Stability improvement of the fatty acid binding protein Sm14 from S. mansoni by Cys replacement Structural and functional characterization of a vaccine candidate

The Schistosoma mansoni fatty acid binding protein (FABP), Sm14, is a vaccine candidate against, S. mansoni and F. hepatica. Previously, we demonstrated the importance of a correct fold to achieve protection in immunized animals after cercariae challenge [[10]. C.R.R. Ramos, R.C.R. Figueredo, T.A. Pertinhez, M.M. Vilar, A.L.T.O. Nascimento, M. Tendler, I. Raw, A. Spisni, P.L. Ho, Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein: structural, functional and immunoprotection analysis. J. Biol. Chem. 278 (2003) 12745-12751.]. Here we show that the reduction of vaccine efficacy over time is due to protein dimerization and subsequent aggregation. We produced the mutants Sm14-M20(C62S) and Sm14-M20(C62V) that, as expected, did not dimerize in SDS-PAGE. Molecular dynamics calculations and unfolding experiments highlighted a higher structural stability of these mutants with respect to the wild-type. In addition, we found that the mutated proteins, after thermal denaturation, refolded to their active native molecular architecture as proved by the recovery of the fatty acid binding ability. Sm14-M20(C62V) turned out to be the more stable form over time, providing the basis to determine the first 3D solution structure of a Sm14 protein in its apo-form. Overall, Sm14-M20(C62V) possesses an improved structural stability over time, an essential feature to preserve its immunization capability and, in experimentally immunized animals, it exhibits a protection effect against S. mansoni cercariae infections comparable to the one obtained with the wild-type protein. These facts indicate this protein as a good lead molecule for large-scale production and for developing an effective Sm14 based anti-helminthes vaccine.

Plasma rotation measurement in small tokamaks using an optical spectrometer and a single photomultiplier as detector.

The method for plasma rotation measurement in the tokamak TCABR is reported in this article. During a discharge, an optical spectrometer is used to scan sequentially spectral lines of plasma impurities and spectral lines of a calibration lamp. Knowing the scanning velocity of the diffraction grating of the spectrometer with adequate precision, the Doppler shifts of impurity lines are determined. The photomultiplier output voltage signals are recorded with adequate sampling rate. With this method the residual poloidal and toroidal plasma rotation velocities were determined, assuming that they are the same as those of the impurity ions. The results show reasonable agreement with the neoclassical theory and with results from similar tokamaks.

Gene structure and M20T polymorphism of the Schistosoma mansoni Sm14 fatty acid-binding protein Molecular, functional, and immunoprotection analysis

The Schistosoma mansoni Sm14 antigen belongs to the fatty acid-binding protein family and is considered a vaccine candidate against at least two parasite worms, Fasciola hepatica and S. mansoni. Here the genomic sequence and the polymorphism of Sm14 have been characterized for the first time. We found that the conserved methionine at position 20 is polymorphic, being exchangeable with threonine (M20T). To evaluate the function of the amino acid residue at this position, we have also constructed the mutant Sm14-A20 besides the two native isoforms (Sm14-M20 and Sm14-T20). The three purified recombinant His6-tagged Sm14 proteins (rSm14-M20, rSm14-T20, and rSm14-A20) present a predominant â-barrel structure as shown by CD spectroscopy. Thermal and urea unfolding studies evidenced a higher structural stability of rSm14-M20 over the other forms (rSm14M20>rSm14-T20>rSm14-A20). All of the Sm14 proteins were able to bind 11-(dansylamino)undecanoic acid (DAUDA) without substantial difference in the binding affinity. However, rSm14-M20 exhibited a higher affinity for natural fatty acids than the rSm14-T20 and rSm14-A20 proteins as judged by competitive experiments against DAUDA (rSm14-M20>rSm14-T20> rSm14-A20). The rSm14-M20 or rSm14-T20 isoforms but not the rSm14-A20 mutant was able to induce significant protection against S. mansoni cercariae challenge in immunized mice. The level of protection efficacy correlates with the extent of structure stability of the recombinant Sm14 isoforms and mutant.

Pilot study of a novel treatment for androgenetic alopecia using enriched cell culture medium: clinical trials.

