A chromatographic method for the production of a human immunoglobulin G solution for intravenous use

Immunoglobulin G (IgG) of excellent quality for intravenous use was obtained from the cryosupernatant of human plasma by a chromatographic method based on a mixture of ion-exchange, DEAE-Sepharose FF and arginine Sepharose 4B affinity chromatography and a final purification step by Sephacryl S-300 HR gel filtration. The yield of 10 experimental batches produced was 3.5 g IgG per liter of plasma. A solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1% Triton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates. Anticomplementary activity was 0.3 CH50/mg IgG and prekallikrein activator levels were less than 5 IU/ml. Stability at 37ºC for 30 days in the liquid state was satisfactory. IgG was stored in flasks (2.5 g/flask) at 4 to 8ºC. All the characteristics of the product were consistent with the requirements of the 1997 Pharmacopée Européenne.

High quality human immunoglobulin G purified from Cohn fractions by liquid chromatography

In order to obtain intravenous immunoglobulin G (iv IgG) of high quality from F-I+II+III or F-II+III pastes prepared by the Cohn method, we developed a chromatography process using ion exchange gels, Q-Sepharose FF and CM-Sepharose FF, and Sephacryl S-300 gel filtration. Viral inactivation was performed by incubating the preparation with pepsin at pH 4.0 at 35oC for 18 h. The characteristics of 28 batches produced by us were: yield 4.3 ± 0.2 g/l plasma, i.e., a recovery of 39.1 ± 1.8%; IgG subclasses distribution: IgG1 = 58.4%, IgG2 = 34.8%, IgG3 = 4.5% and IgG4 = 2.3%; IgG size distribution was 98.4% monomers, 1.2% dimers and 0.4% polymers and protein aggregates; anticomplement activity was less than 0.5 CH50/mg IgG, and prekallikrein activator activity (PKA) was less than 5 IU/ml. These characteristics satisfied the requirements of the European Pharmacopoea edition, and the regulations of the Brazilian Health Ministry (M.S. Portaria No. 2, 30/10/1998).

Association between EcoRI fragment-length polymorphism of the immunoglobulin lambda variable 8 (IGLV8) gene family with rheumatoid arthritis and systemic lupus erythematosus

The human immunoglobulin lambda variable 8 (IGLV8) subgroup is a gene family containing three members, one of them included in a monomorphic 3.7-kb EcoRI genomic fragment located at the major lambda variable locus on chromosome 22q11.1 (gene IGLV8a, EMBL accession No. Z73650) at 100% frequency in the normal urban population. The second is a polymorphic RFLP allele included in a 6.0-kb EcoRI fragment at 10% frequency, and the third is located in a monomorphic 8.0-kb EcoRI fragment at 100% frequency, the last being translocated to chromosome 8q11.2 and considered to be an orphan gene. Our Southern blot-EcoRI-RFLP studies in normal individuals and in patients with rheumatoid arthritis (RA) or with systemic lupus erythematosus (SLE), using a specific probe for the IGLV8 gene family (probe pVL8, EMBL accession No. X75424), have revealed the two monomorphic genomic fragments containing the IGLV8 genes, i.e., the 3.7-kb fragment from chromosome 22q11.1 and the 8.0-kb fragment from 8q11.2, both occurring at 100% frequency (103 normal individuals, 48 RA and 28 SLE patients analyzed), but absence of the 6.0-kb IGLV8 polymorphic RFLP allele in all RA or SLE patients. As expected, the frequency of the 6.0-kb allele among the normal individuals was 10%. These findings suggest an association between the absence of the 6.0-kb EcoRI fragment and rheumatoid arthritis and systemic lupus erythematosus.

Detection of immunoglobulin G in the lung and liver of hamsters with visceral leishmaniasis

Several organs are affected in visceral leishmaniasis, not only those rich in mononuclear phagocytes. Hypergammaglobulinemia occurs during visceral leishmaniasis; anti-Leishmania antibodies are not primarily important for protection but might be involved in the pathogenesis of tissue lesions. The glomerulonephritis occurring in visceral leishmaniasis has been attributed to immune complex deposition but in other organs the mechanism has not been studied. In the current study we demonstrated the presence of IgG in the lung and liver of hamsters with visceral leishmaniasis. Hamsters were injected intraperitoneally with 2 x 10(7) amastigotes of Leishmania (Leishmania) chagasi and the presence of IgG in the liver and lung was evaluated at 7, 15, 30, 45, 80 and 102 days postinfection (PI) by immunohistochemistry. The parasite burden in the spleen and liver increased progressively during infection. We observed a deposit of IgG from day 7 PI that increased progressively until it reached highest intensity around 30 and 45 days PI, declining at later times. The IgG deposits outlined the sinusoids. In the lung a deposit of IgG was observed in the capillary walls that was moderate at day 7 PI, but the intensity increased remarkably at day 30 PI and declined at later times of infection. No significant C3 deposits were observed in the lung or in the liver. We conclude that IgG may participate in the pathogenesis of the inflammatory process of the lung and liver occurring in experimental visceral leishmaniasis and we discuss an alternative mechanism other than immune complex deposition.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.

