Use of Nanostructured Silica SBA-15 as an Oral Vaccine Adjuvant to Control Mycoplasma hyopneumoniae in Swine Production.

Mycoplasma hyopneumoniae is a difficult-to-control bacterium since commercial vaccines do not prevent colonization and excretion. The present study aimed to evaluate the performance of an orally administered vaccine composed of antigens extracted from Mycoplasma hyopneumoniae and incorporated into mesoporous silica (SBA-15), which has an adjuvant-carrier function, aiming to potentiate the action of the commercial intramuscular vaccine. A total of 60 piglets were divided into four groups (n = 15) submitted to different vaccination protocols as follows, Group 1: oral SBA15 + commercial vaccine at 24 days after weaning, G2: oral vaccine on the third day of life + vaccine commercial vaccine at 24 days, G3: commercial vaccine at 24 days, and G4: commercial vaccine + oral vaccine at 24 days. On the first day, the piglets were weighed and, from the third day onwards, submitted to blood collections for the detection and quantification of anti-Mycoplasma hyopneumoniae IgG. Nasal swabs were collected to monitor IgA by ELISA, and oropharyngeal swabs were used to assess the bacterial load by qPCR. Biological samples were collected periodically from the third day of life until the 73rd day. At 41 days of life, 15 individuals of the same age, experimentally challenged with an inoculum containing M. hyopneumoniae, were co-housed with the animals from groups (1 to 4) in a single pen to increase the infection pressure during the nursery period. At 73 days, all piglets were euthanized, and lungs were evaluated by collecting samples for estimation of bacterial load by qPCR. Quantitative data obtained from physical parameters and laboratory investigation were analyzed by performing parametric or non-parametric statistical tests. Results indicate that animals from G2 showed smaller affected lung areas compared to G3. Animals from G2 and G4 had a low prevalence of animals shedding M. hyopneumoniae at 61 days of age. Additionally, no correlation was observed between lung lesions and M. hyopneumoniae load in lung and BALF samples in animals that received the oral vaccine, while a strong correlation was observed in other groups. In the present study, evidence points to the effectiveness of the oral vaccine developed for controlling M. hyopneumoniae in pig production under field conditions.

Nanostructured SBA-15 silica: An effective protective vehicle to oral hepatitis B vaccine immunization.

Due to its physicochemical properties, nanostructured mesoporous SBA-15 silica shows great potential as a vaccine adjuvant. This study evaluated the capacity of SBA-15 to encapsulate/adsorb the recombinant purified HBsAg from the Hepatitis B virus and the immunoresponsiveness of mice orally immunized with HBsAg inside SBA-15. A simulation of small angle X-ray scattering experimental results, together with the nitrogen adsorption isotherms data, allowed to determine the appropriate mass ratio of HBsAg:SBA-15, indicating antigen encapsulation into SBA-15 macroporosity. This was also evaluated by bicinchoninic acid assay and gel electrophoresis. The recruitment of inflammatory cells, an increase in production of specific antibodies, and the non-influence of silica on TH1 or TH2 polarization were observed after oral immunization. Besides, SBA-15 enhanced the phagocytosis of ovalbumin by dendritic cells, an important key to prove how this adjuvant works. Thus, it seems clear that the nanostructured SBA-15 is an effective and safe adjuvant for oral immunizations.

Ordered mesoporous silicas for potential applications in solid vaccine formulations.

