Biodistribution and toxicity of epitope-functionalized dextran iron oxide nanoparticles in a pregnant murine model.

In pursuit of a preventive therapeutic for maternal autoantibody-related (MAR) autism, we assessed the toxicity, biodistribution, and clearance of a MAR specific peptide-functionalized dextran iron oxide nanoparticle system in pregnant murine dams. We previously synthesized ~15 nm citrate-coated dextran iron oxide nanoparticles (DIONPs), surface-modified with polyethylene glycol and MAR peptides to produce systems for nanoparticle-based autoantibody reception and entrapments (SNAREs). First, we investigated their immunogenicity and MAR lactate dehydrogenase B antibody uptake in murine serum in vitro. To assess biodistribution and toxicity, as well as systemic effects, we performed in vivo clinical and post mortem pathological evaluations. We observed minimal production of inflammatory cytokines-interleukin 10 (IL-10) and IL-12 following in vitro exposure of macrophages to SNAREs. We established the maximum tolerated dose of SNAREs to be 150 mg/kg at which deposition of iron was evident in the liver and lungs by histology and magnetic resonance imaging but no concurrent evidence of liver toxicity or lung infarction was detected. Further, SNAREs exhibited slower clearance from the maternal blood in pregnant dams compared to DIONPs based on serum total iron concentration. These findings demonstrated that the SNAREs have a prolonged presence in the blood and are safe for use in pregnant mice as evidenced by no associated organ damage, failure, inflammation, and fetal mortality. Determination of the MTD dose sets the basis for future studies investigating the efficacy of our nanoparticle formulation in a MAR autism mouse model.

How microwave treatment of gluten affects its toxicity for celiac patients? A study on the effect of microwaves on the structure, conformation, functionality and immunogenicity of gluten.

The microwave heating of wheat kernels, flour, and gluten, has attracted attention lately because it has been claimed to abolish gluten toxicity for celiac patients. Nevertheless, contradictory results have been reported regarding the effect on gluten celiac-immunotoxicity. In order to better understand the effect of the microwave treatment on gluten structure, conformation, functionality and celiac-immunotoxicity, a central composite design with two factors, power level, and treatment time, was used to investigate a possible quadratic and interaction effects between both factors. Extractable gliadins content was affected by the power and time in a linear and quadratic fashion; extractable glutenins were not affected. Gluten secondary structure was affected by the microwave treatment and related to the polymer's disaggregation phenomenon observed. In fact, the microwave treatment increased the amount of potentially toxic epitopes released after peptic and tryptic digestion, showing inefficiency as a treatment to detoxify the gluten for celiac disease patients.

Identifying polyglutamine protein species in situ that best predict neurodegeneration.

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by expanded polyQ and disappears in higher-molecular-weight aggregated forms, such as inclusion bodies. These results suggest that protein monomers and possibly small oligomers containing expanded polyQ stretches can adopt a conformation that is recognized by 3B5H10 and is toxic or closely related to a toxic species.

Callose deposition: a multifaceted plant defense response.

Callose deposition in Arabidopsis has emerged as a popular model system to quantify activity of plant immunity. However, there has been a noticeable rise in contradicting reports about the regulation of pathogen-induced callose. To address this controversy, we have examined the robustness of callose deposition under different growth conditions and in response to two different pathogen-associated molecular patterns, the flagellin epitope Flg22 and the polysaccharide chitosan. Based on a commonly used hydroponic culture system, we found that variations in growth conditions have a major impact on the plant's overall capacity to deposit callose. This environmental variability correlated with levels of hydrogen peroxide (H2O2) production. Depending on the growth conditions, pretreatment with abscissic acid stimulated or repressed callose deposition. Despite a similar effect of growth conditions on Flg22- and chitosan-induced callose, both responses showed differences in timing, tissue responsiveness, and colocalization with H2O2. Furthermore, mutant analysis revealed that Flg22- and chitosan-induced callose differ in the requirement for the NADPH oxidase RBOHD, the glucosinolate regulatory enzymes VTC1 and PEN2, and the callose synthase PMR4. Our study demonstrates that callose is a multifaceted defense response that is controlled by distinct signaling pathways, depending on the environmental conditions and the challenging pathogen-associated molecular pattern.

