses (up to 50 Gy) reduced the amount of sarcoplasmatic reticulum and condensed the myofibrils, as shown by electron microscopy 3 days post-irradiation. After that, contractility of smooth muscle strips was reduced. Ca2+ handling was altered after irradiation, as shown in fura-2 loaded cells, with elevated basal intracellular Ca2+, reduced amount of intrareticular Ca2+, and reduced capacitive Ca2+ entry. Radiation also induced apoptosis, judged from flow cytometry of cells loaded with proprium iodide. Electron microscopy showed that radiation caused condensation of chromatin in dense masses around the nuclear envelope, the presence of apoptotic bodies, fragmentation of the nucleus, detachment of cells from their neighbors, and reductions in cell volume. Radiation also caused activation of caspase 12. Apoptosis was reduced by the administration of the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone methyl ester (Z-VAD-FMK) during the 3 day period after irradiation, and by the chelator of intracellular Ca2+, 1,2-bis(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA), from 1 h before until 2 h after irradiation. BAPTA also reduced the effects of radiation on contractility, basal intracellular Ca2+, amount of intrareticular Ca2+, capacitative Ca2+ entry, and apoptosis. In conclusion, the effects of gamma radiation on contractility, Ca2+ handling, and apoptosis appear due to a toxic action of intracellular Ca2+. Ca2+-induced damage to the sarcoplasmatic reticulum seems a key event in impaired Ca2+ handling and apoptosis induced by γ-radiation. © 2008 Elsevier B.V. All rights reserved.

Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia

Bites from snake (Bothrops genus) cause local tissue damage and systemic complications, which include alterations such as hemostatic system and acute renal failure (ARF). Recent studies suggest that ARF pathogenesis in snakebite envenomation is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (BlV) in the renal perfusion system and in cultured renal tubular cells of the type MDCK (Madin-Darby Canine kidney). BlV (10 μg/mL) reduced the perfusion pressure at 90 and 120 min. The renal vascular resistance (RVR) decreased at 120 min of perfusion. The effect on urinary flow (UF) and glomerular filtration rate (GFR) started 30 min after BlV infusion, was transient and returned to normal at 120 min of perfusion. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. The treatment with BlV caused decrease in cell viability to the lowest concentration tested with an IC50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. However, a cell death process may involve apoptosis in lower concentrations. BlV treatment (1.25 μg/mL) led to significant depolarization of the mitochondrial membrane potential and, indeed, we found an increase in the expression of cell death genes in the lower concentrations tested. The venom also evoked an increase in the cytosolic Ca2+ in a concentration dependent manner, indicating that Ca2+ may participate in the venom of B. leucurus effect. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is a result of the direct nephrotoxicity which may involve the cell death mechanism. © 2012.

Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Infrared LED irradiation photobiomodulation of oxidative stress in human dental pulp cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Studies of natural killer cells in patients with paracoccidioidomycosis

The number and activity of natural killer (NK) cells were studied in 34 untreated patients with paracoccidioidomycosis, 20 with the chronic form of the disease and 14 with the acute form. NK cells were detected with monoclonal antibody Leu-11c and the cytotoxic activity was measured using a single cell assay against K562 target cells. Both groups of patients had an increased number of circulating NK cells, their cytotoxic activity being significantly lower than in the healthy controls. These findings may be of importance in the immunological disturbances associated with paracoccidioidomycosis since NK cells exert important immune effector functions and may play a role in resistance against Paracoccidioides brasiliensis.

In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors

Lima S.A.F., Wodewotzky T.I., Lima-Neto J.F., Beltrao-Braga P.C.B. & Alvarenga F.C.L. 2012. [In vitro differentiation of mesenchimal stem cells of dogs into osteogenic precursors.] Diferenciacao in vitro de celulas-tronco mesenquimais da medula ossea de caes em precursores osteogenicos. Pesquisa Veterinaria Brasileira 32(5):463-469. Departamento de Reproducao Animal e Radiologia Veterinaria, Faculdade de Medicina Veterinaria e Zootecnia, Universidade Estadual Paulista, Campus de Botucatu, Distrito de Rubiao Junior s/n, Botucatu, SP 18618-970, Brazil. E-mail: silviavet@usp.br The aim of our research was to evaluate the potential for osteogenic differentiation of mesenchimal stem cells (MSC) obtained from dog bone marrow. The MSC were separated using the Ficoll method and cultured under two different conditions: DMEM low glucose or DMEM/F12, both containing L-glutamine, 20% of FBS and antibiotics. MSC markers were tested, confirming CD44+ and CD34- cells with flow cytometry. For osteogenic differentiation, cells were submitted to four different conditions: Group 1, same conditions used for primary cell culture with DMEM supplemented media; Group 2, same conditions of Group 1 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. Group 3, Cells cultured with supplemented DMEM/F12 media, and Group 4, same conditions as in Group 3 plus differentiation inductors Dexametazone, ascorbic acid and beta-glicerolphosphate. The cellular differentiation was confirmed using alizarin red and imunostaining with SP7/Osterix antibody. We observed by alizarin staining that calcium deposit was more evident in cells cultivated in DMEM/F12. Furthermore, by SP/7Osterix antibody immunostaining we obtained 1:6 positive cells when using DMEM/F12 compared with 1:12 for low-glucose DMEM. Based on our results, we conclude that the medium DMEM/F12 is more efficient for induction of differentiation of mesenchymal stem cells in canine osteogenic progenitors. This effect is probably due to the greater amount of glucose in the medium and the presence of various amino acids.

Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Dendritic cells (DC) represent a heterogeneous cell family of major importance for innate immune responses against pathogens and antigen presentation during infection, cancer, allergy and autoimmunity. The aim of the present study was to characterize canine DC generated in vitro with respect to their phenotype, responsiveness to toll-like receptor (TLR) ligands and T-cell stimulatory capacity. DC were derived from monocytes (MoDC) and from bone marrow hematopoietic cells cultured with either Flt3-ligand (FL-BMDC) or with GM-CSF (GM-BMDC). All three methods generated cells with typical DC morphology that expressed CD1c, CD11c and CD14, similar to macrophages. However, CD40 was only found on DC, CD206 on MPhi and BMDC, but not on monocytes and MoDC. CD1c was not found on monocytes but on all in vitro differentiated cells. FL-BMDC and GM-BMDC were partially positive for CD4 and CD8. CD45RA was expressed on a subset of FL-BMDC but not on MoDC and GM-BMDC. MoDC and FL-DC responded well to TLR ligands including poly-IC (TLR2), Pam3Cys (TLR3), LPS (TLR4) and imiquimod (TLR7) by up-regulating MHC II and CD86. The generated DC and MPhi showed a stimulatory capacity for lymphocytes, which increased upon maturation with LPS. Taken together, our results are the basis for further characterization of canine DC subsets with respect to their role in inflammation and immune responses.

Impact of culture conditions on the proliferative lifespan of human T cells in vitro

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.

Growth hormone regulates growth hormone receptor gene transcription in primary human thyroid cells

In this study the regulation of GH-receptor gene (GHR/GHBP) transcription by different concentrations of GH (0, 12.5, 25, 50, 150, 500 ng/ml) with and without variable TSH concentrations (0.5, 2, 20 mU/l) in primary human thyroid cells cultured in serum-free hormonally-defined medium was studied. The incubation time was 6 h and GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification at hourly intervals. Correlating with the GH-concentrations added a constant and significant increase of GHR/GHBP gene transcription was found. After the addition of 12.5 ng/ml GH, GHR/GHBP mRNA concentration remained constant over the incubation period of 6 h but in comparison with the experiments where no GH was added there was a significant change of GHR/GHBP mRNA expression. Following the addition of 25 ng/ml GH a slight but further increase of GHR/GHBP transcription products was seen which increased even more in the experiments where higher GH concentrations were used. These data focusing on GHR/GHBP gene transcription derived from cDNA synthesis and quantitative PCR amplification were confirmed by run-on experiments. Furthermore, cycloheximide did not affect these changes supporting the notion that GH stimulates GHR/GHBP gene transcription directly. In a second set of experiments, in combination with variable TSH levels, identical GH concentrations were used and no difference in either GHR/GHBP mRNA levels or in transcription rate (run-on experiments) could be found. In conclusion, we report data showing that primary thyroid cells express functional GH-receptors in which GH has a direct and dose dependent effect on the GHR/GHBP gene transcription. Furthermore, TSH does not a have a major impact on GHR/GHBP gene regulation.

Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients

FOXP3-expressing naturally occurring CD4(+)CD25(high) T regulatory cells (Treg) are relevant in the control of autoimmunity, and a defect in this cell population has been observed in several human autoimmune diseases. We hypothesized that altered functions of peripheral Treg cells might play a role in the immunopathogenesis of myasthenia gravis, a T cell-dependent autoimmune disease characterized by the presence of pathogenic autoantibodies specific for the nicotinic acetylcholine receptor. We report in this study a significant decrease in the in vitro suppressive function of peripheral Treg cells isolated from myasthenia patients in comparison to those from healthy donors. Interestingly, Treg cells from prednisolone-treated myasthenia gravis patients showed an improved suppressive function compared with untreated patients, suggesting that prednisolone may play a role in the control of the peripheral regulatory network. Indeed, prednisolone treatment prevents LPS-induced maturation of monocyte-derived dendritic cells by hampering the up-regulation of costimulatory molecules and by limiting secretion of IL-12 and IL-23, and enhancing IL-10. In addition, CD4(+) T cells cultured in the presence of such tolerogenic dendritic cells are hyporesponsive and can suppress autologous CD4(+) T cell proliferation. The results shown in this study indicate that prednisolone treatment promotes an environment that favors immune regulation rather than inflammation.

Toxic effects of brake wear particles on epithelial lung cells in vitro

ABSTRACT: BACKGROUND: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. RESULTS: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. CONCLUSION: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.