In an effort to develop efficient vaccine formulations, the use of ordered mesoporous silica (SBA-15) as an antigen carrier has been investigated. SBA-15 has required properties such as high surface area and pore volume, including narrow pore size distribution to protect antigens inside its matrix. This study aimed to examine the impact of solvent removal methods, specifically freeze-drying and evaporation on the intrinsic properties of an immunogenic complex. The immunogenic complexes, synthesized and incorporated with BSA, were characterized by various physicochemical techniques. Small Angle X-ray Scattering measurements revealed the characteristic reflections associated to pure SBA-15, indicating the preservation of the silica mesostructured following BSA incorporation and the formation of BSA aggregates within the macropore region. Nitrogen Adsorption Isotherm measurements demonstrated a decrease in surface area and pore volume for all samples, indicating that the BSA was incorporated into the SBA-15 matrix. Fluorescence spectroscopy evidenced that the tryptophan residues in BSA inside SBA-15 or in solution displayed similar spectra, showing the preservation of the aromatic residues' environment. The Circular Dichroism spectra of BSA in both conditions suggest the preservation of its native secondary structure after the encapsulation process. The immunogenic analysis with the detection of anti-BSA IgG did not give any significant difference between the non-dried, freeze-dried or evaporated groups. However, all groups containing BSA and SBA-15 showed results almost three times higher than the groups with pure BSA (control group). These facts indicate that none of the BSA incorporation methods interfered with the immunogenicity of the complex. In particular, the freeze-dried process is regularly used in the pharmaceutical industry, therefore its adequacy to produce immunogenic complexes was proved Furthermore, the results showed that SBA-15 increased the immunogenic activity of BSA.

Evaluation of tumor growth remission in a murine model for subcutaneous solid tumors - Benefits of associating the antitumor agent crotamine with mesoporous nanosilica particles to achieve improved dosing frequency and efficacy.

Crotamine is a highly cationic polypeptide first isolated from South American rattlesnake venom, which exhibits affinity for acidic lysosomal vesicles and proliferating cells. This cationic nature is pivotal for its in vitro cytotoxicity and in vivo anticancer actions. This study aimed to enhance the antitumor efficacy of crotamine by associating it with the mesoporous SBA-15 silica, known for its controlled release of various chemical agents, including large proteins. This association aimed to mitigate the toxic effects while amplifying the pharmacological potency of several compounds. Comprehensive characterization, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential analysis, confirmed the successful association of crotamine with the non-toxic SBA-15 nanoparticles. The TEM imaging revealed nanoparticles with a nearly spherical shape and variations in uniformity upon crotamine association. Furthermore, DLS showed a narrow unimodal size distribution, emphasizing the formation of small aggregates. Zeta potential measurements indicated a distinct shift from negative to positive values upon crotamine association, underscoring its effective adsorption onto SBA-15. Intraperitoneal or oral administration of crotamine:SBA-15 in a murine melanoma model suggested the potential to reduce the frequency of crotamine doses without compromising efficacy. Interestingly, while the oral route enhanced the antitumor efficacy of crotamine, pH-dependent release from SBA-15 was observed. Thus, associating crotamine with SBA-15 could reduce the overall required dose to inhibit solid tumor growth, bolstering the prospect of crotamine as a potent anticancer agent.

Efficacy evaluation of a novel oral silica-based vaccine in inducing mucosal immunity against Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae, the main etiological agent of Porcine Enzootic Pneumonia, is widely spread in swine production worldwide. Its prevention is of great interest for the productive system, since its colonization in the lung tissue leads to intense production losses. This study aimed to compare the M. hyopneumoniae shedding and acute-phase response in 30 pigs submitted to different vaccination protocols: an experimental oral vaccine using a nanostructured mesoporous silica (SBA-15) as adjuvant (n = 10); an intramuscular commercially available vaccine at 24 days of age (n = 10); and a control group (n = 10) following experimental challenge with M. hyopneumoniae. Laryngeal and nasal swabs were collected weekly and oral fluids were collected at 7, 10, 14, 17, 23, 28, 35, 42, and 49 days post-infection to monitor pathogen excretion by qPCR. Nasal swabs were also used to detect anti-M. hyopneumoniae IgA by ELISA. Blood samples were collected for monitoring acute phase proteins. The antibody response was observed in both immunized groups seven days after vaccination, while the control group became positive for this immunoglobulin at 4 weeks after challenge. Lung lesion score was similar in the immunized groups, and lower than that observed in the control. SBA-15-adjuvanted oral vaccine provided immunological response, decreased shedding of M. hyopneumoniae and led to mucosal protection confirmed by the reduced pulmonary lesions. This study provides useful data for future development of vaccines against M. hyopneumoniae.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a "flip-flop" phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter.