Androgenetic Alopecia (AA) afflicts a large part of the population and of the many treatments available today none is completely satisfactory. Testing the efficacy and safety of a novel topical treatment for AA which is based on cell culture medium supplemented with insulin, thyroxin and growth hormone (CCM). The 48 participants classified as androgenetic alopecia Type II, III or IV on the Hamilton scale, concluded a randomized, vehicle-controlled, double-blind trial of 6 months duration. Under occlusive cover the gel was self applied for at least 3 hours daily. Evaluation was based on hair counts, investigator global assessment and participants self-administered questionnaire. Cessation of hair loss was reported by most participants within 28 weeks, and further confirmed by the hair count (HC) in ~80% of participants. Moreover, as early as 4 months after the start of the treatment, a time dependent increase of up to 50% in HC was observed. The average change in HC between the two groups differed significantly (p=0.007), with values of 4.1% for control and 13.8% for CCM. Following 4 months of treatment, a time dependent increase in HC (>10%) above minimal was observed in 55% of the CCM and 25% of the control and this trend continued. At 6 months 63% of the CCM and 33% of the control group exhibited increase of HC higher than 10%. The average increase in HC in the CCM and the control groups was 17.1% and 8.9% respectively (p=0.035). Self evaluation questionnaires revealed a time dependent increase in satisfaction in the CCMusers compared to the control. While the average score at T2 was similar in CCM and control (2.7 and 2.6 respectively), the score at T6 in the CCM increased to 5.9 and decreased to -0.4 in the control (p=0.007). Global-clinical evaluation following six months treatment revealed significantly (p=0.02) more hair loss in the control group (40%) compared to the CCM (7%) treated group. CCM was found effective in treating androgenetic alopecia in men. It induced cessation of hair loss, increased rate of hair growth and appearance of new hair. No side effects were reported or observed.

Vaccination against Fasciola hepatica infection using a Schistosoma mansoni defined recombinant antigen, Sm14

Fasciola hepatica is the causative agent of fasciolosis in many areas in America, Europe, Africa, Asia and Australia. There is an urgent need for improved methods to control the parasite's transmission. We describe the use of an experimental vaccine based on a recombinant antigen cloned from another parasite, Schistosoma mansoni (Sm14), that induces high levels of cross protection in mice against both S. mansoni and F. hepatica. Sheep and mice vaccinated with Sm14 were significantly protected against challenge infection with metacercariae of Fasciola hepatica and were completely free of the histopathological hepatic damage related to liver fluke infection. The vaccine will provide a valuable new tool to aid in transmission control of this economically important disease

Preliminary analysis of Sm14 in distinct fractions of Schistosoma mansoni adult worm extract.

In previous studies it was shown that the recombinant molecule, r-Sm14, induces high levels of protection against Schistosoma mansoni infection in two outbred animal models and immune crossprotection against infection by Fasciola hepatica in Swiss outbred mice. r-Sm14 was derived from a living worm extract, called SE, and is being developed as the molecular basis of an anti-helminth bivalent vaccine against the two parasites, for medical and veterinary application. Present data refer to SDS-PAGE and Western Blotting analysis of four different preparations of S. mansoni adult worms focusing Sm14 identification. The extracts correspond to the initial fraction of the SE extraction process, containing products released by living worms (SEi); SE2, reextraction of adult worms in PBS; and SE of separated male and female adult worms. In all extracts it was possible to detect the component of 14 kDa, that was recognized by specific anti-rSm14 antibody raised in rabbits.

r-Sm14 - pRSETA efficacy in experimental animals.

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system.

Improved vitality of experimental random dorsal skin flaps in rats treated with enriched cell culture medium.

A defined, serum-free cell culture medium supplemented with nonsteroidal anabolic hormones, insulin, thyroxin, and growth hormone was found to accelerate wound healing by stimulating vascularized granulation tissue formation, epithelialization, and angiogenesis. The aim of this work was to study the effect of cell culture medium on the survival rate of cephalically based random dorsal skin flaps in an animal model. A total of 77 Sprague-Dawley rats were randomized into five treatment groups: pharmacologic delay with cell culture medium, flap enhancement with cell culture medium, surgical delay, biological delay with saline, and control. Statistically significant differences in distal flap necrosis were found among all groups (p<0.003). The rats treated with cell culture medium before flap elevation showed a significant increase in flap viability: a survival rate of 83 percent, compared with the control group, which demonstrated a survival rate of only 58 percent (p<0.0001). The surgical delay and the groups treated with cell culture medium yielded similar results with no significant difference between them. This study indicates that preoperative injection of cell culture medium may play a role in decreasing skin flap necrosis.

