Outcome of challenge with Coxsackievirus B4 in young mice after maternal infection with the same virus during gestation

Enteroviral infections go usually unnoticed, even during pregnancy, yet some case histories and mouse experiments indicate that these viruses may be transmitted vertically. More frequently, however, transmission occurs by (fecal) contamination during and shortly after birth. The aim of this study was to investigate the effect of maternal infection in mice (1) on gravidity outcome and (2) on subsequent challenge of the offspring with the same virus. CD1 outbred female mice were infected by the oral route with coxsackievirus B4 strain E2 or mock-infected at days 4, 10, or 17 of gestation. Weight and signs of sickness were noted daily. Pups were infected at day 25 after birth (4 days postweaning). Organs (brain, pancreas, and heart) were analyzed for viral RNA and histopathology. We observed that maternal infection at day 4 or day 17 of gestation had little effect on pregnancy outcome, whereas infection at day 10 affected dams and/or offspring. Infection of pups resulted in severe inflammation of the pancreas, but only when dams were previously infected, especially at day 17. The blood glucose levels were elevated. Because no trace of infection was found at the time of challenge, a role for immunopathology is suggested.     

Burkholderia cenocepacia lectin A binding to heptoses from the bacterial lipopolysaccharide

Bacteria from the Burkholderia cepacia complex (Bcc) cause highly contagious pneumonia among cystic fibrosis (CF) patients. Among them, Burkholderia cenocepacia is one of the most dangerous in the Bcc and is the most frequent cause of morbidity and mortality in CF patients. Indeed, it is responsible of "cepacia syndrome", a deadly exacerbation of infection, that is the main cause of poor outcomes in lung transplantation. Burkholderia cenocepacia produces several soluble lectins with specificity for fucosylated and mannosylated glycoconjugates. These lectins are present on the bacterial cell surface and it has been proposed that they bind to lipopolysaccharide epitopes. In this work, we report on the interaction of one B. cenocepacia lectin, BC2L-A, with heptose and other manno configured sugar residues. Saturation transfer difference NMR spectroscopy studies of BC2L-A with different mono- and disaccharides demonstrated the requirement of manno configuration with the hydroxyl or glycol group at C6 for the binding process. The crystal structure of BC2L-A complexed with the methyl-heptoside confirmed the location of the carbohydrate ring in the binding site and elucidated the orientation of the glycol tail, in agreement with NMR data. Titration calorimetry performed on monosaccharides, heptose disaccharides and bacterial heptose-containing oligosaccharides and polysaccharides confirmed that bacterial cell wall contains carbohydrate epitopes that can bind to BC2L-A. Additionally, the specific binding of fluorescent BC2L-A lectin on B. cenocepacia bacterial surface was demonstrated by microscopy.     

Plant Aurora kinases play a role in maintenance of primary meristems and control of endoreduplication

? The conserved family of Aurora kinases has multiple functions during mitosis. The roles of plant Aurora kinases have been characterized using inhibitor treatments. ? We down-regulated Aurora kinases in Arabidopsis thaliana using RNA interference (RNAi). We carried out a detailed phenotypic analysis of Aurora RNAi plants, biochemical and microscopic studies of AtAurora1 kinase together with AtTPX2 (targeting protein for Xklp2) and γ-tubulin. ? Cell division defects were observed in plants with reduced expression of Aurora kinases. Furthermore, the maintenance of primary meristems was compromised and RNAi seedlings entered endoreduplication prematurely. AtAurora1, its activator AtTPX2, and γ-tubulin were associated with microtubules in vitro; they were attached to regrowing kinetochore microtubules and colocalized on spindle microtubules and with a subset of early phragmoplast microtubules. Only the AtAurora1 kinase was translocated to the area of the cell plate. ? RNAi silencing of Aurora kinases showed that, in addition to their function in regulating mitosis, the kinases are required for maintaining meristematic activity and controlling the switch from meristematic cell proliferation to differentiation and endoreduplication. The colocalization and co-fractionation of AtAurora1 with AtTPX2, and γ-tubulin on microtubules in a cell cycle-specific manner suggests that AtAurora1 kinase may function to phosphorylate substrates that are critical to the spatiotemporal regulation of acentrosomal microtubule formation and organization.     

The metabolism of flubendazole and the activities of selected biotransformation enzymes in Haemonchus contortus strains susceptible and resistant to anthelmintics

SUMMARY Haemonchus contortus is one of the most pathogenic parasites of small ruminants (e.g. sheep and goat). The treatment of haemonchosis is complicated because of recurrent resistance of H. contortus to common anthelmintics. The aim of this study was to compare the metabolism of the anthelmintic drug flubendazole (FLU) and the activities of selected biotransformation enzymes towards model xenobiotics in 4 different strains of H. contortus: the ISE strain (susceptible to common anthelmintics), ISE-S (resistant to ivermectin), the BR strain (resistant to benzimidazole anthelmintics) and the WR strain (resistant to all common anthelmintics). H. contortus adults were collected from the abomasums from experimentally infected lambs. The in vitro as well as ex vivo experiments were performed and analysed using HPLC with spectrofluorimetric and mass-spectrometric detection. In all H. contortus strains, 4 different FLU metabolites were detected: FLU with a reduced carbonyl group (FLU-R), glucose conjugate of FLU-R and 2 glucose conjugates of FLU. In the resistant strains, the ex vivo formation of all FLU metabolites was significantly higher than in the susceptible ISE strain. The multi-resistant WR strain formed approximately 5 times more conjugates of FLU than the susceptible ISE strain. The in vitro data also showed significant differences in FLU metabolism, in the activities of UDP-glucosyltransferase and several carbonyl-reducing enzymes between the susceptible and resistant H. contortus strains. The altered activities of certain detoxifying enzymes might protect the parasites against the toxic effect of the drugs as well as contribute to drug-resistance in these parasites.     

