Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of ß-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1ß. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Unravelling the Immunotoxicity of Polycaprolactone Nanoparticles-Effects of Polymer Molecular Weight, Hydrolysis, and Blends.

Poly-ε-caprolactone (PCL) is a biodegradable polyester that has FDA and CE approval as a medical device. Nonetheless, the lack of toxicity exhibited by the polymer cannot be extrapolated to its nanomaterial conformation. Despite PCL-based NPs being widely studied in the biomedical field for their advantages as controlled drug delivery systems, little data describe PCL NPs' toxicity, particularly immunotoxicity. This work assessed different PCL-based delivery systems intended for protein delivery regarding their immunotoxicity and hemocompatibility. Two different molecular weight PCL polymers were used, as well as blends with chitosan and glucan. Results showed that the presence of NaOH during the production of PCL2 NPs and PCL2/glucan NPs induced PCL alkali hydrolysis, generating more reactive groups (carboxyl and hydroxyl) that contributed to an increased toxicity of the NPs (higher reduction in peripheral blood mononuclear cell viability and lower hemocompatibility). PCL2/glucan NPs showed an anti-inflammatory activity characterized by the inhibition of LPS stimulated nitric oxide (NO) and TNF-α. In conclusion, generalizations among different PCL NP delivery systems must be avoided, and immunotoxicity assessments should be performed in the early stage of product development to increase the clinical success of the nanomedicine.

Optimization of Chitosan-α-casein Nanoparticles for Improved Gene Delivery: Characterization, Stability, and Transfection Efficiency.

Among non-viral vectors, the cationic polymer chitosan has gained attention as a gene delivery system. We hypothesized that the addition of casein into the nanoparticle's structure would facilitate a proper gene transfer. The work herein presented aimed to optimize the production method of chitosan-casein nanoparticles (ChiCas NPs) and to test their ability as a gene delivery system. ChiCas NPs formulation optimization was carried out by analyzing several characteristics such as NP size, zeta potential, and chitosan and casein incorporation efficacy. The best formulation developed presented small and homogenous particle size (around 335 nm) and positive zeta potential (≈ + 38 mV), and showed to be stable for 34 weeks both, at 4°C and 20°C. The particles were further used to entrap or to adsorb DNA and form NPs-DNA complexes. In vitro transfection studies, carried out in COS-7 cells, suggested a low transfection efficiency of the different NPs:DNA ratios tested, comparatively to the positive control. Nonetheless, we could observe that the complexes with larger sizes presented better transfection results than those with smaller diameters. To conclude, ChiCas NPs have great technological potential since the preparation process is very simple, and the DNA incorporation efficacy is very high and shows to be physically very stable. The NPs:DNA ratio still needs to be optimized with the aim of achieving better transfection results and being able to anticipate a high gene expression on DNA-based vaccination studies.

Radiographic Reference Values for the Cardiac Silhouette in Bonelli's Eagle (Aquila fasciata).

Radiographs are an important diagnostic tool available in wildlife hospitals to evaluate the size of the avian heart. Despite the large variety wild birds in the Iberian peninsula, clinical studies addressing these species are lacking. To establish reference values for cardiac size in the Bonelli's eagle (Aquila fasciata), ventrodorsal radiographs of 20 healthy birds were obtained, and the width of the cardiac silhouette, sternum, thorax, coracoid, and hepatic silhouette were measured. The ratios between cardiac width and other mentioned indices were calculated. Results showed that cardiac silhouette width should occupy 81%-93% of sternal width, 48%-57% of thoracic width, and 506%-673% of coracoid width. The cardiac silhouette width was strongly correlated with sternal and thoracic widths. A moderate correlation was found between the width of the heart and the width of the coracoid. No significant correlation was found between width of the cardiac silhouette and the hepatic silhouette. These results support that sternal and thoracic widths should be used preferentially when evaluating the width of the cardiac silhouette in this species. The values obtained in this study can be used as a reference for normal cardiac size when evaluating radiographs of Bonelli's eagles.