Métodos analíticos para detecção de glúten em alimentos: [revisão] / Analytical methods for detecting gluten in foods: [review]

O tratamento para a doença celíaca (DC) consiste em dieta livre das prolaminas: gliadina, hordeina, secalina e avenina existentes no trigo, centeio, cevada e aveia. A Comissão do Codex Alimentarius(FAO/WHO) definiu o limite de 200 ppm (mg/kg) de glúten para o alimento ser considerado livre desse produto. A revisão de 2004 do Codex Alimentarius sugeriu o limite de 20 ppm para produtos naturalmente sem glúten e de 200 ppm para produtos derivados de ingredientes não fonte de glúten, porém esses limites estão ainda em discussão. Entre os métodos analíticos para detectar ou determinar glúten/gliadina têm sido empregadas as técnicas de: espectrometria de massa, cromatografia líquida, análise de DNA do trigo e imununoenzimáticos. O método oficial adotado pela Association of Official Analytical Chemistry(AOAC) é o ELISA baseado no anticorpo monoclonal para gliadina. O Codex Alimentarius endossou temporariamente, o R5 ELISA como Método Tipo I. O R5 ELISA utiliza anticorpo monoclonal para o pentapeptídeo tóxico existente na gliadina, hordeina e secalina. O ELISA, em função de sua maior sensibilidade e apropriado limite de detecção (1,5 ppm de gliadina), é considerado superior às demais técnicas. A presença de pequenos fragmentos de proteína existentes em prolaminas hidrolisadas devem ser avaliados por métodos baseados em DNA.

Treatment of celiac disease (CD) is consisted of a diet free of prolamins as gliadin, hordein, secalin, and avenin existing in wheat, barley, rye, and oats. The Codex Alimentarius Commission (FAO/WHO) has defined the maximum gluten content of 200 ppm (mg/kg) to consider as gluten-free products. The Codex Commission Review 2004 recommended the limits of 20 mg/kg dry mass for naturally gluten- free products, and 200 mg/kg for food derived from gluten - free products. Among analytical methods for detecting or determining glúten/gliadin the following techniques have been used: mass spectrometry,high performance-liquid chromatography, wheat DNA analysis by PCR, and immunoenzyme assay. ELISA based on monoclonal antibody for ω gliadin has been the technique officially recommended by the Association of Official Analytical Chemistry (AOAC). R5-ELISA has got a provisory approval from the Codex Alimentarius as type I method. R5-ELISA is based on the use of a monoclonal antibody for pentapeptide occurring in gliadin, hordein, secalin, and avenin, and this assay presents sensitivity and limit detection (1.5 ppm gliadin) rates superior in comparison to other techniques. Although, the qualitative analysis for detecting he presence of small fragment of protein in hydrolized prolamine should be assessed by means of DNA-based techniques.

Encephalitogenic epitopes of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein for experimental allergic encephalomyelitis induction in Biozzi ABH (H-2Ag7) mice share an amino acid motif.

Myelin basic protein (MBP) and synthetic MBP peptides were screened for their ability to induce experimental allergic encephalomyelitis in Biozzi ABH (H-2Ag7) mice. In contrast to the failure of native MBP to induce experimental allergic encephalomyelitis, the use of overlapping MBP peptides revealed epitopes within MBP 12-26 and MBP 21-35, which induced mild disease. In comparison with disease induced by spinal cord homogenate or peptides of myelin oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP), the low incidence indicates that, at least in ABH mice, MBP is a minor encephalitogen. However, the data suggest the presence of a peptide core between MBP 21-26 (HARHGF), which contains similar elements to the previously defined encephalitogenic MOG 1-22 and PLP 56-70 peptides. The fine specificity of these epitopes was further investigated using frame-shifted peptides, which indicated cores between MOG 9-15 (GYPIRAL) and PLP 62-68 (NVIHAFQ). Based on these pathogenic peptides, a putative H-2Ag7 binding motif is suggested that contains a series of hydrophobic, basic, small, and large hydrophobic residues within a 6 to 7 amino acid core. The core and particular importance of these four residues in PLP 56-70 was confirmed in vitro using amino acid substitution studies. These findings support many of the predictions made by computer modeling of peptide:H-2Ag7 interactions. This may have relevance in the design of strategies in the treatment of experimental autoimmune diseases in animals that express this haplotype.

