César Milstein / César Milstein

Resumen El presente comentario, escrito en virtud del decreto del gobierno nacional que designó a 2021 como año de homenaje al Dr. César Milstein, es una visión personal sobre el significado del desarrollo de los anticuerpos monoclonales, del aporte que brindó y brinda a la ciencia y tecnología actual y del contexto del correspondiente Premio Nobel en el ámbito de la Inmunología y las Ciencias Médicas. El objetivo de este artículo es transmitir, a modo de homenaje, algunas de mis experiencias en la especialidad y la relación profesional y personal con el creador de la producción de anticuerpos monoclonales por fusión de células somáticas, tecnología que continúa realizando grandes aportes al conocimiento.

Abstract The present commentary, which was written by virtue of the national government decree that designated 2021 as the year of the tribute to Dr. César Milstein, is a personal vision of the meaning of the development of monoclonal antibodies, of the contribution that he has always made to the current science and technology, and of the context of the corresponding Nobel Prize within the sphere of Immunology and the Medical Sciences. The aim of this article is to share, as a tribute, some of my experiences in the specialty and my professional and personal relationship with the creator of the immense breakthrough still being delivered by his development.

Resumo O presente comentário, escrito em virtude do decreto do governo nacional que nomeou o ano 2021 como ano de homenagem ao Dr. César Milstein, é uma visão pessoal sobre o significado do desenvolvimento dos anticorpos monoclonais, da contribuição que ofereceu e oferece à ciência e tecnologia atual e do contexto do correspondente Prêmio Nobel no âmbito da Imunologia e das Ciências Médicas. O objetivo deste artigo é transmitir, à maneira de homenagem, algumas das minhas experiências na especialidade e na relação profissional e pessoal com o criador do imenso avanço que continua nos oferecendo seu descobrimento.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

Avances en las inmunoterapias para alergias alimentarias / New insights in immunotherapies for food allergies / Novas perspectivas em imunoterapias para alergias alimentares

Las enfermedades alérgicas son las inmunopatologías que con mayor prevalencia se presentan en el mundo. Pueden o no estar mediadas por anticuerpos IgE, sin embargo estas últimas son las que más intensamente se han estudiado por el riesgo que presentan para la vida del paciente. Si bien el único tratamiento que logra revertir estos mecanismos es la no exposición al alergeno, esto no siempre es posible. Por esta razón, y a partir del mayor conocimiento alcanzado del sistema inmune de mucosas junto al desarrollo de modelos animales de alergia, existe un marcado interés en la especialidad para el desarrollo de inmunoterapias que controlen y reviertan el estado de alergia. A partir de los ensayos pre-clínicos en animales y la aplicación de protocolos terapéuticos en ensayos clínicos, se han desarrollado terapias mucosales que logran inducir mecanismos de tolerancia específicos del alergeno, los cuales son capaces de revertir la sensibilización alérgica. Dado que el principal escollo siguen siendo las reacciones adversas inducidas durante el tratamiento, se requiere profundizar los estudios para desarrollar protocolos terapéuticos más seguros. En este punto la medicina traslacional encuentra un campo próspero para fortalecer las interacciones entre la ciencia básica, la aplicada y la clínica.

Allergic diseases are the most prevalent immunopathologies worldwide. Although different mechanisms -IgE-independent or IgE-dependent- can be involved in the immunopathogenesis, the latter are the most studied reactions since they can be life-threatening. Nowadays, allergen avoidance is the unique effective treatment for allergic patients. However, this is rather difficult to implement. For this reason, and based on the new insights into the mucosal immune system and the development of animal models of allergy, there is an increasing interest in developing novel therapies to control or reverse allergic disorders. Pre-clinical studies and clinical trials have been successful to prove that immunotherapies may accomplish mucosal mechanisms of allergen-specific tolerance, which are able to revoke the allergic sensitization. Since the main obstacle in these therapies still has adverse reactions induced during treatment, further studies are required to explore safe and effective therapeutic protocols. At this point, translational medicine is a flourishing field in the areas of basic science, applied science, and clinical research.