Adjuvant Activity of Poly-ε-caprolactone/Chitosan Nanoparticles Characterized by Mast Cell Activation and IFN-γ and IL-17 Production.

Polymeric nanoparticles (NPs) are extremely attractive vaccine adjuvants, able to promote antigen delivery and in some instances, exert intrinsic immunostimulatory properties that enhance antigen specific humoral and cellular immune responses. The poly-ε-caprolactone (PCL)/chitosan NPs were designed with the aim of being able to combine the properties of the 2 polymers in the preparation of an adjuvant for the hepatitis B surface antigen (HBsAg). This article reports important results of an in vitro mechanistic study and immunization studies with HBsAg associated with different concentrations of the nanoparticles. The results revealed that PCL/chitosan NPs promoted mast cell (MC) activation (ß-hexosaminidase release) and that its adjuvant effect is not mediated by the TNF-α secretion. Moreover, we demonstrated that HBsAg loaded PCL/chitosan NPs, administered through the subcutaneous (SC) route, were able to induce higher specific antibody titers without increasing IgE when compared to a commercial vaccine, and that the IgG titers are nanoparticle-dose dependent. The results also revealed the NPs' capability to promote a cellular immune response against HBsAg, characterized by the production of IFN-γ and IL-17. These results demonstrated that PCL/chitosan NPs are a good hepatitis B antigen adjuvant, with direct influence on the intensity and type of the immune response generated.

The Inclusion of Chitosan in Poly-ε-caprolactone Nanoparticles: Impact on the Delivery System Characteristics and on the Adsorbed Ovalbumin Secondary Structure.

This report extensively explores the benefits of including chitosan into poly-ε-caprolactone (PCL) nanoparticles (NPs) to obtain an improved protein/antigen delivery system. Blend NPs (PCL/chitosan NPs) showed improved protein adsorption efficacy (84%) in low shear stress and aqueous environment, suggesting that a synergistic effect between PCL hydrophobic nature and the positive charges of chitosan present at the particle surface was responsible for protein interaction. Additionally, thermal analysis suggested the blend NPs were more stable than the isolated polymers and cytotoxicity assays in a primary cell culture revealed chitosan inclusion in PCL NPs reduced the toxicity of the delivery system. A quantitative 6-month stability study showed that the inclusion of chitosan in PCL NPs did not induce a change in adsorbed ovalbumin (OVA) secondary structure characterized by the increase in the unordered conformation (random coil), as it was observed for OVA adsorbed to chitosan NPs. Additionally, the slight conformational changes occurred, are not expected to compromise ovalbumin secondary structure and activity, during a 6-month storage even at high temperatures (45°C). In simulated biological fluids, PCL/chitosan NPs showed an advantageous release profile for oral delivery. Overall, the combination of PCL and chitosan characteristics provide PCL/chitosan NPs valuable features particularly important to the development of vaccines for developing countries, where it is difficult to ensure cold chain transportation and non-parenteral formulations would be preferred.

Weight gain in the first two years of life, asthma and atopy: the SCAALA cohort study.

OBJECTIVE: To evaluate the association between weight gain in the first two years of life and the occurrence of wheezing, asthma, serum IgE, skin reactivity and pulmonary function. DESIGN: Cohort study. SETTING: The metropolitan region of Salvador, Bahia, Brazil. SUBJECTS: The association was studied between 1997 and 2005 in 669 children up to 11 years of age. Data were collected on asthma and risk factors, both current factors and those present in the first years of life. Weight gain was considered fast when the Z-score was >0·67. Poisson regression was used in the multivariate statistical analysis. RESULTS: Wheezing was reported in 25·6 % of the children. Weight gain was considered fast (Z-score >0·67) in 29·6 % of the children and slow (Z-score <-0·67) in 13·9 %. Children in the slow weight gain group had 36 % fewer symptoms of asthma (prevalence ratio = 0·65; 95 % CI 0·42, 0·99). CONCLUSIONS: Slower weight gain in the early years of life may constitute a protective factor against symptoms of asthma. The relevance of this finding for public health is not yet certain, since it is known that children with slow and fast weight gain may be more likely to develop adverse health consequences related to both these situations.