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

Evaluation of NDEL1 oligopeptidase activity in blood and brain in an animal model of schizophrenia: effects of psychostimulants and antipsychotics.

Nuclear distribution element-like 1 (NDEL1) enzyme activity is important for neuritogenesis, neuronal migration, and neurodevelopment. We reported previously lower NDEL1 enzyme activity in blood of treated first episode psychosis and chronic schizophrenia (SCZ) compared to healthy control subjects, with even lower activity in treatment resistant chronic SCZ patients, implicating NDEL1 activity in SCZ. Herein, higher NDEL1 activity was observed in the blood and several brain regions of a validated animal model for SCZ at baseline. In addition, long-term treatment with typical or atypical antipsychotics, under conditions in which SCZ-like phenotypes were reported to be reversed in this animal model for SCZ, showed a significant NDEL1 activity reduction in blood and brain regions which is in line with clinical data. Importantly, these results support measuring NDEL1 enzyme activity in the peripheral blood to predict changes in NDEL1 activity in the CNS. Also, acute administration of psychostimulants, at levels reported to induce SCZ-like phenotype in normal rat strains, increased NDEL1 enzyme activity in blood. Therefore, alterations in NDEL1 activity after treatment with antipsychotics or psychostimulants may suggest a possible modulation of NDEL1 activity secondary to neurotransmission homeostasis and provide new insights into the role of NDEL1 in SCZ pathophysiology.

The Crotoxin:SBA-15 Complex Down-Regulates the Incidence and Intensity of Experimental Autoimmune Encephalomyelitis Through Peripheral and Central Actions.

Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 µg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.

Evaluation of a diphtheria and tetanus PLGA microencapsulated vaccine formulation without stabilizers.

Polymeric microspheres containing diphtheria and tetanus toxoids were prepared without protein stabilizers. A vaccine containing 2 Lf(tetanus) and 0.4 Lf(diphtheria) was injected either in BALB/c mice or in guinea-pigs. As control, a group received the alum-adsorbed unencapsulated toxoids. In mice, on day 44 one group and control received a booster and at day 111 the other group received the same booster dose. Before de booster, all groups had very low neutralizing antibodies, as determined by Toxin binding inhibition assay. One week after booster all groups had high antibody titers, especially those immunized with microencapsulated vaccine, which were at least 5 times higher than those immunized with alum vaccine for both antigens. Besides, guinea pigs receiving lower dose had antibodies titers as high as 60 UI/mL, and 30 times higher than those immunized with alum vaccine. Therefore by using an encapsulated vaccine without any kind of protein stabilizer we were able to induce in vivo protective responses irrespective of observed in vitro protein degradation by HPLC. Manipulating the vaccination schedule at the same time to the toxoids encapsulation does not only increase the antibody titers but also their specificity.

3D visualisation of hepatitis B vaccine in the oral delivery vehicle SBA-15.

Developing a technology that enables oral vaccines to work efficiently remains a considerable effort since a number of difficulties must be addressed. The key objective being to ensure the safe passage through the harsh conditions within the gastrointestinal tract, promoting delivery that induces enhanced immune response. In the particular case of hepatitis B, the oral formulation in the nanostructured silica SBA-15 is a viable approach. As a result of its porous structure, low toxicity and structural stability, SBA-15 is capable to protect and release the hepatitis B surface antigen (HBsAg), used in the vaccination scheme, at the desired destination. Furthermore, when compared to the currently used injection based delivery method, better or similar antibody response has been observed. However, information about the organisation of the antigen protein remains unknown. For instance, HBsAg is too large to enter the 10 nm ordered mesopores of SBA-15 and has a tendency to agglomerate when protected by the delivery system. Here we report on the pH dependence of HBsAg aggregation in saline solution investigated using small angle X-rays scattering that resulted in an optimisation of the encapsulation conditions. Additionally, X-ray microscopy combined with neutron and X-ray tomography provided full 3D information of the HBsAg clustering (i.e. agglomeration) inside the SBA-15 macropores. This method enables the visualisation of the organisation of the antigen in the interior of the delivery system, where agglomerated HBsAg coexists with its immunological effective uniformly distributed counterpart. This new approach, to be taken into account while preparing the formulation, can greatly help in the understanding of clinical studies and advance new formulations.

Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

BACKGROUND: The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. METHODS: Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. RESULTS AND DISCUSSION: We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. CONCLUSION: Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth.

Paracoccin is an N-acetyl-glucosamine-binding lectin from Paracoccidioides brasiliensis, which can be obtained in small amounts either from culture supernatants or yeast cell extracts. In the present work, immunoelectron microscopy with mouse anti-paracoccin IgG localized the antigen to the cell wall of P. brasiliensis yeast forms. Paracoccin interacted with chitin, and colocalized with beta-1,4-homopolymer of GlcNAc to the budding sites of P. brasiliensis yeast cell. In order to evaluate the role of paracoccin on fungal growth, yeast cells were cultivated in the presence of anti-paracoccin antibodies. A significant reduction of both colony forming units and individual yeast cells was observed as well as morphological alterations such as smaller colonies and cells more loosely aggregated than in control cultures without the antibody. A role of paracoccin on the cell wall organization was reinforced by alterations in the labeling pattern of chitin when yeasts were treated with anti-paracoccin antibodies. Binding of specific antibodies to paracoccin may disrupt the paracoccin/chitin interactions, resulting in the inhibition of P. brasiliensis growth.

The origin of prostate gland-secreted IgA and IgG.

The prostate secretes immunoglobulin (Ig) A (IgA) and IgG; however, how immunoglobulins reach the secretion, where the plasma cells are located, whether immunoglobulins are antigen-specific and where activation of the adaptive response occurs are still unknown. Immune cells, including CD45RA+ cells, were scattered in the stroma and not organized mucosae-associated lymphoid-tissue. IgA (but not IgG) immunostaining identified stromal plasma cells and epithelial cells in non-immunized rats. Injected tetramethylrhodamine-IgA transcytosed the epithelium along with polymeric immunoglobulin receptor. Oral immunization with ovalbumin/mesopourous SBA-15 silica adjuvant resulted in more stromal CD45RA+/IgA+ cells, increased content of ovalbumin-specific IgA and IgG, and the appearance of intraepithelial CD45RA+/IgG+ cells. An increased number of dendritic cells that cooperate in other sites with transient immunocompetent lymphocytes, and the higher levels of interleukin-1ß, interferon-γ and transforming growth factor-ß, explain the levels of specific antibodies. Nasal immunization produced similar results except for the increase in dendritic cells. This immunomodulatory strategy seems useful to boost immunity against genitourinary infections and, perhaps, cancer.

Identification of snake bradykinin-potentiating peptides (BPPs)-simile sequences in rat brain--Potential BPP-like precursor protein?

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.

Antibody responsiveness during immunization and challenge of genetically modified antibody responder mice with murine hepatitis virus 3.