As doenças alérgicas são as imunopatologias mais prevalentes em todo o mundo. Embora possam estar mediadas ou não por anticorpos IgE, estas últimas são as reacções mais intensamente estudadas, devido ao risco que apresentam para a vida do paciente. Ainda que o único tratamento eficaz para reverter este mecanismos seja a não exposição dos pacientes ao alergeno, isto nem sempre é possível. Por este motivo, e com base nas novas perspectivas sobre o sistema imune de mucosas, junto com o desenvolvimento de modelos e para o animais de alergia, existe um interesse crescente na especialidade para o desenvolvimento de imunoterapias que controlem e revertam o estado de alergia. A partir de estudos pré-clínicos em animais e a aplicação de protocolos terapêuticos em ensaios clínico, foram desenvolvidas terapias mucosas que conseguem induzir mecanismos de tolerância específicos do alergeno, que são capazes de reverter a sensibilização alérgica. Devido a que o principal obstáculo nestas terapias continuam sendo as reações adversas induzidas durante o tratamento, é necessário realizar mais estudos para desenvolver protocolos terapêuticos mais seguros. Neste ponto, medicina translacional é um campo próspero para fortalecer as interações entre a ciência básica, a aplicada e a clínica.

The Major Soybean Allergen Gly m Bd 28K Induces Hypersensitivity Reactions in Mice Sensitized to Cow's Milk Proteins.

Reactions to soy have been reported in a proportion of patients with IgE-mediated cow's milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics' tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP.

Brucella abortus Invasion of Osteocytes Modulates Connexin 43 and Integrin Expression and Induces Osteoclastogenesis via Receptor Activator of NF-κB Ligand and Tumor Necrosis Factor Alpha Secretion.

Osteoarticular brucellosis is the most common localization of human active disease. Osteocytes are the most abundant cells of bone. They secrete factors that regulate the differentiation of both osteoblasts and osteoclasts during bone remodeling. The aim of this study is to determine if Brucella abortus infection modifies osteocyte function. Our results indicate that B. abortus infection induced matrix metalloproteinase 2 (MMP-2), receptor activator for NF-κB ligand (RANKL), proinflammatory cytokines, and keratinocyte chemoattractant (KC) secretion by osteocytes. In addition, supernatants from B. abortus-infected osteocytes induced bone marrow-derived monocytes (BMM) to undergo osteoclastogenesis. Using neutralizing antibodies against tumor necrosis factor alpha (TNF-α) or osteoprotegerin (OPG), RANKL's decoy receptor, we determined that TNF-α and RANKL are involved in osteoclastogenesis induced by supernatants from B. abortus-infected osteocytes. Connexin 43 (Cx43) and the integrins E11/gp38, integrin-α, integrin-ß, and CD44 are involved in cell-cell interactions necessary for osteocyte survival. B. abortus infection inhibited the expression of Cx43 but did not modify the expression of integrins. Yet the expression of both Cx43 and integrins was inhibited by supernatants from B. abortus-infected macrophages. B. abortus infection was not capable of inducing osteocyte apoptosis. However, supernatants from B. abortus-infected macrophages induced osteocyte apoptosis in a dose-dependent manner. Taken together, our results indicate that B. abortus infection could alter osteocyte function, contributing to bone damage.

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions.

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-ß were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-ß-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-ß-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

Cross-reactivity between the soybean protein p34 and bovine caseins.

PURPOSE: Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity. METHODS: In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model. RESULTS: P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-γ remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34. CONCLUSIONS: Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34.

Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy.

Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow's milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.

Unlipidated outer membrane protein Omp16 (U-Omp16) from Brucella spp. as nasal adjuvant induces a Th1 immune response and modulates the Th2 allergic response to cow's milk proteins.

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow's Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.

Brucella abortus invasion of synoviocytes inhibits apoptosis and induces bone resorption through RANKL expression.

