Paralytic shellfish toxins (PSTs) are a group of toxins that cause paralytic shellfish poisoning through blockage of voltage-gated sodium channels. PSTs are produced by prokaryotic freshwater cyanobacteria and eukaryotic marine dinoflagellates. Proliferation of toxic algae species can lead to harmful algal blooms, during which seafood accumulate high levels of PSTs, posing a health threat to consumers. The existence of PST-transforming enzymes was first remarked due to the divergence of PST profiles and concentrations between contaminated bivalves and toxigenic organisms. Later, several enzymes involved in PST transformation, synthesis and elimination have been identified. The knowledge of PST-transforming enzymes is necessary for understanding the processes of toxin accumulation and depuration in mollusk bivalves. Furthermore, PST-transforming enzymes facilitate the obtainment of pure analogues of toxins as in natural sources they are present in a mixture. Pure compounds are of interest for the development of drug candidates and as analytical reference materials. PST-transforming enzymes can also be employed for the development of analytical tools for toxin detection. This review summarizes the PST-transforming enzymes identified so far in living organisms from bacteria to humans, with special emphasis on bivalves, cyanobacteria and dinoflagellates, and discusses enzymes’ biological functions and potential practical applications.
Overall, laboratory water quality analysis must have stability in their results, especially in laboratories accredited by ISO 17025. Accredited parameters should be strictly reliable. Using control charts to ascertain divergences between results is thus very useful. The present work applied a methodology of analysis of results through control charts to accurately monitor the results for a wastewater treatment plant. The parameters analyzed were pH, BOD5, COD, total suspended solids, and total phosphorus. The stability of the results was analyzed from the control charts and 30 analyses performed in the last 12 months. From the results, it was possible to observe whether the results are stable, according to the rehabilitation factor that cannot exceed WN = 1.00 and the efficiency of removal of pollutants that remained above 70% for all parameters. The method of determining the technological reliability and stability of the treatment station using control charts is an efficient tool for detecting any instability in the results. These results help to monitor the results of the analyses more clearly and thus enable a rapid response to possible disturbances and maintain the quality of the analysis control, as well as determining the accreditation entities.
Background The pathogenesis of systemic sclerosis (SSc) is largely unknown, although proinflammatory cytokines are considered to play an important role. Aims Investigate the pattern of expression of proinflammatory cytokines by peripheral blood (PB) Th17 cell and IL-2 and IL-17 relative gene expression in SSc and to explore their clinical associations. Methods This study included 41 SSc patients and 20 age- and sex-matched healthy controls (HC). SSc patients were classified according to LeRoy et al. as having limited cutaneous SSc (lSSc, n = 29) or diffuse cutaneous SSc (dSSc, n = 12). A further subdivision was made based upon clinical parameters of disease duration and clinical manifestations. Intracellular expression of IL-17, IL-2, TNF-α and IFN-γ in Th17 cells was assessed by flow cytometry. A detailed functional activity characterisation of Th17 cells was performed in a single multicolour data file obtained after merging the original six-colour-staining (eight-parameter) data files from each sample using the Infinicyt (Cytogonos) software. Relative gene expression of IL-2 and IL-17 in total PB cells was determined by qRT-PCR Results No differences were observed concerning the frequency of Th17 cells or Tc17 cells between SSc patients and HC. Th17 cells from both diffuse and limited SSc showed a higher expression of IL-2 (particularly in dSSc) than Th17 cells from HC. Similar observations were made for TNF-α expression but it only reached statistical significance in lSSc. Likewise, an increased frequency of Tc17 cells expressing IL-2 in lSSc was also observed. Eight distinct Th17 cell subpopulations according to their pattern of cytokine expression (IL-17, IL-2, TNF-α and IFN-γ) were identify after merging data files. A higher frequency of Th17 cells simultaneously producing simultaneously IL17, IL-2 and TNF-α was observed in lSSc. On the other hand a significantly lower frequency of Th17 cells simultaneously producing IL-17, TNF-α and IFN-γ was also observed in the same group. The percentage of Th17 cells producing only TNF-α or IFN-γ was significantly decreased in SSc patients compared to HC group. Moreover a higher IL-17 mRNA expression in total PB cells was observed in SSc patients and no differences were found concerning IL-2 mRNA levels. Conclusions These results suggest that Th17 cells are functionally altered in SSc patients, exhibiting a higher ability to simultaneously produce IL-17, IL-2 and TNF-α. We also observed in SSc patients increased levels of IL-17 mRNA expression in total PB cells. These data suggest that Th17 cells may be involved in pathophysiology and/or disease progression of SSc.
