Development of a Glycocluster Adjuvant Mimicking Natural Yeast beta-(1,2)-Structures

Allergy is characterized by T helper (Th) 2-type immune response after encounter with an allergen leading to subsequent immunoglobulin (Ig) E-mediated hypersensitivity reaction and further allergic inflammation. Allergen-specific immunotherapy (SIT) balances the Th2-biased immunity towards Th1 and T regulatory responses. Adjuvants are used in allergen preparations to intensify and modify SIT. β-(1,2)-oligomannoside constituents present in Candida albicans (C. albicans) cell wall possess Th1-type immunostimulatory properties. The aim of this thesis was to develop a β-(1,2)-linked carbohydrate compound with known structure and anti-allergic properties to be applied as an adjuvant in SIT. First the immunostimulatory properties of various fungal extracts were studied. C. albicans appeared to be the most promising Th1-inducing extract, which led to the synthesis of various mono- or divalent oligomannosides designed on the basis of C. albicans. These carbohydrates did not induce strong cytokine production in human peripheral blood mononuclear cell (PBMC) cultures. In contrast to earlier reports using native oligosaccharides from C. albicans, synthetic -(1,2)-linked mannotetraose did not induce any tumor necrosis factor production in murine macrophages. Next, similarities with synthesized divalent mannosides and the antigenic epitopes of β-(1,2)-linked C. albicans mannan were investigated. Two divalent compounds inhibited specific IgG antibodies binding to below 3 kDa hydrolyzed mannan down to the level of 30–50% showing similar antigenicity to C. albicans. Immunomodulatory properties of synthesized carbohydrate assemblies ranging from mono- to pentavalent were evaluated. A trivalent acetylated dimannose (TADM) induced interleukin-10 (IL-10) and interferon-γ responses. TADM also suppressed birch pollen induced IL-4 and IL-5 responses in allergen (Bet v) stimulated PBMCs of birch pollen allergic subjects. This suppression was stronger with TADM than with other used adjuvants, immunostimulatory oligonucleotides and monophosphoryl lipid A. In a murine model of asthma, the allergen induced inflammatory responses could also be suppressed by TADM on cytokine and antibody levels.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52%) and cirrhosis in 11/52 (21%) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

Role of immunoglobulin E and mast cells in murine models of asthma

Immunoglobulin E (IgE) and mast cells are believed to play important roles in allergic inflammation. However, their contributions to the pathogenesis of human asthma have not been clearly established. Significant progress has been made recently in our understanding of airway inflammation and airway hyperresponsiveness through studies of murine models of asthma and genetically engineered mice. Some of the studies have provided significant insights into the role of IgE and mast cells in the allergic airway response. In these models mice are immunized systemically with soluble protein antigens and then receive an antigen challenge through the airways. Bronchoalveolar lavage fluid from mice with allergic airway inflammation contains significant amounts of IgE. The IgE can capture the antigen presented to the airways and the immune complexes so formed can augment allergic airway response in a high-affinity IgE receptor (FcepsilonRI)-dependent manner. Previously, there were conflicting reports regarding the role of mast cells in murine models of asthma, based on studies of mast cell-deficient mice. More recent studies have suggested that the extent to which mast cells contribute to murine models of asthma depends on the experimental conditions employed to generate the airway response. This conclusion was further supported by studies using FcepsilonRI-deficient mice. Therefore, IgE-dependent activation of mast cells plays an important role in the development of allergic airway inflammation and airway hyperresponsiveness in mice under specific conditions. The murine models used should be of value for testing inhibitors of IgE or mast cells for the development of therapeutic agents for human asthma.

Modulation of B lymphocyte function by an aqueous fraction of the ethanol extract of Cissampelos sympodialis Eichl (Menispermaceae)