Arthritis is one of the most common complications of human active brucellosis, but its pathogenic mechanisms have not been completely elucidated. In this paper, we describe the role of synoviocytes in the pathogenesis of brucellar arthritis. Our results indicate that Brucella abortus infection inhibited synoviocyte apoptosis through the upregulation of antiapoptotic factors (cIAP-2, clusterin, livin, and P21/CIP/CDNK1A). In contrast, infection did not change the expression of proteins that have been involved in apoptosis induction such as Bad, Bax, cleaved procaspase 3, CytC, and TRAIL, among others; or their expression was reduced, as occurs in the case of P-p53(S15). In addition, B. abortus infection induced upregulation of adhesion molecules (CD54 and CD106), and the adhesion of monocytes and neutrophils to infected synoviocytes was significantly higher than to uninfected cells. Despite this increased adhesion, B. abortus-infected synoviocytes were able to inhibit apoptosis induced by supernatants from B. abortus-infected monocytes and neutrophils. Moreover, B. abortus infection increased soluble and membrane RANKL expression in synoviocytes that further induced monocytes to undergo osteoclastogenesis. The results presented here shed light on how the interactions of B. abortus with synovial fibroblasts may have an important role in the pathogenesis of brucellar arthritis.

In vivo evidence of cross-reactivity between cow's milk and soybean proteins in a mouse model of food allergy.

BACKGROUND: Cow's milk allergy (CMA) is an important problem worldwide and the development of an in vivo system to study new immunotherapeutic strategies is of interest. Intolerance to soybean formula has been described in CMA patients, but it is not fully understood. In this work, we used a food allergy model in BALB/c mice to study the cross-reactivity between cow's milk protein (CMP) and soy proteins (SP). METHODS: Mice were orally sensitized with cholera toxin and CMP, and then challenged with CMP or SP to induce allergy. Elicited symptoms, plasma histamine, humoral and cellular immune response were analyzed. Th1- and Th2-associated cytokines and transcription factors were assessed at mucosal sites and in splenocytes. Cutaneous tests were also performed. RESULTS: We found that the immediate symptoms elicited in CMP-sensitized mice orally challenged with SP were consistent with a plasma histamine increase. The serum levels of CMP-specific IgE and IgG1 antibodies were increased. These antibodies also recognized soy proteins. Splenocytes and mesenteric lymph node cells incubated with CMP or SP secreted IL-5 and IL-13. mRNA expression of Th2-associated genes (IL-5, IL-13, and GATA-3) was upregulated in mucosal samples. In addition, sensitized animals exhibited positive cutaneous tests after the injection of CMP or SP. CONCLUSIONS: We demonstrate that CMP-sensitized mice, without previous exposure to soy proteins, elicited hypersensitivity signs immediately after the oral administration of SP, suggesting that the immunochemical cross-reactivity might be clinically relevant. This model may provide an approach to further characterize cross-allergenicity phenomena and develop new immunotherapeutic treatments for allergic patients.

Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice.

The immunogenicity and protective efficacy of recombinant SurA (rSurA) and rDnaK from Brucella spp. were evaluated in BALB/c mice. Immunization with rSurA in adjuvant induced a vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. In addition, after in vitro stimulation with rSurA, spleen cells from rSurA-immunized mice produced interleukin-2 (IL-2), interferon (IFN)-gamma, IL-4 and IL-5. Immunization with rDnaK plus adjuvant induced a strong humoral response resulting in similar anti-rDnaK IgG titers than immunization with rDnaK alone. IgG2a titers predominated over IgG1 in mice injected with rDnaK alone or rDnaK plus adjuvant. Spleen cells from mice immunized with rDnaK plus adjuvant secreted IFN-gamma and IL-2 upon stimulation with rDnaK and induced a specific cytotoxic response. On the contrary, mice immunized with rDnaK alone did not exhibit a specific T helper or cytotoxic response in vitro. Mice given rSurA or rDnaK with adjuvant exhibited a significant degree of protection whereas immunization with rDnaK alone induced a low but still statistically significant level of protection against B. abortus infection. All studied vaccines were less protected than mice immunized with H38 or B. abortus strain 19 control vaccines. Altogether these results suggest that rSurA or rDnaK induce partial protection against B. abortus infection and could be useful candidates for the development of subunit vaccines against brucellosis.

In vitro presentation of gliadin-derived peptides by different cell lines.