A model of chronic IgE-mediated food allergy in ovalbumin-sensitized mice

Food allergy is most frequently the result of IgE-mediated hypersensitivity reactions. Here, we describe a chronic model in which some of the intestinal and systemic consequences of continuous egg white solution ingestion by ovalbumin-sensitized eight-week-old BALB/c mice, 6 animals per group, of both sexes, were investigated. There was a 20% loss of body weight that began one week after antigen exposure and persisted throughout the experiment (3 weeks). The sensitization procedure induced the production of anti-ovalbumin IgG1 and IgE, which were enhanced by oral antigen exposure (129% for IgG1 and 164% for IgE, compared to sensitization values). Intestinal changes were determined by jejunum edema at 6 h (45% Evans blue extravasation) and by a significant eosinophil infiltration with a peak at 48 h. By day 21 of continuous antigen exposure, histological findings were mild, with mast cell hyperplasia (100%) and increased mucus production (483%). Altogether, our data clearly demonstrate that, although immune stimulation was persistently occurring in response to continuous oral antigen exposure, regulatory mechanisms were occurring in the intestinal mucosa, preventing overt pathology. The experimental model described here reproduces the clinical and pathological changes of mild chronic food allergy and may be useful for mechanistic studies of this common clinical condition.

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.

Humoral and cellular immune responses to Blomia tropicalis and concanavalin A-binding fractions in atopic patients

Blomia tropicalis, Dermatophagoides pteronyssinus and D. farinae are prevalent house dust mites. Concanavalin A-binding components derived from B. tropicalis (Bt-ConA extract) are highly immunogenic in allergic diseases. The aim of the present study was to evaluate the humoral and cellular immune responses to B. tropicalis in mite-sensitized patients. A total of 137 patients with allergic rhinitis with/without asthma and 109 non-atopic subjects were selected and analyzed by the skin prick test, and for total serum IgE and specific IgE levels to both Bt-total and Bt-ConA extracts, their proliferative response and cytokine (IFN-γ and IL-5) production by peripheral blood mononuclear cells (PBMC) stimulated with both extracts. Skin prick test showed that 70% of the patients were sensitized to Bt (Bt+) and similar levels of specific IgE to Bt-total and Bt-ConA extracts were demonstrable in Bt+ patients. Significant PBMC proliferation was observed in response to Bt-total extract in Bt+, but not in Bt- patients and non-atopic subjects (P < 0.001). Bt-ConA extract induced increased proliferative responses in all patient groups compared to medium alone (P < 0.05), but these responses were significantly decreased in the presence of the mannopyranoside ConA inhibitor (P < 0.05). Significant IFN-γ production was observed after Bt-ConA stimulation of Bt+ patients (P < 0.05), while Bt-total extract had no effect. IL-5 production was consistently detected in Bt+ patients after allergen-specific stimulation or with no stimulus, indicating that PBMC from allergic patients are prone to produce Th2 profile cytokines, spontaneously or inductively by allergen restimulation. These data showed that ConA-binding components isolated from B. tropicalis may contain relevant antigens that are involved in both humoral and cellular immune responses. However, without an additional purification procedure to eliminate the residual contamination with ConA, its use in immunotherapeutic procedures cannot be recommended.

IgE cross-reactivity between Lolium multiflorum and commercial grass pollen allergen extracts in Brazilian patients with pollinosis

Lolium multiflorum (Lm) grass pollen is the major cause of pollinosis in Southern Brazil. The objectives of this study were to investigate immunodominant components of Lm pollen allergens and the cross-reactivity of IgE with commercial grass pollen allergen extracts. Thirty-eight serum samples from patients with seasonal allergic rhinitis (SAR), 35 serum samples from patients with perennial allergic rhinitis (PAR) and 30 serum samples from non-atopic subjects were analyzed. Allergen sensitization was evaluated using skin prick test and serum IgE levels against Lm pollen extract were determined by ELISA. Inhibition ELISA and immunoblot were used to evaluate the cross-reactivity of IgE between allergens from Lm and commercial grass pollen extracts, including L. perenne (Lp), grass mix I (GI) and II (GII) extracts. IgE antibodies against Lm were detected in 100% of SAR patients and 8.6% of PAR patients. Inhibition ELISA demonstrated IgE cross-reactivity between homologous (Lm) and heterologous (Lp or GII) grass pollen extracts, but not for the GI extract. Fifteen IgE-binding Lm components were detected and immunoblot bands of 26, 28-30, and 32-35 kDa showed >90% recognition. Lm, Lp and GII extracts significantly inhibited IgE binding to the most immunodominant Lm components, particularly the 55 kDa band. The 26 kDa and 90-114 kDa bands presented the lowest amount of heterologous inhibition. We demonstrated that Lm extract contains both Lm-specific and cross-reactive IgE-binding components and therefore it is suitable for measuring quantitative IgE levels for diagnostic and therapeutic purposes in patients with pollinosis sensitized to Lm grass pollen rather than other phylogenetically related grass pollen extracts.

