Biological activity of metal-edds (ethylenediaminedisuccinate) complexes in K562 and PBMC cells

The effect of S,S-ethylenediaminedisuccinic acid (edds) on the quenching of metal-catalyzed (metal = Mn, Fe, Co, Ni, Cu, Zn) oxidation of ascorbic acid was tested in vitro via oxidation of the fluorescent probe 1,2,3-dihydrorhodamine dihydrochloride. The pro-oxidant activity of iron was not fully suppressed, even at a four-fold molar excess of the ligand. The effect of serum on the toxicity to peripheral blood mononuclear cells (PBMC) and K562 cells was investigated. The cytotoxic effect of Fe-edds was abrogated in the presence of Trolox or serum proteins. The probable pathways of cell toxicity were investigated through blocking of the monocarboxylate transporters (MCT) in association with cell cycle studies by flow cytometry. Cells treated with metal complexes and alpha-cyano-4-hydroxycinnamic acid, a known MCT inhibitor, showed recovery of viability, suggesting that MCT proteins may be involved in the internalization of metal-edds complexes. The free acid induced cell cycle arrest in G0/G1 (PBMC) and S (K562) phases, suggesting direct DNA damage or interference in DNA replication.

Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia

The detection of minimal residual disease (MRD) is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL) providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC) with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM) and peripheral blood (PB) of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d) 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28). MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758) (Testudines, Cheloniidae)

Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA) for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae) Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Horizontal subsurface flow constructed wetlands for mitigation of ametryn-contaminated water

The feasibility of using constructed wetlands (CWs) for the mitigation of pesticide runoff has been studied in the last decade. However, a lack of related data was verified when subsurface flow constructed wetlands (SSF CWs) are considered for this purpose. In the present work, SSF CWs were submitted to continuous ametryn addition and evaluated during an I I-week period, with the aim of determining the feasibility of these systems for mitigation of contaminated water. Ametryn was not added to one CW cell in order to provide a control for the experiments. Monitoring of treatment performance was executed by standard water quality parameters, ametryn chromatography quantification and macrophyte (Typha latifolia L) nutritional and agronomic property analysis. Results indicated that 39% of the total initially added amount of ametryn was removed, transferred or transformed. Herbicide metabolism and mineralisation were carried out by chemical and biological mechanisms. No statistic differences were observed in nutritional contents found in the T. latifolia crops of the CWs after the experimental period. Moreover, the biomass production (one valuable source of renewable energy) was equal to 3.3 t.ha(-1) (dry matter) in wetland cells. It was concluded that constructed wetland systems are capable of mitigating water contaminated with ametryn, acting as buffer filters between the emission sources and the downstream superficial water bodies.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Renal hypothermia: Experience in pigs and clinical trial

Purpose: This study was designed to compare the effectiveness of two methods of inducing renal hypothermia through laparoscopy in pigs and humans. Materials and Methods: Twelve pigs were divided into four groups of three animals each. Both kidneys of the animals in Groups A, B, and C were submitted to pelvic irrigation with cold saline (4 degrees C) for 20 minutes, with flow rates of 5 mL/min, 10 mL/min, and 15 mL/min, respectively. In Group D renal hypothermia was induced by intracorporeal ice slush applied to the surface for 20 minutes. All maneuvers were performed laparoscopically and renal cortex temperature was measured by a thermocouple needle. Five human patients also underwent laparoscopic partial nephrectomy due to renal cell carcinoma. In one case renoprotection was induced by retrograde endoscopic cold saline perfusion at a flow rate of 10 mL/min. In the remaining four patients we induced renal hypothermia via laparoscopic application of ice slush. The renal temperature of the human patients was also monitored using a thermocouple needle. Results: In the pigs, at 20 minutes of renal pelvis perfusion the mean renal temperature, the temperature drop, and saline flow per gram of kidney were: Group A, -29.5 degrees C +/- 1.1 (-6.3 degrees C; 0.10 mL); Group B, -22.8 degrees C +/- 1.1 (-13.1 degrees C; 0.22 mL); and Group C, -21.1 degrees C +/- 0.9 (-14.9 degrees C; 0.31 mL). In Group D the mean renal cortex temperature at 20 minutes was 13.6 degrees C +/- 1.2, a drop of -22.5 degrees C. There were striking differences among the groups (P < 0.0001). The laparoscopic partial nephrectomy was uneventful in all five human patients. The lowest renal cortex temperature was 32.5 degrees C, seen in the patient who submitted to pelvic irrigation with cold saline, and the mean temperature drop was 19.1 degrees C +/- 2.5 degrees C in the patients who submitted to ice slush-induced renal hypothermia. Conclusions: Induction of renal hypothermia using intracorporeal ice slush confers lower kidney temperatures than endoscopically-induced cold saline perfusion.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