BACKGROUND: Gliadin peptide presentation and T-cell activation are critical events in the pathogenesis of celiac disease. Several studies have been performed to identify the toxic gliadin peptides but the complexity of the antigenic fraction makes this analysis difficult. In this work, an in vitro model for the analysis of gliadin peptide presentation is studied. METHODS: The human cell lines U937 and THP-1 (monocytic), DUCAF and VAVY (immortalised B cells) and HT-29 and Caco-2 (intestinal epithelial cells) were incubated with biotin-labelled gliadin (bG). FITC-labelled streptavidin was used to detect biotinylated peptides at the cell surface by flow cytometry. RESULTS: All cell lines tested showed a fluorescence signal derived from bG, that was highest when cells were stimulated with IFN-gamma for 48 h. Time course experiments performed using THP-1 cells showed that after 4-h incubation, almost a maximal signal can be reached. THP-1 cells incubated at 4 degrees C or after paraformaldehyde fixation showed a substantial signal reduction, suggesting that metabolic activity was necessary for the detection of the maximal fluorescence signal at the cell surface. The presence of HLA class II-bound biotinylated peptides was observed in cell lysates of THP-1 cells incubated with bG. CONCLUSIONS: In all cell lines tested, a specific biotin-peptide-derived signal was observed. This was increased after IFN-gamma treatment and decreased after fixation or incubation at low temperature. The signal was higher in monocytic and B-cell lines than in the epithelial cell lines. The use of this procedure could be a useful tool to study the in vitro processing and presentation of naturally gliadin-derived peptides.

Desarrollo de un ELISA de alta detectabilidad y especificidad para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos / Development of a quantitative ELISA to determination of gladins in food aimed to coeliac patients

La enfermedad celíaca (EC) es una enfermedad gastrointestinal crónica de muy alta incidencia en nuestro país. Se estima que puede afectar 1 de cada 300 habitantes. En individuos susceptibles, la patología es provocada por la ingestión de mínimas cantidades de prolaminas de los cereales: trigo, triticale, cebada y centeno. Su único tratamiento es una estricta dieta libre de dichas proteínas. La Organización Mundial de la Salud (OMS) establece que un alimento puede ser considerado como apto para consumo por enfermos celíacos sólo si su contenido de gluten es inferior a 1 mg/100 g de producto seco. La detección precisa de estas proteínas requiere entonces de métodos de alta detectabilidad y especificidad para discriminar entre las proteínas nocivas y las de otros vegetales frecuentemente usados como reemplazo en la formulación de los alimentos para estos pacientes. En este trabajo, se muestra el desarrollo de un ELISA con anticuerpos policlonales, para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos. Se empleó un diseño de ELISA competitivo secuencial con un antisuero obtenido en conejos que detecta selectivamente las prolaminas tóxicas. Este inmunoensayo presenta un nivel de detección de 0,1 mg de gluten/100 g de producto y cumple con los niveles de detectabilidad aconsejados por la OMS. Mediante el ELISA descripto se han analizado una gran variedad de muestras comerciales, pudiendo cuantificar las prolaminas incluso en alimentos que sufrieron tratamientos térmicos durante su fabricación

Desarrollo de un ELISA de alta detectabilidad y especificidad para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos / Development of a quantitative ELISA to determination of gladins in food aimed to coeliac patients

La enfermedad celíaca (EC) es una enfermedad gastrointestinal crónica de muy alta incidencia en nuestro país. Se estima que puede afectar 1 de cada 300 habitantes. En individuos susceptibles, la patología es provocada por la ingestión de mínimas cantidades de prolaminas de los cereales: trigo, triticale, cebada y centeno. Su único tratamiento es una estricta dieta libre de dichas proteínas. La Organización Mundial de la Salud (OMS) establece que un alimento puede ser considerado como apto para consumo por enfermos celíacos sólo si su contenido de gluten es inferior a 1 mg/100 g de producto seco. La detección precisa de estas proteínas requiere entonces de métodos de alta detectabilidad y especificidad para discriminar entre las proteínas nocivas y las de otros vegetales frecuentemente usados como reemplazo en la formulación de los alimentos para estos pacientes. En este trabajo, se muestra el desarrollo de un ELISA con anticuerpos policlonales, para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos. Se empleó un diseño de ELISA competitivo secuencial con un antisuero obtenido en conejos que detecta selectivamente las prolaminas tóxicas. Este inmunoensayo presenta un nivel de detección de 0,1 mg de gluten/100 g de producto y cumple con los niveles de detectabilidad aconsejados por la OMS. Mediante el ELISA descripto se han analizado una gran variedad de muestras comerciales, pudiendo cuantificar las prolaminas incluso en alimentos que sufrieron tratamientos térmicos durante su fabricación (AU)