Exploring the immunomodulatory properties of glucan particles in human primary cells.

The immunomodulatory properties of ß-glucans have sparked interest among various medical fields. As vaccine adjuvants, glucan particles offer additional advantages as antigen delivery systems. This study reported the immunomodulatory properties of glucan particles with different size and chemical composition. The effect of glucan microparticles (GPs) and glucan nanoparticles (Glu 130 and 355 NPs) was evaluated on human immune cells. While GPs and Glu 355 NPs demonstrated substantial interaction with Dectin-1 receptor on monocytes, Glu 130 NPs exhibited reduced activation of this receptor. This observation was substantiated by blocking Dectin-1, resulting in inhibition of reactive oxygen species production induced by GPs and Glu 355 NPs. Notably, monocyte-derived dendritic cells (moDCs) stimulated by Glu 355 NPs exhibited phenotypic and functional maturation, essential for antigen cross-presentation. The immunomodulatory efficacy was investigated using an autologous mixed lymphocyte reaction (AMLR), resulting in considerable rates of lymphocyte proliferation and an intriguing profile of cytokine and chemokine release. Our findings highlight the importance of meticulously characterizing the size and chemical composition of ß-glucan particles to draw accurate conclusions regarding their immunomodulatory activity. This in vitro model mimics the human cellular immune response, and the results obtained endorse the use of ß-glucan-based delivery systems as future vaccine adjuvants.

Hospitalizations in Brazil according to National Health Survey estimates, 2013 and 2019.

OBJECTIVE: To compare the profile and prevalence of hospitalizations in Brazil based on estimates from the National Health Survey (PNS), 2013 and 2019. METHODS: A cross-sectional study that used data from the 2013 PNS and the 2019 PNS. The outcome was having been hospitalized for 24 hours or more in the last 12 months. We calculated the proportion of the population in different categories of age group, presence or absence of chronic diseases, and perception of health status. We estimated the total number of hospitalizations and the proportion corresponding to each category of age group, chronic disease, and perceived health status. We calculated the prevalence of hospitalization according to geographic, socioeconomic, and health conditions. We compared the estimates of two editions of the PNS using Student's t-test for independent samples. We considered significant differences when the p-value was less than 0.01. And finally, we compared hospitalization estimates with administrative data to assess data consistency. RESULTS: We observed that the proportion of chronically ill people in the population increased from 15.04% to 31.48%. This group was responsible for 36.76% of the total number of hospitalizations in 2013 and 57.61% in 2019. The prevalence of hospitalizations increased significantly between the two surveys and the increases were higher in the Southeast region and among people who have private health insurance. A discrepancy was found between administrative data and survey estimates. Obstetric hospitalizations and health insurance hospitalizations were underestimated. CONCLUSION: There was an increase in overall hospitalization rates in the period between the PNS 2013 and PNS 2019, especially among people with better access to health services. The hospitalization profile also changed-in the 2013 PNS, hospitalizations of people without chronic diseases predominated. This was reversed in PNS 2019.

Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

Overweight, asthma symptoms, atopy and pulmonary function in children of 4-12 years of age: findings from the SCAALA cohort in Salvador, Bahia, Brazil.

OBJECTIVE: To evaluate the association between overweight and the occurrence of asthma and atopy in a cohort of children of 4-12 years of age living in the city of Salvador in 2005. DESIGN: Cross-sectional study nested in a cohort. SETTING: The metropolitan region of Salvador, Bahia, Brazil. SUBJECTS: The study included 1129 children of 4-12 years age who presented complete information on the variables used here. Skin tests for allergy, spirometry, faecal parasitology, serum IgE and anthropometric surveys were conducted. Poisson's multivariate regression was adopted. RESULTS: Wheezing was found in 29·1% and asthma in 22·8% of children, both conditions being more common in those under 6 years of age and 34% more common in overweight children (prevalence ratio (PR) = 1·34; 95% CI 1·07, 1·67) following adjustment. The ratio between forced expiratory volume in 1s and forced vital capacity was associated with overweight (PR = 1·35; 95% CI 1·11, 1·61). No statistically significant association was found between overweight and allergen-specific IgE or with wheezing. CONCLUSIONS: These results are in agreement with the hypothesis that overweight is associated with asthma and pulmonary function, even following adjustment for intervening variables known to be associated with the pathogeny of asthma.

