Manejo clínico y diagnóstico para pacientes con enfermedades de inmunodeficiencia primaria por déficit de producción de anticuerpos / Clinical management and diagnosis for patients with primary immunodeficiency diseases due to deficiency of antibody production

1. INTRODUCCIÓNLas inmunodeficiencias primarias son un grupo de más de 400 enfermedades, en las cuales el sistema inmune pierde sus funciones de reconocimiento de patógenos o funciona de forma inapropiada. Algunas de ellas son relativamente comunes; mientras otras son raras. Estas enfermedades son en ocasiones de por vida, debilitantes y costosas1,2.Sin embargo, muchos progresos se han hecho desde su des-cripción original en el año de 1952. Se han dado grandes pasos en cuanto a su entendimiento de las Inmunodeficiencias Pri-marias a nivel genético, de sus características, y tratamiento. Algunos tipos afectan un único tipo de célula; otros afectan más de un componente del sistema inmune2,3.Tomando en cuenta que la aproximación es entre 1-2% de la población, a nivel país se puede decir que un aproximado entre 170 000 a 340 000 pacientes en el país no cuentan con un diagnóstico y muchos mueren por falta de este. El número de afiliados al Instituto Ecuatoriano de Seguridad Social hasta julio de 2021 es de 3 672,611 por lo que se considera que un estimado de 36 726 a 73 452 pacientes podrían presentar este tipo de enfermedades y requerir de atención por infecciones a repetición, enfermedad autoinmune y enfermedades linfopro-liferativas, además de que sin un tratamiento específico po-drían fallecer debido a infecciones graves o tener discapacidad permanente, lo que implica mayor carga para el sistema de Seguridad Social en subsidios y menores ingresos. Ecuador, cuenta con 86 pacientes diagnosticados, según la base de datos de la Sociedad Latino-Americana de Inmunodeficiencias4.Algunas terapias, como la de reemplazo para inmunoglobu-linas, a la que es tributaria más del 60% de estas patologías permite que la esperanza de vida y la morbilidad casi alcancen a aquellos que no presentan la enfermedad5­7.

1. INTRODUCTIONPrimary immunodeficiencies are a group of more than 400 diseases, in which the immune system loses its pathogen recog-nition functions or functions inappropriately. Some of them are relatively common, while others are rare. These diseases are sometimes lifelong, debilitating, and costly1,2. However, much progress has been made since its original description in 1952. Great strides have been made in understanding Primary Immunodeficiencies at the genetic level, their characteristics, and treatment. Some types affect only one type of cell; others affect more than one component of the immune system2,3. Considering that the approximation is between 1 to 2% of the population, at the country level we could say that approximately between 170 000 to 340 000 patients in the country do not have a diagnosis and many die due to lack of it. The number of social security affiliates until July 2021 is 3 672,611, so we could consider that approximately 36 726 to 73 452 patients could present this type of disease and require care for recurrent infections, autoimmune disease and lymphoproliferative diseases, in addition to the fact that without specific treatment they could die due to serious infections or have permanent disability, which implies a greater burden for the social security system in subsidies and lower income. Currently the country has 86 diagnosed patients, according to the database of the Latin American Society of Immunodeficiencies4. Many of the therapies, such as immunoglobulin replacement therapy, to which more than 60% of these pathologies are de-pendent, allow life expectancy and morbidity to almost reach those who do not have the disease 5­7.

Health assessment of gasoline and fuel oxygenate vapors: immunotoxicity evaluation.

Female Sprague Dawley rats were exposed via inhalation to vapor condensates of either gasoline or gasoline combined with various fuel oxygenates to assess potential immunotoxicity of evaporative emissions. Test articles included vapor condensates prepared from "baseline gasoline" (BGVC), or gasoline combined with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA). Target concentrations were 0, 2000, 10,000 or 20,000mg/mg(3) administered for 6h/day, 5days/week for 4weeks. The antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocyte (sRBC), was used to determine the effects of the gasoline vapor condensates on the humoral components of the immune system. Exposure to BGVC, G/MTBE, G/TAME, and G/TBA did not result in significant changes in the IgM AFC response to sRBC, when evaluated as either specific activity (AFC/10(6) spleen cells) or as total spleen activity (AFC/spleen). Exposure to G/EtOH and G/DIPE resulted in a dose-dependent decrease in the AFC response, reaching the level of statistical significance only at the high 20,000mg/m(3) level. Exposure to G/ETBE resulted in a statistically significant decrease in the AFC response at the middle (10,000mg/m(3)) and high (20,000mg/m(3)) exposure concentrations.

Evaluation of an intragastric challenge model for Shigella dysenteriae 1 in rhesus monkeys (Macaca mulatta) for the pre-clinical assessment of Shigella vaccine formulations.