Relativistic plasma viscosity of the Burnett kind.

Hydrodynamic equations to describe relativistic and ultrarelativistic plasma dynamics were obtained by Dzhavakhishvili and Tsintsadze [Sov. Phys. JETP 37, 666 (1973)] using the Chapman and Enskog scheme to solve the relativistic kinetic equations for the different plasma species. This approach leads to a representation of the particle viscosities in the Navier-Stokes form and, therefore, some relevant physical processes, such as the Burnett type of particle viscosity, cannot be properly dealt with in this scheme. In this paper we employ the extended Grad method to derive hydrodynamic equations which include ultrarelativistic viscosities of the Burnett type, i.e., viscosities that depend not only on derivatives of the particle macroscopic velocities but also on derivatives of particle heat fluxes.

Static-electric field induction by a silicone cushion for the treatment of hypertrophic and keloid scars.

Silicone gel and silicone occlusive sheeting are widely used at present for the treatment of hypertrophic and keloid scars, without any scientific explanation as to their mode of action. In a recent paper the possibility was raised that static electricity generated by friction-activated silicone sheeting could be the reason for this effect, and that it can, with time, cause involution of hypertrophic and keloid scars. The objective of this study was to test this hypothesis and to observe whether a continuous and also an increased negatively charged static-electric field will shorten the treatment period. A device to implement these requirements gradually evolved over a 5-year period. A number of prototypes were tested until the final product was attained. Some of the patients in this study were treated initially with a silicone sponge inserted in the cushion. Later this version was changed to the final design described herein. A silicone cushion was developed with the purpose of increasing a negative static-electric charge to accelerate the regression process. The cushion is custom-made using a silicone occlusive sheeting envelope of 0.75-mm thickness, which does not deteriorate with use, and is partially filled with high viscosity silicone oil. Its edges are sealed, and its size is designed to extend a little beyond the scarred area. Static electricity readings, generated by activating the cushion by pumping action with the fingers, stretching or deforming the cushion, are invariably much higher when compared with those obtained with silicone occlusive sheeting and silicone gel sheeting. The interaction between the negatively charged ions of the cushion and the ionic charges of the tissue fluids may be the critical factor in achieving hypertrophic and keloid scars involution. Of the 30 patients enrolled in the study, 3 patients dropped out. Treatment with the silicone cushions yielded 63.3 percent cessation of itching and burning followed by pallor and flattening of the scar, some markedly so, over a few weeks to 6-month period. An additional 26.6 percent had their scars resolved in up to 12 months of treatment. Good contact of the cushion over the scar has been shown to be important in this clinical trial, and much creativity is needed for making elastic strap bindings that ensure this contact. The clinical trials extended over a 12-month period. Ten patients (33.3 percent) who had recalcitrant scars with little response to the use of the silicone cushion were given intralesional corticosteroid injections, in addition to the continued use of the cushion, resulting in a fairly rapid resolution of these scars over a period of months to a year.

Contrasting genomic variability between clones from field isolates and laboratory populations of Schistosoma mansoni.

The extent of genomic variability of clones of Schistosoma mansoni obtained from field isolates was compared with that of strains that have been laboratory maintained. Analysis was undertaken using randomly amplified polymorphic DNAs (RAPDs) generated with three primers. Phenograms showing the similarity among the clones were constructed. The data showed that while the laboratory strain is highly homogeneous the clones derived from the field populations were highly variable with 43% of RAPDs exhibiting polymorphisms among 23 clones. Clones isolated from the same infected individual were always more closely grouped than clones from different individuals. The data clearly demonstrated that earlier analyses of the genomic variability in S. mansoni have underestimated this phenomenon due to the failure to examine field isolates.