Chitosan-coated PLGA nanoparticles for the nasal delivery of ropinirole hydrochloride: In vitro and ex vivo evaluation of efficacy and safety.

Nose-to-brain delivery is an attractive route for direct drug delivery to the central nervous system (CNS), avoiding hepatic first-pass metabolism and solving blood-brain barrier passage issues. Therefore, the aim of the present study was the development of PLGA and PLGA/chitosan (chit) nanoparticles (NPs) with mucoadhesive properties, able to encapsulate ropinirole hydrochloride (RH), an anti-Parkinsonian dopaminergic agonist, and suitable to promote RH delivery across the nasal mucosa. NPs produced by nanoprecipitation showed spherical shape and a mean average size of 98.8 nm and 468.0 nm (PLGA and PLGA/chit, respectively). RH loaded PLGA/chit NPs showed a complete release of the drug in simulated nasal electrolyte solution (SNES) over the period of 24 h and increased the permeation of RH through sheep nasal mucosa by 3.22-fold in comparison to PLGA NPs. None of RH loaded NPs induced hemolysis in whole blood or the production of reactive oxygen species (ROS) in Raw 264.7 cells. On their turn, PLGA/chit NPs decreased cell viability of Raw 264.7 cells and Peripheral Blood Mononuclear Cells (PBMCs) in a concentration-dependent manner. These results revealed that, particularly PLGA/chit NPs, could be a valuable carrier for the delivery of RH to the CNS, opening a new path for Parkinson's disease therapy.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 µg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

A Methodological Safe-by-Design Approach for the Development of Nanomedicines.

Safe-by-Design (SbD) concepts foresee the risk identification and reduction as well as uncertainties regarding human health and environmental safety in early stages of product development. The EU's NANoREG project and further on the H2020 ProSafe initiative, NanoReg2, and CALIBRATE projects have developed a general SbD approach for nanotechnologies (e.g., paints, textiles, etc.). Based on it, the GoNanoBioMat project elaborated a methodological SbD approach (GoNanoBioMat SbD approach) for nanomedicines with a focus on polymeric nanobiomaterials (NBMs) used for drug delivery. NBMs have various advantages such as the potential to increase drug efficacy and bioavailability. However, the nanoscale brings new challenges to product design, manufacturing, and handling. Nanomedicines are costly and require the combination of knowledge from several fields. In this paper, we present the GoNanoBioMat SbD approach, which allows identifying and addressing the relevant safety aspects to address when developing polymeric NBMs during design, characterization, assessment of human health and environmental risk, manufacturing and handling, and combines the nanoscale and medicine field under one approach. Furthermore, regulatory requirements are integrated into the innovation process.

Chitosan Plus Compound 48/80: Formulation and Preliminary Evaluation as a Hepatitis B Vaccine Adjuvant.

Current vaccine research is mostly based on subunit antigens. Despite the better toxicity profile of these antigens they are often poorly immunogenic, so adjuvant association has been explored as a strategy to obtain a potent vaccine formulation. Recently, mast cell activators were recognized as a new class of vaccine adjuvants capable of potentiating mucosal and systemic immune responses. In this study, a co-adjuvanted delivery system was developed and characterized, combining the mast cell activator C48/80 with chitosan nanoparticles (Chi-C48/80 NPs), and the results were compared with plain chitosan nanoparticles. The adsorption of model antigens onto the NP surface as well as the biocompatibility of the system was not affected by the incorporation of C48/80 in the formulation. The stability of the nanoparticles was demonstrated by studying the variation of size and zeta potential at different times, and the ability to be internalized by antigen presenting cells was confirmed by confocal microscopy. Vaccination studies with hepatitis B surface antigen loaded Chi-C48/80 NPs validated the adjuvanticity of the delivery system, demonstrating for the first time a successful association between a mast cell activator and chitosan nanoparticles as a vaccine adjuvant for hepatitis B virus, applied to a nasal vaccination strategy.