The aim of this study was to evaluate some immunological patterns involved in natural and acquired resistance against MHV3 using the original model of genetically modified lines of mice selected for high (HIII) and low (LIII) antibody responsiveness. As previously shown, a lower pre-existing anti-MHV antibody level was found in susceptible HIII mice as compared to resistant LIII mice. Mortality rates of the F1 (H x L) hybrids and F2 and backcross segregants reflected co-dominance of both characters and the survivors had higher preexisting anti-MHV antibody titers. The present data show that both lines had the potential to synthesize antibodies and that the resistance acquired by the susceptible HIII mice paralleled the antibody synthesis. Nevertheless, higher antibody titers were necessary to confer resistance in HIII mice than in LIII ones. When compared to uvMHV3, a single immunization with a related infectious MHV strain induced a higher antibody synthesis and led the HIII mice to resist the MHV3 challenge. A direct correlation between the antibody level and resistance to infection was always observed in HIII mice. Although mounting a Th2 response as indicated by IgG1 responses, they were also able to readily synthesize large amounts of IgG2a antibodies after immunization or during infection, reflecting a Th1 response. The transfer of anti-MHV antibodies to susceptible HIII mice was capable of conferring resistance to MHV3, providing the antibodies were present before virus infection and in large amounts. The resistance and the survival time of these animals increased with the level of antibody administered. If these direct and clear data suggest that HIII mice can acquire resistance through antibodies, the basis of the resistance of the resistant LIII mice may rely on mechanisms less dependent on antibodies.

A universal polysaccharide conjugated vaccine against O111 E. coli.

E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.

A rational design for the nanoencapsulation of poisonous animal venoms in liposomes prepared with natural phospholipids.

Liposomes have been used since the 1970's to encapsulate drugs envisaging enhancement in efficacy and therapeutic index, avoidance of side effects and increase in the encapsulated agent stability. The major problem when encapsulating snake venoms is the liposomal membrane instability caused by venom phospholipases. Here the results obtained encapsulating Crotalus durissimus terrificus and a pool of Bothropic venoms within liposomes (LC and LB, respectively) used to produce anti-venom sera are presented. The strategy was to modify the immunization protocol to enhance antibody production and to minimize toxic effects by encapsulating inactivated venoms within stabilized liposomes. Chemically modified venoms were solubilized in a buffer containing an inhibitor and a chelating agent. The structures of the venoms were analyzed by UV, CD spectroscopy and ELISA. In spite of the differences in the helical content between natural and modified venoms, they were recognized by horse anti-sera. To maintain long-term stability, mannitol was used as a cryoprotectant. The encapsulation efficiencies were 59 % (LB) and 99 % (LC), as followed by filtration on Sephacryl S1000. Light scattering measurements led us to conclude that both, LB (119 ±47 nm) and LC (147±56 nm) were stable for 22 days at 4 °C, even after lyophilization. Genetically selected mice and mixed breed horses were immunized with these formulations. The animals did not show clinical symptoms of venom toxicity. Both, LB and LC enhanced by at least 30 % the antibody titers 25 days after injection and total IgG titers remained high 91 days after immunization. The liposomal formulation clearly exhibited adjuvant properties.

Premolis semirufa (Walker, 1856) envenomation, disease affecting rubber tappers of the Amazon: searching for caterpillar-bristles toxic components.

BACKGROUND: The caterpillar of the moth Premolis semirufa (Lepidoptera: Arctiidae), commonly named Pararama, is endemic of the Amazon basin. Accidental contact with these caterpillar bristles causes local symptoms such as intense heat, pain, edema and itching which last for three to seven days; however, after multiples contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite of the public health hazard of Premolis semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the biological and immunochemical characteristics of the caterpillar's bristles components. Analysis of the bristles extract in in vitro assays revealed the presence of proteolytic and hyaluronidase activities but no phospholipase A(2) activity. In vivo, it was observed that the bristles extract is not lethal but can induce an intense inflammatory process, characterized by the presence of neutrophils in the paw tissues of injected mice. Furthermore, the bristles components stimulated an intense and specific antibody response but autoantibodies such as anti-DNA or anti-collagen type II were not detected. CONCLUSION: The results suggest that Premolis semirufa caterpillar bristles secretion contains a mixture of different enzymes that may act together in the generation and development of the clinical manifestations of the Pararama envenomation. Moreover, the high immunogenicity of the caterpillar bristles components, as shown by the generation of high antibody titers, may also contribute to the induction and establishment of the inflammatory disease.