Background: The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs). Methods: Lineages of hFTs were expanded, had their karyotype analyzed, were characterized by flow cytometry and underwent in vitro adipogenic, chondrogenic, osteogenic, and myogenic differentiation. Results: Here we show for the first time that hFTs, which are discarded after some gynecological procedures, are a rich additional source of MSCs, which we designated as human tube MSCs (htMSCs). Conclusion: Human tube MSCs can be easily isolated, expanded in vitro, present a mesenchymal profile and are able to differentiate into muscle, fat, cartilage and bone in vitro.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Low-Level Laser Irradiation (InGaAlP-660 nm) Increases Fibroblast Cell Proliferation and Reduces Cell Death in a Dose-Dependent Manner

Background and Objective: Impaired cell metabolism and increased cell death in fibroblast cells are physiological features of chronic tendinopathy. Although several studies have shown that low-level laser therapy (LLLT) at certain parameters has a biostimulatory effect on fibroblast cells, it remains uncertain if LLLT effects depend on the physiological state. Study Design/Material and Methods: High-metabolic immortal cell culture and primary human keloid fibroblast cell culture were used in this study. Trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine cell viability and proliferation. Propidium iodide stain was used for cell-cycle analysis by flow cytometry. Laser irradiation was performed daily on three consecutive days with a GaAlAs 660-nm laser (mean output: 50 mW, spot size 2 mm(2), power density = 2.5 W/cm(2)) and a typical LLLT dose and a high LLLT dose (irradiation times: 60 or 420 s; fluences: 150 or 1050 J/cm(2); energy delivered: 3 or 21 J). Results: Primary fibroblast cell culture from human keloids irradiated with 3 J showed significant proliferation by the trypan blue exclusion test (p < 0.05), whereas the 3T3 cell culture showed no difference using this method. Propidium iodide staining flow cytometry data showed a significant decrease in the percentage of cells being in proliferative phases of the cell cycle (S/g(2)/M) when irradiated with 21 J in both cell types (hypodiploid cells increased). Conclusions: Our data support the hypothesis that the physiological state of the cells affects the LLLT results, and that high-metabolic rate and short-cell-cycle 3T3 cells are not responsive to LLLT. In conclusion, LLLT with a dose of 3 J reduced cell death significantly, but did not stimulate cell cycle. A LLLT dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10. RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Complex Oscillatory Response of a PEM Fuel Cell Fed with H(2)/CO and Oxygen

Oscillatory kinetics is commonly observed in the electrocatalytic oxidation of most species that can be used in fuel cell devices. Examples include formic acid, methanol, ethanol, ethylene glycol, and hydrogen/carbon monoxide mixtures, and most papers refer to half-cell experiments. We report in this paper the experimental investigation of the oscillatory dynamics in a proton exchange membrane (PEM) fuel cell at 30 degrees C. The system consists of a Pt/C cathode fed with oxygen and a PtRu (1:1)/C anode fed with H(2) mixed with 100 ppm of CO, and was studied at different cell currents and anode flow rates. Many different states including periodic and nonperiodic series were observed as a function of the cell current and the H(2)/CO flow rate. In general, aperiodic/chaotic states were favored at high currents and low flow rates. The dynamics was further characterized in terms of the relationship between the oscillation amplitude and the subsequent time required for the anode to get poisoned by carbon monoxide. Results are discussed in terms of the mechanistic aspects of the carbon monoxide adsorption and oxidation. (C) 2010 The Electrochemical Society. [DOI: 10.1149/1.3463725] All rights reserved.

An environmentally friendly flow system for high-sensitivity spectrophotometric determination of free chlorine in natural waters

A green and highly sensitive analytical procedure was developed for the determination of free chlorine in natural waters, based on the reaction with N,N-diethyl-p-phenylenediamine (DPD). The flow system was designed with solenoid micro-pumps in order to improve mixing conditions by pulsed flows and to minimize reagent consumption as well as waste generation. A 100-cm optical path flow cell based on a liquid core waveguide was employed to increase sensitivity. A linear response was observed within the range 10.0 to 100.0 mu g L(-1), with the detection limit, coefficient of variation and sampling rate estimated as 6.8 mu g (99.7% confidence level), 0.9% (n = 20) and 60 determinations per hour, respectively. The consumption of the most toxic reagent (DPD) was reduced 20,000-fold and 30-fold in comparison to the batch method and flow injection with continuous reagent addition, respectively. The results for natural and tap water samples agreed with those obtained by the reference batch spectrophotometric procedure at the 95% confidence level. (C) 2010 Elsevier By. All rights reserved.