Resumen La infección por SARS-CoV-2 es hoy el principal problema de salud pública en el mundo. No es claro el papel de las citoquinas en la fisiopatología del COVID-19,que en algunos individuos presenta una progresión rápida, severa y mortal asociada con proinflamación sistémicos relacionada con coagulopatías y fallas multiorgánicas. En este estudio, evaluamos los niveles séricos de citoquinas y su correlación con IgM, IgG e IgA, en 24 muestras de individuos positivos y 8 muestras de individuos negativos, para SARS-CoV-2. Hallamos concentraciones significativamente menores de IFN-g, TNF, IL-2 e IL-4 y un aumento significativo de IL-6 en el grupo de infectados hospitalizados respecto a los no infectados, así como una tendencia significativa al aumento, para IgG e IgA en el mismo grupo de individuos, respecto a infectados asintomáticos. Nuestros datos soportan el papel de la IL-6 en la severidad de la enfermedad destacando su potencial papel como biomarcador en la prognosis de esta patología. También, soportan la hipótesis sobre la función de los anticuerpos en el control efectivo del patógeno; se observa una respuesta inmune humoral más débil, frente a la proteína de la nucleocápside viral, en individuos con un mejor curso de la enfermedad.

Abstract The emergency caused by the infection in humans of SARS-COV-2 and the clinical syndrome resulting from the infection (COVID-19) is a major public health crisis with global repercussions. Currently, the role of different cytokine profiles in the infection pathophysiology and its outcome remains unclear despite the coordina ted efforts of the scientific community. COVID-19 shows a rapid progression where the disease severity and mortality are linked to systemic pro-inflammatory pro cesses associated to a dysregulation in the cytokine production balance, resulting in blood clothing disorders and multiorgan failure. Here we evaluate the serum concentration for a cytokine panel as well as the antibody titers of IgM, IgG and IgA from 24 individuals who tested positive for SARS-CoV-2 by RT-PCR (divided into three separate groups according to disease severity) and eight RT-PCR-negative controls. Significantly lower concentrations of IFN-g, TNF, IL-2 and IL-4, and a higher production of IL-6 were observed in hospitalized COVID-19 patients when compared to SARS-CoV-2-negative individuals. Furthermore, a significant and sustained increase in the levels of IgG and IgA was found for the group of hospitalized patients compared to asymptomatic SARS-CoV-2-positive individuals. Our data support previous findings on the role of cytokines like IL-6 in the severity of the disease and highlight their potential use as biomarkers for the prognosis of COVID-19. Finally, we provide evidence supporting the potential function of the antibody response in the effective control of the virus, showing that a somehow weaker humoral immune response can be associated to milder forms of COVID-19.

Resumen El uso terapéutico de insulina exógena de origen animal para el tratamiento de la diabetes mellitus se ha asociado con la inducción de anticuerpos antiinsulínicos, los cuales alterarían la seguridad y la eficacia de esta formulación. Con la introducción de la tecnología del ADN recombinante y la producción de insulinas con secuencias similares a la humana, se redujo, pero no se eliminó, la aparición de dichos anticuerpos. En el marco del análisis de la inmunogenicidad inducida por las proteínas terapéuticas y con el objetivo de evaluar el perfil de inmunidad humoral de tres formulaciones comerciales de insulina exógena humana (insulina regular, insulina Neutral protamine Hagedorn [NPH], e insulina glargina), se estudió la presencia de anticuerpos antiinsulínicos en 29 muestras de suero de pacientes con diabetes tipo 2, y se caracterizó su reactividad cruzada, su perfil según la subclase de IgG y su capacidad para formar complejos inmunitarios. Los pacientes se clasificaron en tres grupos según su tratamiento: a) insulina regular y NPH (n=10), b) insulina NPH (n=9) y c) glargina (n=10). La detección, caracterización y evaluación de las subclases de IgG se hizo mediante ELISA indirecto, y la detección de complejos inmunitarios constituidos por anticuerpos antiinsulínicos e insulina exógena, mediante ELISA de captura. Los resultados permitieron evidenciar que: i) cada formulación de insulina es reconocida de manera diferencial por los anticuerpos antiinsulínicos, lo cual sugiere un mayor potencial inmunogénico de la insulina NPH y uno menor de la insulina glargina; ii) el perfil según la subclase de IgG de los anticuerpos antiinsulínicos en humanos es diferencial para cada formulación, siendo predominante la subclase IgG3 para la insulina NPH en pacientes en tratamiento con insulina regular y NPH; iii) la presencia de anticuerpos antiinsulínicos no se asoció con alteraciones de los parámetros metabólicos analizados, y iv) los complejos inmunitarios constituidos por anticuerpos antiinsulínicos e insulina exógena no se detectaron con la metodología utilizada en los sueros de los pacientes con diabetes tipo 2 estudiados. Los resultados obtenidos permiten concluir que existen diferencias entre el perfil inmunogénico de las insulinas evaluadas, lo cual es importante en el análisis de la inmunogenicidad de dichas formulaciones en cuanto a su seguridad y eficacia.