Cissampelos sympodialis Eichl species are used in folk medicine for the treatment of asthma, arthritis and rheumatism. In the present study, we investigated the immunomodulatory effect of an aqueous fraction of a 70% (v/v) ethanol extract of C. sympodialis leaves on B lymphocyte function. The hydroalcoholic extract inhibited the in vitro proliferative response of resting B cells induced by LPS (IC50 = 17.2 µg/ml), anti-delta-dextran (IC50 = 13.9 µg/ml) and anti-IgM (IC50 = 24.3 µg/ml) but did not affect the anti-MHC class II antibody-stimulated proliferative response of B cell blasts obtained by stimulation with IL-4 and anti-IgM. Incubation with the hydroalcoholic extract used at 50 µg/ml induced a 700% increase in intracellular cAMP levels. IgM secretion by resting B cells (obtained from normal mice) and polyclonally activated B cells (obtained from Trypanosoma cruzi-infected animals) was inhibited by the hydroalcoholic extract. The latter were more sensitive to the hydroalcoholic extract since 6.5 µg/ml induced a 20% inhibition in the response of cells from normal mice while it inhibited the response of B cells from infected animals by 75%. The present data indicate that the alcoholic extract of C. sympodialis inhibited B cell function through an increase in intracellular cAMP levels. The finding that the hydroalcoholic extract inhibited immunoglobulin secretion suggests a therapeutic use for the extract from C. sympodialis in conditions associated with unregulated B cell function and enhanced immunoglobulin secretion. Finally, the inhibitory effect of the hydroalcoholic extract on B cells may indicate an anti-inflammatory effect of this extract.

Comparison of different monoclonal antibodies against immunosuppressive proteins of Ascaris suum

The extract of Ascaris suum suppresses the humoral and cellular immune responses to unrelated antigens in the mouse. In order to further characterize the suppressive components of A. suum, we produced specific monoclonal antibodies which can provide an important tool for the identification of these proteins. The A. suum immunosuppressive fractions isolated by gel filtration from an extract of adult worms were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells and the cloned hybrid cells obtained were screened to determine the specificity of secreted antibodies. Three monoclonal antibodies named MAIP-1, MAIP-2 and MAIP-3 were selected and were shown to react with different epitopes of high molecular weight proteins from the A. suum extract. All antibody molecules have kappa-type light chains but differ in heavy chain isotype. MAIP-1 is a mouse IgM, MAIP-2 is an IgA immunoglobulin and MAIP-3 is an IgG1 immunoglobulin and they recognize the antigen with affinity constants of 1.3 x 10(10) M-1, 7.1 x 10(9) M-1 and 3.8 x 10(7) M-1, respectively. The proteins recognized by these monoclonal antibodies (PAS-1, PAS-2 and PAS-3) were purified from the crude extract by affinity chromatography and injected with ovalbumin in BALB/c mice in order to determine their suppressive activity on heterologous antibody production. It was demonstrated that these three proteins are able to significantly suppress anti-ovalbumin antibody secretion, with PAS-1 being more efficient than the others.

Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody

Pituitary adenomas sometimes show rapid growth and recurrence, and about one third invade the structures surrounding the sella turcica. In an attempt to determine aggressive behavior at an early stage, we used the MIB-1 antibody to identify the Ki-67 antigen. The present study was designed to evaluate pituitary adenomatous tissue in terms of secretion and proliferation and to correlate the Ki-67 index with hormone phenotype and invasive behavior. Material from 159 patients submitted to one or more resections of pituitary adenomas was evaluated. Forty-two non-secretory adenomas and 43 adenomas immunoreactive for growth hormone, 19 for prolactin, 18 for growth hormone and prolactin, 16 for adrenocorticotropic hormone (ACTH), and 21 cases of plurihormonal/gonadotropin adenomas were detected by immunohistochemistry. The MIB-1 antibody was positive in 139 samples and the Ki-67 index ranged from 0.16 to 15.48% (mean = 1.22 ± 2.09%), with no significant difference between genders, age groups, or secretory and non-secretory status. The Ki-67 index was higher in ACTH-secreting adenomas. Invasive pituitary adenomas had a significantly higher Ki-67 index (2.01 ± 3.15%) than macroadenomas with or without supra-sellar extension (1.12 ± 1.87%; P = 0.02). The index was not significantly different in the subgroup of adenomas with invasion of the cavernous sinus compared to groups with other types of invasion. We conclude that tumoral proliferative activity evaluated by the detection of the Ki-67 antigen is significantly higher in invasive than noninvasive adenomas, information which can be useful in therapeutic postoperative management since index cut-off values associated with aggressive behavior can be established.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (≥1.0 μg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (≥0.15 μg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (≥1.0 μg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