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.

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.

Increased immunoglobulin production in silver catfish (Rhamdia quelen) exposed to agrichemicals

Fish vaccination has been increasingly exploited as a tool to control pathogen infection. The production of immunoglobulin following vaccination might be affected by several factors such as management procedures, water temperature, and the presence of xenobiotics. In the present study, we aimed to investigate the kinetics of immunoglobulin production in silver catfish (Rhamdia quelen) inoculated with inactivated Aeromonas hydrophila and kept at two different water temperatures (17.4±0.4° or 21.3±0.3°C). The effect of a second antigen inoculation and exposure of fish to sublethal concentrations of the herbicides atrazine and glyphosate at 10% of the lethal concentration (LC50-96h) on specific serum antibodies were also investigated. Antibodies to A. hydrophila were detected as early as 7 days post-inoculation and increased steadily up to 35 days. The kinetics of antibody production were similar in fish kept at 17.4±0.4° and 21.3±0.3°C, and reinoculation of antigen at 21 days after priming failed to increase specific antibody levels. Intriguingly, we found that, in fish exposed to atrazine and glyphosate, the secretion of specific antibodies was higher than in non-exposed inoculated fish. These findings are important for the design of vaccines and vaccination strategies in Neotropical fish species. However, because atrazine and glyphosate are widespread contaminants of soil and water, their immune-stimulating effect could be harmful, in that fish living in herbicide-contaminated water might have increased concentrations of nonspecific antibodies that could mediate tissue injury.

Modeling of allergen proteins found in sea food products

Shellfish are a source of food allergens, and their consumption is the cause of severe allergic reactions in humans. Tropomyosins, a family of muscle proteins, have been identified as the major allergens in shellfish and mollusks species. Nevertheless, few experimentally determined three-dimensional structures are available in the Protein Data Base (PDB). In this study, 3D models of several homologous of tropomyosins present in marine shellfish and mollusk species (Chaf 1, Met e1, Hom a1, Per v1, and Pen a1) were constructed, validated, and their immunoglobulin E binding epitopes were identified using bioinformatics tools. All protein models for these allergens consisted of long alpha-helices. Chaf 1, Met e1, and Hom a1 had six conserved regions with sequence similarities to known epitopes, whereas Per v1 and Pen a1 contained only one. Lipophilic potentials of identified epitopes revealed a high propensity of hydrophobic amino acids in the immunoglobulin E binding site. This information could be useful to design tropomyosin-specific immunotherapy for sea food allergies.

Doença alérgica polínica: polens alergógenos e seus principais alérgenos

Pacientes com alergia a pólen de gramíneas, comumente denominada polinose, freqüentemente apresentam reatividade a alérgenos de pólen de inúmeras gramíneas devido às reatividades cruzadas entre anticorpos IgE dirigidos contra proteínas presentes nos polens de gramíneas. Nesse contexto, pólen de Italian ryegrass (Lolium multiflorum), ou azevém anual, gramínea da família Poaceae cultivada no Sul do Brasil, é considerado o principal agente sensibilizante em pacientes com polinose. Nessa região, o azevém é capaz de produzir grande quantidade de pólen. Adicionalmente, outras gramíneas da família Poaceae crescem naturalmente no Sul, entretanto, sem relevância clínica. Extratos de pólen derivados de gramíneas homólogas ou heterólogas são freqüentemente empregados no diagnóstico e tratamento da alergia sazonal a pólen, sendo que para esses fins não se encontra comercialmente disponível no Brasil extrato padronizado de pólen de L. multiflorum. Futuros estudos serão importantes para melhor caracterizar a reatividade cruzada entre alérgenos de pólen de L. multiflorum e alérgenos de outras gramíneas com o objetivo de aprimorar o diagnóstico e imunoterapia de pacientes com alergia a pólen causada por L. multiflorum.

Análise de citocinas pela RT-PCR em pacientes com rinite alérgica

Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.