Abstract The therapeutic use of exogenous insulin of animal origin for the treatment of diabetes mellitus has been associated with the development of insulin antibodies, which raises concerns regarding the safety and efficacy of this formulation. With the advent of recombinant DNA technology and the generation of insulin with human-like sequences, the production of insulin antibodies has been reduced but not eliminated. As part of the analysis of immunogenicity induced by therapeutic proteins and to evaluate the immunogenic profile of three commercial formulations of human exogenous insulin (regular, neutral protamine Hagedorn (NPH), and glargine) we evaluated the presence of insulin antibodies in 29 serum samples from volunteers with type 2 diabetes, and we characterized their cross-reactivity, IgG subclass profile, and ability to form immune complexes. To this end, volunteers were classified into three groups according to their current insulin therapy: a) Regular and NPH insulin (n=10); b) NPH (n=9), and c) glargine (n=10). For the detection, characterization, and evaluation of the IgG subclasses we used an indirect ELISA test, and for the detection of immune complexes formed by insulin antibodies and exogenous insulin, capture ELISA. Our results evidenced that: i) Each insulin formulation was recognized differentially by the insulin antibodies suggesting a greater immunogenic potential for NPH insulin and a lower one for glargine insulin; ii) the IgG subclasses profile of insulin antibodies in humans was different for each formulation; the IgG3 subclass for NPH insulin was predominant in volunteers treated with regular and NPH insulin; iii) the presence of insulin antibodies was not associated with alterations in the metabolic parameters analyzed, and iv) immune complexes composed of exogenous insulin antibodies were not detected in the sera of diabetic volunteers. The results obtained in this study allowed us to conclude that there are differences between the immunogenic profile of the insulin formulations tested, which has significant value in the analysis of their immunogenicity with regard to their safety and efficacy.

RESUMO O resultado clínico da infecção por espécies de Leishmania do subgênero Viannia no modelo de hamster (Mesocricetus auratus) tem se mostrado diferente, dependendo do protocolo experimental. O peso corporal tem sido um importante determinante da evolução clínica da infecção em hamsters com leishmaniose visceral, mas sua importância como parâmetro clínico em hamsters com leishmaniose cutânea não é conhecido. Neste estudo, a evolução clínica da infecção com L. (V) panamensis foi avaliada em jovens e adultos hamsters machos durante 11 semanas, comparando os parâmetros clínicos tais como a atitude, a temperatura, a frequência respiratória, a aparência das fezes, e o peso corporal entre infectado e grupos não infectados. Os resultados mostraram que o peso corporal diminuiu em hamsters adultos após infecção por L. (V) panamensis. Esta observação suporta a utilização do peso corporal, como um parâmetro adicional para definir a administração ou o tratamento de leishmaniose cutânea em hamsters adultos infectados usados como modelo animal experimental para a leishmaniose.

SUMMARY The clinical outcome of infection with Leishmania species of the subgenus Viannia in hamster model (Mesocricetus auratus) has shown to be different depending on experimental protocol. Body weight has been a relevant determinant of the clinical outcome of the infection in hamsters with visceral leishmaniasis but its importance as a clinical parameter in hamsters with cutaneous leishmaniasis is not known. In this study, the clinical evolution of infection with L. (V) panamensis was evaluated in juvenile and adult male hamsters during 11 weeks by comparing clinical parameters such as attitude, temperature, respiratory rate, appearance of the stool, and body weight between infected and non-infected groups. Results showed that body weight decreased in adult hamsters after infection by L. (V) panamensis; this observation supports the use of body weight as an additional parameter to define the management or treatment of cutaneous leishmaniasis in infected adult hamsters used as an animal experimental model for leishmaniasis.