Evaluation of the immune humoral response of Brazilian patients with Rubinstein-Taybi syndrome

Rubinstein-Taybi syndrome (RTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, mental and growth deficiency, and recurrent respiratory infections. RTS has been associated with CREBBP gene mutations, but EP300 gene mutations have recently been reported in 6 individuals. In the present study, the humoral immune response in 16 RTS patients with recurrent respiratory infections of possible bacterial etiology was evaluated. No significant differences between patients and 16 healthy controls were detected to explain the high susceptibility to respiratory infections: normal or elevated serum immunoglobulin levels, normal salivary IgA levels, and a good antibody response to both polysaccharide and protein antigens were observed. However, most patients presented high serum IgM levels, a high number of total B cell and B subsets, and also high percentiles of apoptosis, suggesting that they could present B dysregulation. The CREBBP/p300 family gene is extremely important for B-cell regulation, and RTS may represent an interesting human model for studying the molecular mechanisms involved in B-cell development.

The inhibitory effect of photodynamic therapy and of an anti-VCAM-1 monoclonal antibody on the in vivo growth of C6 glioma xenografts

We investigated the effect of photodynamic therapy (PDT) and of an anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody on the in vivo growth of C6 glioma. Seven days after inoculation with C6 cells, adult male Wistar rats weighing 280-300 g with MRI-confirmed glioma were randomly assigned to 4 groups (N = 15 per group): PDT + VCAM-1 antibody group; PDT group; VCAM-1 antibody group; control group. Eight days after inoculation, hematoporphyrin monomethyl ether (HMME) was administered as a photosensitizer and PDT was performed at 630 nm (illumination intensity: 360 J/cm²) for 10 min. VCAM-1 antibody (50 µg/mL) was then administered (0.5 mL) through the tail vein every other day from day 8 to day 16. At day 21, 5 rats in each group were sacrificed and cancers were harvested for immunohistochemistry and Western blot assay for the detection of VCAM-1, and TUNEL assay was used to detect apoptosis. Survival and tumor volume were recorded in the remaining 10 rats in each group. In the PDT group, tumor growth was significantly suppressed (67.2%) and survival prolonged (89.3%), accompanied by an increase in apoptosis (369.5%), when compared to control. Furthermore, these changes were more pronounced in the PDT + VCAM-1 antibody group. After PDT, VCAM-1 expression was markedly increased (121.8%) and after VCAM-1 monoclonal antibody treatment, VCAM-1 expression was significantly reduced (58.2%). PDT in combination with VCAM-1 antibody can significantly inhibit the growth of C6 glioma and prolong survival. This approach may represent a promising strategy in the treatment of glioma.

Do whole-body vibration exercise and resistance exercise modify concentrations of salivary cortisol and immunoglobulin A?

A single bout of resistance exercise (RE) induces hormonal and immune responses, playing an important role in a long-term adaptive process. Whole-body vibration (WBV) has also been shown to affect hormonal responses. Evidence suggests that combining WBV with RE may amplify hormonal and immune responses due to the increased neuromuscular load. Therefore, the aim of this study was to evaluate salivary cortisol (Scortisol) and salivary IgA (SIgA) concentrations following a RE session combined or not with WBV. Nine university students (22.9 ± 5.1 years, 175.8 ± 5.2 cm, and 69.2 ± 7.3 kg) performed five sets of squat exercise (70% one-repetition-maximum) combined (R+V30) or not (R) with WBV at 30 Hz. Saliva samples were obtained before and after exercise. Subjects also rated their effort according to the Borg CR-10 scale (RPE). Data were analyzed by a mixed model. RPE was higher after R+V30 (8.3 ± 0.7) compared to R (6.2 ± 0.7). However, Scortisol (pre: 10.6 ± 7.6 and 11.7 ± 7.6, post: 8.3 ± 6.3 and 10.2 ± 7.2 ng/mL for R and R+V30, respectively) and SIgA concentrations (pre: 98.3 ± 22.6 and 116.1 ± 51.2, post: 116.6 ± 64.7 and 143.6 ± 80.5 µg/mL for R and R+V30, respectively) were unaffected. No significant correlations were observed between Scortisol and RPE (r = 0.45, P = 0.22; r = 0.30, P = 0.42, for R and R+V30, respectively). On the basis of these data, neither protocol modified salivary cortisol or IgA, although RPE was higher after R+V30 than R.