Out of control proliferation of toxic phytoplankton, called harmful algal blooms (HABs), have a significant economic impact on bivalve aquaculture and harvesting in coastal waters. Some phytotoxins, such as paralytic shellfish toxins (PSTs), are of concern due to the life-threatening symptoms they can cause. Development of rapid and low-cost screening tools would be a welcome addition to the laboratory methodologies employed in routine monitoring programs. However, most of the assays and biosensors for the screening of PSTs, are restricted to a single target, saxitoxin (STX), which is the most potent PST. The present study aimed at developing an assay for the detection of N-sulfocarbamoyl PST—GTX5, which is one of the most abundant toxins in bivalves during G. catenatum blooms as found on the Portuguese coast. Enzymatic assay employing PSTs’ transforming enzyme—carbamoylase—was proposed. Carbamoylase was extracted and purified from the surf clam S. solida. Carbamoylase displayed similar specificity to both carbamate (STX) and N-sulfocarbamate toxins (GTX5 and C1+2) converting them into decarbamoyl saxitoxin (dcSTX) and decarbamoyl gonyautoxins 2+3 (dcGTX2+3), respectively. The enzymatic assay involved hydrolysis of GTX5 by carbamoylase and quantification of the product of enzymatic reaction, dcSTX, using a potentiometric chemical sensor. A potentiometric sensor with plasticized PVC membrane that displayed sensitivity to dcSTX and selectivity in the presence of GTX5 was employed. Enzymatic assay allowed determination of GTX5 in the concentration range from 0.43 to 3.30 µmolL−1, which encompasses levels of GTX5 in contaminated bivalve extracts with toxicities above PSTs regulatory limits. The feasibility of the carbamoylase-based potentiometric assay for detection of GTX5 was demonstrated.
Background: CD28null T cells are terminally differentiated T cells lacking CD28 co-receptor. These cells display properties of proinflammatory killer cells 1,2 and are suggested to be resistant to apoptosis in vivo 3 . Frequencies of CD28null T cells are increased in various chronic, inflammatory diseases. CD28null T cells dominate both in the affected muscle and peripheral blood of patients with idiopathic inflammatory myopathies 2,4 (myositis), suggesting a role these cells in disease mechanism and muscle pathology. Recently, it was found in our lab that after conventional glucocorticoid treatment, the relative number of regulatory T cells (Tregs) was unchanged or decreased, while the CD28null T cell proportion was mainly increased in muscle tissue of myositis patients (unpublished data). This leads to our working hypothesis that CD28null T cells are resistant to immunosuppression mediated by glucocorticoids in the setting of myositis. Such resistance could also be against Tregs mediated immunosuppression due to distinct phenotype of CD28null T cells. Objectives: The aim of this study was to evaluate the immunosuppressive effects of glucocorticoids and Tregs on CD28null T cells in an in vitro system. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 3 healthy individuals using Ficoll separation. CD3+CD4+CD25++(high) cells were sorted as Tregs by flow cytometry. For glucocorticoid or Tregs mediated T cell suppression assays, PBMCs were stimulated with plate bound α-CD3 antibody in presence of 4uM glucocorticoid (methyl prednisolone sodium succinate) or optimal proportion of Tregs. Up-regulation of the early activation marker CD69 was measured by flow cytometry.Suppression was estimated based on % reduction in CD69 mean fluorescent intensity compared to stimulated control cells. Results: CD4+CD28null T cells (median % suppression: 40.1%) displayed lower sensitivity towards glucocorticoid-mediated suppression compared to CD28+ counterparts (median: 54.7%), seen in all individuals tested. Similarly, CD4+CD28null T cells (median: 17.5%) were less sensitive towards Tregs mediated suppression compared to CD28+ counterparts (median: 34.4%) in all individuals. No clear trend could be observed in CD8 compartment so far. Conclusions: Although, more individuals need to be tested, the above in vitro data support our in vivo findings that CD28null T cells are relatively resistant to glucocorticoid and Tregs mediated immunosuppression. Lower sensitivity of CD28null T cells towards glucocorticoid and Tregs mediated suppression support their treatment resistance nature in myositis and a role in chronic inflammation and autoimmunity.Background: Agonistic autoantibodies against the angiotensin II receptor type 1 (AT1R) and the endothelin receptor type A (ETAR) have been identified in patients suffering from systemic sclerosis (SSc). Objectives: Here we examined the expression of the AT1R and the ETAR in human immune cells and the pathological effects mediated through these receptors by the corresponding autoa...
Background Substantial evidence supports the implication of immune-activated cells, cytokines and chemokines in the pathogenesis of systemic sclerosis (SSc). The frequency of T cells expressing activation markers is increased in the peripheral blood (PB) of SSc patients. Proinflammatory cytokines, such as IL-2, TNF-α and IFN-γ, seem to be mostly involved in immune responses at early stages of the disease. However, discrepancies exist between the results of several studies. Objectives We undertook the present study to investigate the pattern of expression of proinflammatory cytokines by PB Th1 and Tc1 populations and to explore associations with disease duration. Methods Forty SSc patients and 18 healthy controls (HC) were included. All SSc patients fulfilled the American College of Rheumatology Criteria for the classification of SSc (limited cutaneous SSc (lSSc, n=29) or diffuse cutaneous SSc (dSSc, n=11), according to LeRoy et al.). A further subdivision was made, based upon the duration of disease, as early- (n=11) and late-stage (n=30), and these groups were individually compared with HC. A thorough clinical evaluation was performed and registered. The autoantibody profile was collected from medical records. All patients signed an informed consent and provided a PB sample, which was processed to separately analyze the intracellular expression of IL-2, TNF-α and IFN-γ in Th1 and Tc1 cell populations. Data was statistically analyzed using the SPSS® version 20.0 for windows. Mann-Whitney and Kruskal-Wallis tests were used to evaluate differences between groups. Correlations between continuous variables were assessed by Spearman’s correlation coefficient. P values < 0.05 were considered statistically significant. Results The mean age was 56.0±11.9 and 51.7±9.9 years for SSc patients and HC respectively. Females represented 77.5% of SSc and 83.3% of the control group. The mean disease duration was 9.6±8.55 years, the mean mRSS was 11.60±7.65 and the mean disease activity was 2.74±2.47. The frequency of Th1 and Tc1 circulating cells was not statistically different between SSc patients and HC. The percentage of Th1 and Tc1 cells producing TNF-α was significantly higher in late-stage than in early-stage SSc (p=0.034 and p=0.005, respectively). The percentage of Tc1 cells producing IFN-γ was significantly lower in early-stage than in late-stage SSc (p=0.017). No statistically significant differences were observed between early and late-stage SSc, concerning IL-2 expression among Th1 and Tc1 cells and IFN-γ expression among Th1 cells. There were no significant association between disease subset, history of digital necrosis and internal organs’ involvement, mRSS or disease activity and the frequency of IL-2, TNF-α and IFN-γ expression among Th1 or Tc1 cells. Conclusions The frequency of TNF-α-producing Tc1 cells was higher in late-stage SSc. The potential pathogenic relevance of these observations justifies further investigation, concerning the profile of proinflammatory cytokines and their potential involvement in d...
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