[The use of a new strain of Vibrio cholerae O139 as a producer of an enteric chemical vaccine]. / Ispol'zovanie novogo shtamma Vibrio cholerae O139 v kachestve produtsenta énteral'noi khimicheskoi vaktsiny.

Conditions for the submerged cultivation of a strain of V. cholerae O139, were worked out. These conditions ensured a high yield of biomass, soluble O-antigen and exoenzymes (proteinase, phospholipase A) into the culture medium, which exceeded their production by strains of serovar O1, respectively, 2, 3, 4 and 8 times. The preparation, isolated from the culture fluid and lyophilized, contained up to 50% of O-antigen and exoenzymes. In experiments on white mice the preparation exhibited low toxicity (LD50 was equal, on the average, to 1.2 mg) and immunogenicity (ED50 was equal to 3-5 micrograms) with respect to V. cholerae O139, which corresponded to the protective potency of commercial vaccine against V. cholerae O1 infection.

Specific targeting of phototoxic haptenated liposomes to a hapten-specific B cell lymphoma.

A method is reported to eliminate B lymphocytes specific for a haptenated lipid by using the lipid hapten to target a photosensitive drug to them. The photosensitizer eosin was coupled to a phospholipid and incorporated into trinitrophenol (TNP)-bearing small unilamellar vesicles of egg phosphatidylcholine (PC) and cholesterol in order to target the photosensitizer to B lymphoma cells (A20-HL) with TNP-specific membrane IgM receptors in vitro. Exposure of the treated cells to visible light led to an antigen-specific toxic effect indicated by inhibition of cell proliferation. A significantly higher concentration of liposomal eosin was required to inhibit control B cells. These were genetically identical B lymphoma cells (A20-2J) which lack only the DNA for the surface antigen receptor. Furthermore, pretreatment with TNP-conjugated keyhole limpet hemocyanin or anti-IgM antibody abolished the antigen-specific toxic effect, confirming that the TNP-targeted liposomal eosin mediates its effect by binding to the Ig antigen receptors on TNP-specific B cells. Incubation of cells with the TNP-bearing phototoxic liposomes at 4 degrees C instead of 37 degrees C did not alter the antigen-specific targeting effect, suggesting that uptake of the liposomal drug into the cells is not necessary for its toxic effect. Replacement of the liposomal phospholipid (egg PC) with saturated species of PC having higher phase transition temperatures or with sphingomyelin caused a decrease of the antigen-specific effect. These results demonstrate the potential use of antigen-bearing liposomal phototoxic drugs for the purpose of targeting and eliminating B cells with antigen-specific surface Ig receptors.

Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes.

BALB/c mice develop autoimmune myocarditis after immunization with mouse cardiac myosin, whereas C57B/6 mice do not. To define the immunogenicity and pathogenicity of cardiac myosin in BALB/c mice, we immunized mice with different forms of cardiac myosin. These studies demonstrate the discordance of immunogenicity and pathogenicity of myosin heavy chains. The cardiac alpha-myosin heavy chains of BALB/c and C57B/6 mice differ by two residues that are near the junction of the head and rod in the S2 fragment of myosin. Myosin preparations from both strains are immunogenic in susceptible BALB/c as well as in nonsusceptible C57B/6 mice; however, BALB/c myosin induces a greater incidence of disease. To further delineate epitopes of myosin heavy chain responsible for immunogenicity and disease, mice were immunized with fragments of genetically engineered rat alpha cardiac myosin. Epitopes in the region of difference between BALB/c and C57B/6 (residues 735-1032) induce disease in both susceptible and nonsusceptible mice. The data presented here demonstrate that pathogenic epitopes of both mouse and rat myosin residue in the polymorphic region of the S2 subunit. In addition, these studies suggest that polymorphisms in the autoantigen may be part of the genetic basis for autoimmune myocarditis.