Evaluation of detection of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> DNA in animal blood samples by quantitative PCR

Aim of the study: The purpose of this study was to find out the relationship between the phase of infection (acute or persistent) and the ability of quantitative PCR to detect DNA of Toxoplasma gondii in circulating leukocytes in blood. Methodology: Animal serum samples were examined (50 sheep, 47 dogs, 32 dairy cows, 91 wild boars and 36 rabbits) for the occurrence of IgM and IgG antibodies to T. gondii by ELISA. Uncoagulated blood samples from the same animals were examined for the detection of T. gondii DNA in circulating leukocytes by real-time PCR. Results: Only IgM antibodies, characteristic for acute infection, were detected in 45 of the 256 serum samples (17.6%). Only IgG antibodies, corresponding with chronic infection, were detected in 120 of the 256 samples (46.8%). In 91 of the 256 samples (35.5%) neither IgM or IgG were detected by ELISA. For real-time PCR, animals were divided into three groups based on the serological results: (group I — acute infection, group II — chronic infection, and group III — no infection). In group I, the presence of T. gondii DNA was detected in 9 out of 45 samples (20%), whereas in group II only 1 of 120 samples was positive for T. gondii DNA (0.8%). In group III, no DNA of T. gondii (0/91 samples) was detected by real-time PCR. Significance: The proof of DNA by real-time PCR in IgM positive samples was statistically significant in comparison to IgG positive samples (P<0.0001).     

Biotechnological methods of <Emphasis Type="Italic">in vitro</Emphasis> propagation in willows (<Emphasis Type="Italic">Salix</Emphasis> spp.)

Many populations of high-mountainous relic dioecious willows in Central Europe only consist of female individuals and are thus limited in their reproductive potential. We completed micropropagation experiments with shoot apexes and nodal segments of common and endangered willow (Salix) species, which can help to reintroduce autochthonous genotypes to their natural sites. Until recently, cultivation of green young shoot apexes of S. alba and S. lapponum showed the highest percentage of regeneration. We successfully applied the two-times-sterilisation due to high contamination of natural explants. The OK medium was the most efficient culture medium. In vitro propagation of willows with unisexual catkins, anther and ovule cultures were tested and optimised. Isolated anthers were cultivated on selected media and then microcallus and calluses of S. caprea and calluses of S. viminalis were formed on the A medium. Among various tested and optimised media for the ovule culture, the CP medium was the most efficient one. In this case, only the microcalluses of S. viminalis were observed. We developed biotechnological procedures that can be useful in conserving fragmented populations of high-mountainous willows.     

Recombinant α-l-rhamnosidase from Aspergillus terreus in selective trimming of rutin

α-l-Rhamnosidase (EC 3.2.1.40) is a biotechnologically important enzyme used for derhamnosylation of many natural compounds. The extracellular α-l-rhamnosidase was purified from the culture of Aspergillus terreus grown on l-rhamnose-rich medium. This enzyme was found to be thermo- and alkali-tolerant, able to operate at 70 °C and pH 8.0. The α-l-rhamnosidase cDNA was cloned from A. terreus, sequenced, and expressed in the yeast Pichia pastoris as a fully functional protein. The recombinant protein was purified to apparent homogeneity and biochemically characterized. Both the native and the recombinant α-l-rhamnosidases catalyzed the conversion of rutin into quercetin-3-glucopyranoside (isoquercitrin), a pharmacologically significant flavonoid usable in nutraceutics. This procedure has high volumetric productivity (up to 300 g/L) and yields the product void of unwanted quercetin. The significant advantage of our expression system consists in shorter production times, up to fourfold increase in enzyme yields and the absence of unwanted β-d-glucosidase as compared to the native production system. Thanks to its unique properties, this enzyme is applicable in a selective synthesis/hydrolysis of various rhamnose containing structures.     

X-Linked Adrenoleukodystrophy: Molecular and Functional Analysis of the ABCD1 Gene in Argentinean Patients

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease associated with mutations in the ABCD1 gene that encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long-chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues, due to a defect in peroxisomal VLCFA β-oxidation. In the present study, we analyzed 10 male patients and 17 female carriers from 10 unrelated pedigrees with X-ALD from Argentina. By sequencing the ABCD1 we detected 9 different mutations, 8 of which were novel. These new mutations were verified by a combination of methods that included both functional (western blot and peroxisomal VLCFA β-oxidation) and bioinformatics analysis. The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G>C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). In vitro studies suggest that p.Ala19Ser is a polymorphism. Moreover, we identified two novel intronic polymorphisms and two amino acid polymorphisms. In conclusion, this study extends the spectrum of mutation in X-ALD and facilitates the identification of heterozygous females.     

Increased T Regulatory Cells Are Associated with Adverse Clinical Features and Predict Progression in Multiple Myeloma

Background

Regulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions.

Methods

We analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD).

Results

We found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naïve (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (>10 mg/dL), decreased normal plasma cell (≤5%) count and IgA myeloma subtype. We also showed that MM patients with ≥5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pre-treatment.

Conclusions

Our study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression.