SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis.

Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases.

Labyrinthine fenestration for stapes fixation in chronic ear disease others than otosclerosis.

The objective of this study is to assess the results of labyrinthine fenestration for fixed stapes in chronic ear disease. Using a prospective database, pre- and postoperative audiometric data from patients undergoing labyrinthine fenestration for fixation of the stapes in chronic ear disease others than otosclerosis between 2002 and 2012 were evaluated. Twenty-three labyrinthine fenestrations in chronic ear disease were performed (17 malleo-stapedotomies, 4 incus-stapedotomies, 1 neo-malleus-stapedotomy, 1 TORP-stapedotomy). Overall, the mean short-term (2 months) and long-term (42 months) postoperative air-bone gap (0.5-3 kHz) were 17.5 and 16.5 dB, respectively; long-term air-bone gap of <20 dB was obtained in 73 % of patients. There was no significant difference in air-bone gap closure between tympanosclerotic and post inflammatory osteogenic fixation of the stapes (p = 0.267). Hearing benefit success using the 'Belfast rule of the thumb' was achieved in 48 %. Normal bilateral hearing was achieved in 17 % and bilateral symmetric hearing impairment in 26 %. Only in 4 %, bone conduction worsened by more than 5 dB. Labyrinthine fenestration is an option in selected cases of stapes fixation in chronic ear disease and provides hearing gain without significant risk for sensorineural hearing loss. In those already selected cases, hearing benefit success 'Belfast rule of the thumb' is achieved only in half of the cases. This and the possible alternatives, should therefore be discussed preoperatively.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.

Changes in N2 fixation in Stylosanthes scabra derived from tissue culture

Plants were regenerated from leaf-derived callus culture of Stylosanthes scabra, a polyploid legume tolerant to drought and adapted to acid soils. A total of 168 regenerants were planted out in Leonard jars in a complete randomized design. Nitrogen fixation and vegetative growth were indirectly evaluated by shoot dry weight, root dry weight, shoot N content and acetylene reduction activity. The results showed higher variation in the regenerants than in controls not submitted to tissue culture. Significant differences were found for all nitrogen fixation related-traits

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Fuzzy tolerance/equivalence relations from intersection, union and complement operators and their application to a clustering method

This master thesis work introduces the fuzzy tolerance/equivalence relation and its application in cluster analysis. The work presents about the construction of fuzzy equivalence relations using increasing generators. Here, we investigate and research on the role of increasing generators for the creation of intersection, union and complement operators. The objective is to develop different varieties of fuzzy tolerance/equivalence relations using different varieties of increasing generators. At last, we perform a comparative study with these developed varieties of fuzzy tolerance/equivalence relations in their application to a clustering method.

The role of complement in the modulation by fluid-phase IgG of the production of reactive oxygen species by polymorphonuclear leukocytes stimulated with IgG immune complexes

The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMN) can be induced by immune complexes and is an important component of phagocytosis in the killing of microorganisms, but can also be involved in inflammatory reactions when immune complexes are deposited in tissues. We have observed that fluid-phase IgG can inhibit the generation of ROS by rabbit PMN stimulated with precipitated immune complexes of IgG (ICIgG) in a dose-dependent manner, acting as a modulatory factor in the range of physiological IgG concentrations. This inhibitory effect is compatible with the known affinity (Kd) of monomeric IgG for the receptors involved (FcRII and FcRIII). The presence of complement components in the immune complexes results in a higher stimulation of ROS production. In this case, however, there is no inhibition by fluid-phase IgG. The effect of complement is strongly dependent on the presence of divalent cations (Ca2+ or Mg2+) in the medium, whereas the stimulation of ICIgG (without complement) does not depend on these cations. We have obtained some evidence indicating that iC3b should be the component involved in the effect of complement through interaction with the CR3 receptor. The absence of the inhibitory effect of fluid-phase IgG in ROS production when complement is present in the immune complex shows that complement may be important in vivo not only in the production of chemotactic factors for PMN, but also in the next phase of the process, i.e., the generation of ROS.

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

Complement system in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) consists 20% of keratinocytederived non-melanoma skin cancers (NMSC), the incidence of which is increasing globally. cSCC is the most common metastatic skin cancer and it causes approximately 20% of skin cancer-related deaths. At present, there are no molecular markers for predicting which cSCC lesions are aggressive or metastasize rapidly. UV radiation is the most important risk factor for cSCC. During the development of cSCC, normal epidermal keratinocytes are transformed and form actinic keratosis (AK), which progresses to cSCC in situ (cSCCIS, Bowen’s disease) and finally to invasive and metastatic cSCC. Inflammatory factors and cells are a part of cancer microenvironment and cSCC can develop in the chronically irritated skin or in the context of chronic inflammation. The complement system is a central part of innate immunity and it regulates normal immunological and inflammatory processes. In this study, the role of complement system components and inhibitors were studied in the progression of cSCC in culture and in vivo. Elevated expression of complement factor H (CFH), complement factor I (CFI), complement component C3 and complement factor B (CFB) was noted in cSCC cells in culture. The analysis with immunohistochemistry (IHC) revealed that the expression of CFH, CFI, C3 and CFB was specifically noted in tumor cells in vivo. The staining intensity of CFH, CFI, C3 and CFB was also stronger in invasive cSCC than in AK or cSCCIS samples. The knockdown of CFH, CFI and CFB with specific siRNAs decreased cSCC cell viability and migration, whereas the knockdown of C3 reduced only cSCC cell migration. Moreover, the knockdown of CFI, C3 and CFB inhibited growth of cSCC xenograft tumors established in SCID mice in vivo. In these tumors, CFI, C3 and CFB knockdown decreased the number of proliferating cells. Moreover, the knockdown of CFI increased local inflammation and complement activation. This study provides evidence for the roles of CFH, CFI, C3 and CFB in the tumor progression indicating these as molecular biomarkers and putative therapeutic targets of cSCC.

Statistical analysis of event-related potential elicited by verb-complement merge in Brazilian Portuguese

An interesting fact about language cognition is that stimulation involving incongruence in the merge operation between verb and complement has often been related to a negative event-related potential (ERP) of augmented amplitude and latency of ca. 400 ms - the N400. Using an automatic ERP latency and amplitude estimator to facilitate the recognition of waves with a low signal-to-noise ratio, the objective of the present study was to study the N400 statistically in 24 volunteers. Stimulation consisted of 80 experimental sentences (40 congruous and 40 incongruous), generated in Brazilian Portuguese, involving two distinct local verb-argument combinations (nominal object and pronominal object series). For each volunteer, the EEG was simultaneously acquired at 20 derivations, topographically localized according to the 10-20 International System. A computerized routine for automatic N400-peak marking (based on the ascendant zero-cross of the first waveform derivative) was applied to the estimated individual ERP waveform for congruous and incongruous sentences in both series for all ERP topographic derivations. Peak-to-peak N400 amplitude was significantly augmented (P < 0.05; one-sided Wilcoxon signed-rank test) due to incongruence in derivations F3, T3, C3, Cz, T5, P3, Pz, and P4 for nominal object series and in P3, Pz and P4 for pronominal object series. The results also indicated high inter-individual variability in ERP waveforms, suggesting that the usual procedure of grand averaging might not be considered a generally adequate approach. Hence, signal processing statistical techniques should be applied in neurolinguistic ERP studies allowing waveform analysis with low signal-to-noise ratio.

Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production.

Délibération des fermiers généraux sur la fixation des ventes, les apurements et les bons de masse

[Acte. 1710-11-21. Paris]