Mechanisms of evasion of Schistosoma mansoni schistosomula to the lethal activity of complement

Schistosomula of Schistosoma mansoni became resistant to antibody-dependent complement damage in vitro after pre-incubation with normal human erythrocytes (NHuE) whatever the ABO or Rh blood group. Resistant parasites were shown to acquire host decay accelerating factor (DAF) , a 70 kDa glycoprotein attached to the membrane of NHue by a GPI anchor. IgG2a mAb anti-human DAF (IA10) immunoprecipitated a 70 kDa molecule from 125I-labeled schistosomula pre-incubated with NHuE and inhibited their resistance to complement-dependent killing in vtro. Incubationof schistosomula with erytrocytes from patients with paroxsimal nocturnal hemoglobinuria (PNHE) or SRBC, wich are DAF-deficient, did not protect the parasites from complement lesion. Supernatant of 100,000 x g collected from NHuE incubated for 24 h in defined medium was shown to contain a soluble form of DAF and to protect schistosomula from complement killing. Schistosomula treated with trypsin before incubation with NHuE ghosts did not become resistant to complement damage. On the other hand, pre-treatment with chymotrypsin did not interfere with the acquisition of resistance by the schistosomula. These results indicate that, in vitro, NHuE DAF can be transferred to schistosomula in a soluble form and that the binding of this molecule to the parasite surface is dependent upon trypsin-sensitive chymotrypsin-insensitive polipeptide(s) present on the surface of the worm.

Pregnancy-related changes in activity energy expenditure and resting metabolic rate in Switzerland.

BACKGROUND/OBJECTIVES: To measure resting metabolic rate (RMR), activity energy expenditure (AEE), total energy expenditure (TEE) and physical activity pattern, that is, duration and intensity (in metabolic equivalents, METs) of activities performed in late pregnancy compared with postpartum in healthy, well-nourished women living in Switzerland. SUBJECTS/METHODS: Weight, height, RMR, AEE, TEE and physical activity patterns were measured longitudinally in 27 healthy women aged 23-40 years at 38.2+/-1.5 weeks of gestation and 40.0+/-7.2 weeks postpartum. RESULTS: The RMR during late pregnancy was 7480 kJ per day, that is, 1320+/-760 kJ per day (21.4%) higher than the postpartum RMR (P<0.001). Absolute changes in RMR were positively correlated with the corresponding changes in body weight (r=0.61, P<0.001). RMR per kg body weight was similar in late pregnancy vs postpartum (P=0.28). AEE per kg during pregnancy and postpartum was 40+/-13 and 50+/-20 kJ/kg, respectively (P=0.001). There were significant differences in daily time spent at METs<1.5 (1067 vs 998 min, P=0.045), at 2.5< or =METs <3.0 (58 vs 82 min, P=0.002) and METs> or =6 (1 vs 6 min, P=0.014) during pregnancy and postpartum, respectively. CONCLUSIONS: Energy expenditure in healthy women living in Switzerland increases in pregnancy compared with the postpartum state. Additional energy expenditure is primarily attributed to an increase in RMR, which is partly compensated by a decrease in AEE. The decrease in physical activity-related energy costs is achieved by selecting less demanding activities and should be taken into account when defining extra energy requirements for late pregnancy in Switzerland.

Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

A modified assay for measuring thymocyte co-stimulatory activity

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays.

The effect of subminimal inhibitory concentrations of penicillin on growth rate and haemolysin activity of group G Streptococcus

The influence of the subminimal inhibitory concentrations (1/3 and 1/4 of the MIC) of penicillin on growth rate and on haemolysin production of a strain of group G Streptococcus was studied. It was shown that 1/3 of the MIC almost completely inhibited the bacterial growth, but it was not able to inhibit haemolysin activity in the culture supernate. The generation time of bacteria grown in 1/4 of the MIC was approximately twice longer than that of the control culture. In all cultures, the haemolysin, after being produced (or liberated), reached a peak and decreased to low levels, which could suggest that group G Streptococcus produces some end products of metabolism that are able to inhibit haemolysin activity.

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

IRF4 regulates IL-17A promoter activity and controls RORγt-dependent Th17 colitis in vivo.

Background: The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.Methods: IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis.Results: In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL-6 and IL-17 mRNA expression and to a lesser extent with IL-22 mRNA expression in IBD. To further explore the role of IRF4 under in vivo conditions, we studied IRF4-deficient and wildtype mice in experimental colitis. In contrast to DSS colitis, IRF4 deficiency was protective in T-cell-dependent transfer colitis associated with reduced ROR alpha/gamma t levels and impaired IL-6, IL-17a, and IL-22 production, suggesting that IRF4 acts as a master regulator of mucosal Th17 cell differentiation. Subsequent mechanistic studies using database analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays identified a novel IRF4 binding site in the IL-17 gene promoter. Overexpression of IRF4 using retroviral infection induced IL-17 production and IL-17 together with IL-6 induced ROR gamma t expression.Conclusions: IRF4 can directly bind to the IL-17 promotor and induces mucosal ROR gamma t levels and IL-17 gene expression thereby controlling Th17-dependent colitis. Targeting of this molecular mechanism may lead to novel therapeutic approaches in human IBD.

