Long-term Sonographic and Serological Follow-up of Inactive Echinococcal Cysts of the Liver: Hints for a “Watch-and-Wait” Approach

Human cystic echinococcosis is a chronic, complex and neglected infection. Its clinical management has evolved over decades without adequate evaluation of efficacy. Recent expert opinion recommends that uncomplicated inactive cysts of the liver should be left untreated and solely monitored over time (“watch-and-wait” approach). However, clinical data supporting this approach are still scant and published mostly as conference proceedings. In this study, we report our experience with long-term sonographic and serological follow-up of inactive cysts of the liver. From March 1994 to October 2013, 38 patients with 47 liver cysts, diagnosed as inactive without any previous treatment history, were followed with ultrasound and serology at 6–12 months intervals for a period of at least 24 months (median follow-up 51.95 months) in our outpatient clinic. In 97.4% of patients, the cysts remained inactive over time and in only one case was reactivation of the cyst detected. No complications occurred during the time of monitoring. During follow-up, serology tests for CE were negative at diagnosis or became negative in 74.1% and were positive or became positive in 25.9% of cases. Patients with inactive cysts on ultrasound but positive serological tests were also investigated by CT scan (chest and abdomen) to rule out extra-hepatic cyst localization. This study confirms the importance of a stage-specific approach to the management of cystic echinococcosis and supports the use of a monitoring-only approach to inactive, uncomplicated cysts of the liver. It also confirms that serology plays only an ancillary role in the clinical management of these patients, compared to ultrasound and other imaging techniques. The implications of these findings for clinical management and natural history of cystic echinococcosis are discussed.     

The mycoestrogen zearalenone in Portuguese flowing waters and its potential environmental impact

This study provides the first environmental risk assessment (ERA) for zearalenone (ZEN), the only known mycoestrogen, based on a broad-scale investigation on its occurrence in rivers and creeks from Portugal. Water sample filtration and immunoaffinity columns (IAC) clean-up followed by liquid chromatograph with tandem mass spectrometry (LC/MS ⁿ ) provided an analytical method with good analytical performance. ZEN levels were determined for seven Portuguese rivers and one creek, during two different seasons, in a total of 38 samples collected upstream wastewater treatment plants (WWTPs). Overall, 23.7% were contaminated with ZEN at levels ranging between 5.6 and 82.6 ng/L. The highest concentration was observed during spring, although no statistically significant differences were observed between spring and autumn sampling campaigns. The potential ecotoxicological risk from ZEN to different trophic levels of aquatic organisms was evaluated by means of risk quotients (RQs) calculation. Although all the RQs obtained were lower than 1, our results confirm that ZEN is a relatively frequent contaminant in flowing waters in Portugal and might contribute to the overall estrogenic activity in the environment.     

Interphase cytogenetics on paraffin-embedded sections of ovary for detection of genomic constitution in a patient with Turner's syndrome and chromosomal mosaicism

The difficulty of detecting sex chromosome mosaicism cytogenetically hinders the finding of an acceptable explanation for phenotypic-genotypic discrepancy amongst those patients. Fluorescence in situ hybridization (FISH) permits the genomic identification of patients with mosaic karyotypes in interphase nuclei by utilising an X chromosome-specific DNA probe (interphase cytogenetics). We evaluated the efficiency of interphase cytogenetics in the detection of the genomic constitution of the ovary from a patient with Turner's syndrome having mosaicism (46,XX/45,X0) previously established by blood lymphocyte karyotyping. We used a biotin-labelled alphoid repetitive sequence, pBAMX5, specific for the centromeric region of the human X chromosome. Although examination of ovarian sections and blood lymphocytes by FISH showed the presence of both 46,XX and 45,X0 cell lines, the genomic constitution of the germ cells/oocytes in ovarian primordial follicles was shown to be normal (46,XX). Our results (1) show the high applicability of interphase cytogenetics on paraffin sections, (2) indicate the possibility of genomic screening of different tissues that are otherwise not amenable to routine cytogenetic investigation and (3) offer a reliable methodological approach to defining accurate by the percentage of abnormal karyotypes in mosaicism of different organs and non-dividing tissues.     

PbsP,a cell wall‐anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine‐rich region, and a LPXTG cell wall‐anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface‐bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non‐CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.     

Coumarin A/AA induces apoptosis‐like cell death in HeLa cells mediated by the release of apoptosis‐inducing factor

It has been demonstrated that naturally occurring coumarins have strong biological activity against many cancer cell lines. In this study, we assessed the cytotoxicity induced by the naturally isolated coumarin A/AA in different cancer cell lines (HeLa, Calo, SW480, and SW620) and in normal peripheral‐blood mononuclear cells (PBMCs). Cytotoxicity was evaluated using the MTT assay. The results demonstrate that coumarin A/AA was cytotoxic in the four cancer cell lines tested and importantly was significantly less toxic in PBMCs isolated from healthy donors. The most sensitive cancer cell line to coumarin A/AA treatment was Hela. Thus, the programmed cell death (PCD) mechanism induced by this coumarin was further studied in this cell line. DNA fragmentation, histomorphology, cell cycle phases, and subcellular distribution of PCD proteins were assessed. The results demonstrated that DNA fragmentation, but not significant cell cycle disruptions, was part of the PCD activated by coumarin A/AA. Interestingly, it was found that apoptosis‐inducing factor (AIF), a proapoptotic protein of the mitochondrial intermembrane space, was released to the cytoplasm in treated cells as detected by the western blot analysis in subcellular fractions. Nevertheless, the active form of caspase‐3 was not detected. The overall results indicate that coumarin A/AA induces a caspase‐independent apoptotic‐like cell death program in HeLa cells, mediated by the early release of AIF and suggest that this compound may be helpful in clinical oncology. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:263–272, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20288     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Unravelling Glucan Recognition Systems by Glycome Microarrays Using the Designer Approach and Mass Spectrometry

Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.Glucan polysaccharides are polymers of d-glucose with differing linkages in linear or branched sequences. They occur as storage materials in animals, secreted virulence factors of bacteria, and conserved structural components of cell walls of yeasts, fungi, some bacteria, and plants. Polysaccharides of this type are of considerable interest in biology, medicine, and biotechnology and are acknowledged for their immunostimulatory, anticancer, and health-promoting activities (1, 2); for their elicitor activities in defense responses and signaling in plants (3); and for acting as functional ingredients in human nutrition (4). Unraveling recognition systems that mediate these activities is highly desirable as a lead to effective translational applications.Recognition systems involving glucan polysaccharides include those in mammals, such as recognition of fungal β-glucans by Dectin-1, the major receptor of the innate immune system against fungal pathogens (5), and by natural or vaccine-induced protective antifungal antibodies (6, 7); also recognition of mycobacterial α-glucan by the innate immune receptor DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) (8); those in insects, such as the Drosophila Gram-negative binding protein 3 (GNBP3) sensor protein, which binds β-glucans (9); and those in bacteria, such as Brucella abortus, where cyclic β-glucans can serve as virulence factors (10).Another important class of glucan-recognizing proteins comprises noncatalytic carbohydrate-binding modules (CBMs)¹ of bacterial glycoside hydrolases that mediate association with substrate and increase catalytic activity, likely through a targeting mechanism or by driving enzyme specificity (11, 12). Notable examples are CBMs of bacterial cellulolytic enzymes that promote enzymatic deconstruction of intact plant cell walls and that are of industrial significance in the biofuel and bioprocessing sectors (13, 14) and CBMs of rumen or commensal human microbiota with roles in animal and human health (14, 15). CBMs also have roles in other systems: for example, CBM-containing enzymes as virulence factors of bacterial pathogens (16) and CBM-containing human laforin that regulates glycogen metabolism and for which mutations can lead to neurodegenerative disease (17). The number of putative glucan-binding CBMs that have been identified and classified in the Carbohydrate-Active enZyme (CAZy) database (http://www.cazy.org) is expanding, but relatively few have been experimentally investigated for details of carbohydrate binding and fine specificity (11).Searching for and assigning the specificities of glucan-recognizing proteins has thus become increasingly important. It is desirable to have high-throughput and sensitive micro-methods to screen for and characterize ligands for structure–function studies toward effective exploitation in modern therapeutic, nutritional, agricultural, and biofuel-related technologies. Carbohydrate microarrays have served to advance knowledge on specificities of diverse carbohydrate-recognition systems (18–22). Where the desired oligosaccharide probes are unavailable, microarrays need to be generated from ligand-bearing glycomes (23). Using a prototype of such designer microarrays of neoglycolipid (NGL)-probes (23) derived from oligosaccharide fragments of glucans rich in β1,3- or β1,6-linked sequences, we showed that linear β1,3-linked glucose sequences with degree of polymerization (DP) 10 or longer are bound by Dectin-1 (24). Recognition of other types of glucan sequences by Dectin-1 and the applicability of microarrays of diverse gluco-oligosaccharide sequences to other glucan-recognizing proteins required investigation. Cummings, Smith, and colleagues have developed the shotgun strategy (20) to create glycome-scale “gangliome” and “human milk glycome” microarrays. In the shotgun microarrays, the printed probes may not be sequence-defined before array construction and require metadata-assisted glycan sequencing (MAGS), which combines MS analysis (25), binding data with glycan-binding proteins or antibodies, and exoglycosidase treatment after printing (26, 27).Mass spectrometry has become a primary technique in carbohydrate structural analysis (28), and electrospray mass spectrometry (ESI-MS) has been used to provide sequence and partial linkage information on various types of oligosaccharides (29–33). For neutral oligosaccharides, we have found that tandem MS with collision-induced dissociation (CID-MS/MS) in the negative-ion mode is particularly useful and have successfully applied for oligosaccharide chain and blood-group typing (34, 35) and for branching pattern analysis (36).This is because that some important linkages at certain monosaccharide residues can be unambiguously determined with high sensitivity without the need for derivatization and anion complexation as previously recognized, e.g. in the area of gluco-oligosaccharides, Cl⁻-anion adduction has been used to determine sequences of tetrasaccharides of dextran (37).Here, we describe a strategy using the designer approach combined with negative-ion ESI-CID-MS/MS for constructing a microarray of sequence-defined gluco-oligosaccharides representing major sequences in glucans (glucome microarray) as a tool for screening glucan-recognizing proteins and assigning their recognition motifs (Fig. 1). We selected a comprehensive panel of glucan polysaccharides isolated from plants, fungi, and bacteria with different sequences to represent the glucome. We used finely tuned chemical and enzymatic methods to partially depolymerize the polysaccharides and prepare gluco-oligosaccharide fragments with different chain lengths (up to DP-13 or DP-16). We developed a ESI-CID-MS/MS method that enables linkage and sequence determination of linear or branched gluco-oligosaccharides at high-sensitivity and applied this to the sequencing of oligosaccharide fragments prepared. These sequence-defined gluco-oligosaccharides were then converted into NGL probes and used for construction of the microarray. The oligosaccharides encompassed linear sequences with homo (single) linkages: 1,2-, 1,3-, 1,4-, or 1,6- with α or β configurations; and hetero (multiple) linkages: 1,3-, 1,4, or 1,6-; also branched oligosaccharide sequences with 1,3 and 1,6-linkages.Open in a separate windowFig. 1.Neoglycolipid (NGL)-based designer glucome microarray with mass spectrometry as a tool to assign carbohydrate ligands in glucan recognition. Ligand-bearing glucan polysaccharides, described in supplemental Fig. S1 and Table S1, were selected as sources of gluco-oligosaccharides for construction of the microarray. A total of 121 gluco-oligosaccharide fractions were obtained with different DP after partial depolymerization of polysaccharides and fractionation. ESI-CID-MS/MS method was developed using gluco-oligosaccharides with known sequences and applied to determination of sequences of oligosaccharide fragments from polysaccharides. Gluco-oligosaccharides were converted to NGL probes for microarray construction and interrogation with the glucan-recognizing proteins described in supplemental Table S2.To our knowledge, this is the first sequence-defined glycome-scale microarray constructed. We used 12 selected proteins (antibodies and CBMs) known to target α- or β-glucans to validate the approach. We then applied the microarray analysis to Dectin-1 and DC-SIGN, which revealed new insights into the specificities of these signaling molecules of the innate immune system.