Surgical Excision of an Adrenal Neuroblastoma in a Dog.

A 11-month-old, intact male, Rhodesian Ridgeback was presented to the Veterinary Teaching Hospital with signs of inappetence, lethargy, and abdominal pain for 3 days. A large and well-defined abdominal retroperitoneal mass, related with the left kidney, at the expected location of the adrenal gland, was revealed by radiography, ultrasound, and computed tomography. The mass extended caudally to the iliac artery bifurcation, compressing the aorta, caudal vena cava, and both kidneys. Cytology findings were compatible with a malignant round cell tumor. The most probable diagnosis was neuroblastoma. Following a comprehensive discussion with the owners about a treatment plan, surgical excision was performed. Because a wide excision would compromise major vessels, excision was performed after careful dissection of the aorta and vena cava. The left kidney was removed because the proximal ureter could not be separated from the tumor. The animal recovered successfully. Diagnosis was confirmed by histopathology and immunohistochemistry, but the owners decided not to pursue any further treatment. Clinical signs of abdominal pain recurred within 1 month following surgery. Therefore, the animal was euthanized upon the owners' request. This report describes the diagnosis, surgical treatment, and follow-up of a dog with an abdominal peripheral neuroblastoma.

Poly(D,L-Lactic Acid) Nanoparticle Size Reduction Increases Its Immunotoxicity.

Polylactic acid (PLA), a biodegradable and biocompatible polymer produced from renewable resources, has been widely used as a nanoparticulate platform for antigen and drug delivery. Despite generally regarded as safe, its immunotoxicological profile, when used as a polymeric nanoparticle (NP), is not well-documented. Thus, this study intends to address this gap, by evaluating the toxicity of two different sized PLA NPs (PLAA NPs and PLAB NPs), produced by two nanoprecipitation methods and extensively characterized regarding their physicochemical properties in in vitro experimental conditions. After production, PLAA NPs mean diameter (187.9 ± 36.9 nm) was superior to PLAB NPs (109.1 ± 10.4 nm). Interestingly, when in RPMI medium, both presented similar mean size (around 100 nm) and neutral zeta potential, possibly explaining the similarity between their cytotoxicity profile in PBMCs. On the other hand, in DMEM medium, PLAA NPs presented smaller mean diameter (75.3 ± 9.8 nm) when compared to PLAB NPs (161.9 ± 8.2 nm), which may explain its higher toxicity in RAW 264.7. Likewise, PLAA NPs induced a higher dose-dependent ROS production. Irrespective of size differences, none of the PLA NPs presented an inflammatory potential (NO production) or a hemolytic activity in human blood. The results herein presented suggest the hypothesis, to be tested in the future, that PLA NPs presenting a smaller sized population possess increased cytotoxicity. Furthermore, this study emphasizes the importance of interpreting results based on adequate physicochemical characterization of nanoformulations in biological medium. As observed, small differences in size triggered by the dispersion in cell culture medium can have repercussions on toxicity, and if not correctly evaluated can lead to misinterpretations, and subsequent ambiguous conclusions.

Hazard Assessment of Polymeric Nanobiomaterials for Drug Delivery: What Can We Learn From Literature So Far.