Schistosomicidal activity of alkylaminooctanethiosulfuric acids

The schistosomicidal activity of a new series of alkylaminooctanethiosulfuric acids was studied in white Swiss mice infected with the L.E. strain of Schistosoma mansoni (Belo Horizonte, MG, Brazil). In a preliminary screening of six compounds, two derivatives - 2-[(1-methylpropyl)amino]-1-octanethiosulfuric acid and 2-[(1-methylethyl)-amino]-1-octanethiosulfuric acid - given orally in doses of 300 mg/kg/day for five consecutive days, caused interruption of the oviposition and the hepatic shift of more than 90 of the worms. Both compounds caused a significant reduction in worm burden and, interestingly, the female schistosomes were more susceptible. With the therapeutic schedule of two doses of 800 mg/kg over a 20 day interval, the death of almost all the females and about 50 of the males was observed. Female worms recovered from treated mice showed scattered vitteline glands. Results of in vitro experiments against different developmental stages of the parasite revealed the induction of paralysis and damage to the tegument membrane. The drugs presented no toxic effects on the animals.

Barcoding human physical activity to assess chronic pain conditions.

BACKGROUND: Modern theories define chronic pain as a multidimensional experience - the result of complex interplay between physiological and psychological factors with significant impact on patients' physical, emotional and social functioning. The development of reliable assessment tools capable of capturing the multidimensional impact of chronic pain has challenged the medical community for decades. A number of validated tools are currently used in clinical practice however they all rely on self-reporting and are therefore inherently subjective. In this study we show that a comprehensive analysis of physical activity (PA) under real life conditions may capture behavioral aspects that may reflect physical and emotional functioning.¦METHODOLOGY: PA was monitored during five consecutive days in 60 chronic pain patients and 15 pain-free healthy subjects. To analyze the various aspects of pain-related activity behaviors we defined the concept of PA 'barcoding'. The main idea was to combine different features of PA (type, intensity, duration) to define various PA states. The temporal sequence of different states was visualized as a 'barcode' which indicated that significant information about daily activity can be contained in the amount and variety of PA states, and in the temporal structure of sequence. This information was quantified using complementary measures such as structural complexity metrics (information and sample entropy, Lempel-Ziv complexity), time spent in PA states, and two composite scores, which integrate all measures. The reliability of these measures to characterize chronic pain conditions was assessed by comparing groups of subjects with clinically different pain intensity.¦CONCLUSION: The defined measures of PA showed good discriminative features. The results suggest that significant information about pain-related functional limitations is captured by the structural complexity of PA barcodes, which decreases when the intensity of pain increases. We conclude that a comprehensive analysis of daily-life PA can provide an objective appraisal of the intensity of pain.

Fibrin sealant for mesh fixation in endoscopic inguinal hernia repair: is there enough evidence for its routine use?

Fibrin sealing has recently evolved as a new technique for mesh fixation in endoscopic inguinal hernia repair. A comprehensive Medline search was carried out evaluating fibrin sealant for mesh fixation, and finally 12 studies were included (3 randomized trials, 3 nonrandomized trials, and 6 case series). The trials were assessed for operative time, seroma formation, recovery time, recurrence rate, and acute and chronic pain.There was a trend toward decreased operative times for fibrin sealing compared with mechanical stapling; however, the results for seroma formation remained contradictory. The most important finding was the reduced postoperative pain. Recovery times were lower after fibrin sealing and the recurrence rates showed no differences.Fibrin sealing for mesh fixation in the endoscopic inguinal hernia surgery is a promising alternative to mechanical stapling, which can be safely applied. As the overall quality of published data remains poor, further well-designed studies are needed until fibrin sealing can replace mechanical stapling as a new standard for mesh fixation.

Modulatory Role of the Ovarian Function in Neuroimmunoendocrine Axis Activity.

The aim of this study was to evaluate the effect of ovariectomy on the acute-phase response of inflammatory stress. Ex vivo adrenocortical, peripheral mononuclear cell (PMNC) and adipocyte activities were studied in intact and ovariectomized mice. Endotoxemia was mimicked by intraperitoneal administration of bacterial lipopolysaccharide (LPS; 25 mg per mouse) to sham-operated and 21-day ovariectomized mice. Circulating corticosterone, tumor necrosis factor-alpha (TNFalpha) and leptin concentrations were monitored before and 30-120 min after the administration of LPS. Additionally, in vitro experiments were performed with isolated corticoadrenal cells, PMNCs and omental adipocytes from sham-operated and ovariectomized mice incubated with specific secretagogues. The results indicate that while ovariectomy enhanced TNFalpha secretion after in vivo administration of LPS, it reduced corticoadrenal response and abrogated LPS-elicited leptin secretion into the circulation. While the corticoadrenal sensitivity to ACTH stimulation was reduced by ovariectomy, the LPS-induced PMNC response was not affected. Exogenous leptin enhanced baseline PMNC function regardless of surgery. Finally, ovariectomy drastically reduced in vitro adipocyte functionality. Our data support the notion that ovariectomy modified neuroendocrine-immune-adipocyte axis function and strongly suggest that ovarian activity could play a pivotal role in the development of an adequate immune defense mechanism after injury.

A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies

We report the identification of a 48kDa antigen targeted by antibodies which inhibit Plasmodium falciparum in vitro growth by cooperation with blood monocytes in an ADCI assay correlated to the naturally acquired protection. This protein is located on the surface of the merozoite stage of P. falciparum, and is detectable in all isolates tested. Epidemiological studies demonstrated that peptides derived from the amino acid sequence of MSP-3 contain potent B and T-cell epitopes recognized by a majority of individuals living in endemic areas. Moreover human antibodies either purified on the recombinant protein, or on the synthetic peptide MSP-3b, as well as antibodies raised in mice, were all found to promote parasite killing mediated by monocytes.