Short-term and long-term antibody response by mice after immunization against Neisseria meningitidis B or diphtheria toxoid

Serogroup B Neisseria meningitidis (MenB) is a major cause of invasive disease in early childhood worldwide. The only MenB vaccine available in Brazil was produced in Cuba and has shown unsatisfactory efficacy when used to immunize millions of children in Brazil. In the present study, we compared the specific functional antibody responses evoked by the Cuban MenB vaccine with a standard vaccine against diphtheria (DTP: diphtheria, tetanus, pertussis) after primary immunization and boosting of mice. The peak of bactericidal and opsonic antibody titers to MenB and of neutralizing antibodies to diphtheria toxoid (DT) was reached after triple immunization with the MenB vaccine or DTP vaccine, respectively. However, 4 months after immunization, protective DT antibody levels were present in all DTP-vaccinated mice but in only 20% of the mice immunized against MenB. After 6 months of primary immunization, about 70% of animals still had protective neutralizing DT antibodies, but none had significant bactericidal antibodies to MenB. The booster doses of DTP or MenB vaccines produced a significant antibody recall response, suggesting that both vaccines were able to generate and maintain memory B cells during the period studied (6 months post-triple immunization). Therefore, due to the short duration of serological memory induced by the MenB vaccine (VA-MENGOC-BC® vaccine), its use should be restricted to outbreaks of meningococcal disease.

Salivary testosterone and immunoglobulin A were increased by resistance training in adults with Down syndrome

This study was designed to assess the influence of resistance training on salivary immunoglobulin A (IgA) levels and hormone profile in sedentary adults with Down syndrome (DS). A total of 40 male adults with DS were recruited for the trial through different community support groups for people with intellectual disabilities. All participants had medical approval for participation in physical activity. Twenty-four adults were randomly assigned to perform resistance training in a circuit with six stations, 3 days per week for 12 weeks. Training intensity was based on functioning in the eight-repetition maximum (8RM) test for each exercise. The control group included 16 age-, gender-, and BMI-matched adults with DS. Salivary IgA, testosterone, and cortisol levels were measured by ELISA. Work task performance was assessed using the repetitive weighted-box-stacking test. Resistance training significantly increased salivary IgA concentration (P=0.0120; d=0.94) and testosterone levels (P=0.0088; d=1.57) in the exercising group. Furthermore, it also improved work task performance. No changes were seen in the controls who had not exercised. In conclusion, a short-term resistance training protocol improved mucosal immunity response as well as salivary testosterone levels in sedentary adults with DS.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Accelerating food safety and quality control: The use of antibody engineering techniques in the detec-tion of low-molecular weight food contaminants

The increased awareness and evolved consumer habits have set more demanding standards for the quality and safety control of food products. The production of foodstuffs which fulfill these standards can be hampered by different low-molecular weight contaminants. Such compounds can consist of, for example residues of antibiotics in animal use or mycotoxins. The extremely small size of the compounds has hindered the development of analytical methods suitable for routine use, and the methods currently in use require expensive instrumentation and qualified personnel to operate them. There is a need for new, cost-efficient and simple assay concepts which can be used for field testing and are capable of processing large sample quantities rapidly. Immunoassays have been considered as the golden standard for such rapid on-site screening methods. The introduction of directed antibody engineering and in vitro display technologies has facilitated the development of novel antibody based methods for the detection of low-molecular weight food contaminants. The primary aim of this study was to generate and engineer antibodies against low-molecular weight compounds found in various foodstuffs. The three antigen groups selected as targets of antibody development cause food safety and quality defects in wide range of products: 1) fluoroquinolones: a family of synthetic broad-spectrum antibacterial drugs used to treat wide range of human and animal infections, 2) deoxynivalenol: type B trichothecene mycotoxin, a widely recognized problem for crops and animal feeds globally, and 3) skatole, or 3-methyindole is one of the two compounds responsible for boar taint, found in the meat of monogastric animals. This study describes the generation and engineering of antibodies with versatile binding properties against low-molecular weight food contaminants, and the consecutive development of immunoassays for the detection of the respective compounds.