The physicochemical properties of nanobiomaterials, such as their small size and high surface area ratio, make them attractive, novel drug-carriers, with increased cellular interaction and increased permeation through several biological barriers. However, these same properties hinder any extrapolation of knowledge from the toxicity of their raw material. Though, as suggested by the Safe-by-Design (SbD) concept, the hazard assessment should be the starting point for the formulation development. This may enable us to select the most promising candidates of polymeric nanobiomaterials for safe drug-delivery in an early phase of innovation. Nowadays the majority of reports on polymeric nanomaterials are focused in optimizing the nanocarrier features, such as size, physical stability and drug loading efficacy, and in performing preliminary cytocompatibility testing and proving effectiveness of the drug loaded formulation, using the most diverse cell lines. Toxicological studies exploring the biological effects of the polymeric nanomaterials, particularly regarding immune system interaction are often disregarded. The objective of this review is to illustrate what is known about the biological effects of polymeric nanomaterials and to see if trends in toxicity and general links between physicochemical properties of nanobiomaterials and their effects may be derived. For that, data on chitosan, polylactic acid (PLA), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA) and policaprolactone (PCL) nanomaterials will be evaluated regarding acute and repeated dose toxicity, inflammation, oxidative stress, genotoxicity, toxicity on reproduction and hemocompatibility. We further intend to identify the analytical and biological tests described in the literature used to assess polymeric nanomaterials toxicity, to evaluate and interpret the available results and to expose the obstacles and challenges related to the nanomaterial testing. At the present time, considering all the information collected, the hazard assessment and thus also the SbD of polymeric nanomaterials is still dependent on a case-by-case evaluation. The identified obstacles prevent the identification of toxicity trends and the generation of an assertive toxicity database. In the future, in vitro and in vivo harmonized toxicity studies using unloaded polymeric nanomaterials, extensively characterized regarding their intrinsic and extrinsic properties should allow to generate such database. Such a database would enable us to apply the SbD approach more efficiently.

Giardia duodenalis infection and anthropometric status in preschoolers in Salvador, Bahia State, Brazil.

The aim of this study was to estimate the association between Giardia duodenalis infection and anthropometric deficits, as measured by weight-for-age and height-for-age. This cross-sectional study included 629 children from 12 to 48 months of age, selected from 30 geographic areas in the city of Salvador, Bahia State, Brazil. Poisson regression and linear regression were used for the multivariate statistical analyses. G. duodenalis was diagnosed in 13.5% of the children. The children's breastfeeding duration and living conditions (garbage collection and paved streets or sidewalks) modified the effect of G. duodenalis infection on anthropometric status. Among infected children, there were statistically significant associations between weight deficit and shorter breastfeeding (PR=2.22; 95%CI: 1.56-3.14) and inadequate paving of streets and sidewalks (PR=2.00; 95%CI: 1.37-2.92), while height deficit was associated with deficient public garbage collection (PR=2.21; 95%CI: 1.31-2.51). In the linear regression, the association with the anthropometric indicators remained positive and statistically significant. The child's unhealthy living environment aggravated the negative effect of G. duodenalis infection on anthropometric status, and breastfeeding was a protective factor in the outcome.

Oral hepatitis B vaccine: chitosan or glucan based delivery systems for efficient HBsAg immunization following subcutaneous priming.

The World Health Organization encourages "the development of oral formulations to simplify their transport, storage and administration in poor countries", and to "facilitate an effective immunization program to prevent sexually transmitted hepatitis B". Thus, two distinct and promising delivery systems were developed: recombinant hepatitis B surface antigen (HBsAg) encapsulated into alginate-coated chitosan particles (AlgChiPs) and into glucan particles (GPs) mainly composed of ß-1,3-d-glucan. In vitro preliminary studies showed that both could be internalized by peripheral blood mononuclear cells and murine Peyer's patches, an imperative aspect regarding oral immunization. Chitosan particles (ChiPs) have shown interesting immunostimulating properties as mast cells activators. Vaccination studies reveal that three oral immunizations induced serum anti-HBsAg Immunoglobulin G (IgG) in 60 % of the animals and anti-HBsAg secretory IgA in faeces for both formulations. When subcutaneous (SC) priming was done, followed by two oral boosts, all mice were responder and much higher serum anti-HBsAg IgG titers were observed, besides mucosal protective immunity.