Shigellosis is a worldwide disease, characterized by abdominal pain, fever, vomiting, and the passage of blood- and mucus-streaked stools. Rhesus monkeys and other primates are the only animals that are naturally susceptible to shigellosis. A suitable animal model is required for the pre-clinical evaluation of vaccines candidates. In this study, the minimal dose of Shigella dysenteriae1 1617 strain required to produce dysentery in four of five (80% attack rate) monkeys using an escalating dose range for three groups [2 × 10(8) , 2 × 10(9) and 2 × 10(10) colony forming unit (CFU)] was determined. In addition, the monkeys were re-infected. The identified optimal challenge dose was 2 × 10(9) CFU; this dose elicited 60% protection in monkeys when they were re-challenged with a one log higher dose (2 × 10(10) CFU). The challenge dose, 2 × 10(10) CFU, produced severe dysentery in all monkeys, with one monkey dying within 24 h, elicited 100% protection when re-challenged with the same dose. All monkeys exhibited immune responses. This study concludes that the rhesus monkey model closely mimics the disease and immune response seen in humans and is a suitable animal model for the pre-clinical evaluation of Shigella vaccine candidates. Prior infection with the 1617 strain can protect monkeys against subsequent re-challenges with homologous strains.

Assessment of the immunotoxic potential of trichloroethylene and perchloroethylene in rats following inhalation exposure.

The immunotoxic potential of trichloroethylene (TCE) and perchloroethylene (PERC) was assessed after inhalation exposure through the evaluation of the antibody forming cell (AFC) response to sheep red blood cells (SRBC). Female Sprague-Dawley rats were exposed to TCE or PERC vapor at 0, 100, 300, or 1000 ppm for 6 h/day, 5 days/week for 4 weeks (20 exposure days). Additional 0 ppm control groups were included and were dosed with cyclophosphamide via intraperitoneal injection to serve as positive immunosuppressive controls in the SRBC assay. Additional end-points evaluated included liver, kidney, spleen, and thymus weights, hematology, cellular differentials in bronchoalveolar lavage fluid, histopathology of select tissues, and assessment of the phagocytic activity of pulmonary alveolar macrophage (PERC only). Exposure to the high concentration of TCE (1000 ppm) resulted in increases in relative liver and kidney weights and suppression of AFC responses (AFC/spleen and AFC/10(6) spleen cells) by ≈ 70%, while no treatment-related effects were noted at 100 and 300 ppm. Animals exposed to PERC at levels of 300 or 1000 ppm had statistically significant increases in relative liver weights that were accompanied by very slight hypertrophy of the centrilobular hepatocytes. Animals exposed to PERC did not demonstrate a treatment-related effect on the AFC response and no effect was noted on the phagocytic activity of pulmonary alveolar macrophages. The results of these studies indicate that TCE had immunotoxic potential only at high exposure concentrations (1000 ppm), while PERC, at similar exposure concentrations, did not display any evidence of immunotoxicity.

Advances in antibody manufacturing using mammalian cells.

In this review, we describe recent advances in antibody processing technology including: (1) development of proprietary cell lines; (2) improved expression systems optimized by selective technologies to boost underperformers; (3) improved protein-free and serum-free culture media; and (4) attention to glycosylation and other post-translational modifications. Advances in computer technology and sophisticated redesign of bioreactors have been major contributors to the dramatic improvements in antibody yields that have been documented in the last decade. Disposable bioreactor components are now widespread, resulting in improved yields, better quality product and lower costs for producers. Downstream innovations include (1) disposable devices for clarification and purification, (2) improved resins and ligands, and (3) new designs of hardware for improved performance. While there are numerous factors contributing to the increased yields that have been obtained, the most sustained of these is the introduction of disposable technologies on both the upstream and the downstream ends of the process. With the continuing introduction of improved computer technology and technological innovation, there is every reason to believe that quality and quantity of antibody products will continue to improve in the coming years, and supply will be adequate to meet the forthcoming needs of the industry.

Immunological tools for the assessment of both humoral and cellular immune responses in Foxes (Vulpes vulpes) using ovalbumin and cholera toxin B as an antigenic model.

The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed. To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model. Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay. The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization. Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs). Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs. This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia. Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes. These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.

Toxicology and humoral immunity assessment of octamethylcyclotetrasiloxane (D4) following a 28-day whole body vapor inhalation exposure in Fischer 344 rats.

Octamethylcyclotetrasiloxane, D4, is a low viscosity, silicone fluid consisting of four dimethyl-siloxy units ((CH3)2SiO)4 in a cyclic structure. It is primarily used as a building block in the industrial synthesis of long chain silicone polymers. The combination of D4 with decamethylcyclopentasiloxane (D5) is commonly referred to as cyclomethicone which has a wide range of applications as a formulation aid in personal care products. To extend the existing database regarding the biological activities of D4, a 28 day whole body vapor inhalation study was conducted using Fischer 344 rats at 0 (room air), 7, 20, 60, 180 and 540 ppm for 6 hours/day, 5 days/week. Parameters measured included body weights, organ weights, gross pathology, histopathology, serum chemistries, and urinalysis. In addition to these standard toxicological endpoints, the ability of D4 exposed animals to mount an IgM antibody response was evaluated by a splenic antibody forming cell (AFC) assay and a serum enzyme-linked immunosorbant assay (ELISA). The results of this 28-day inhalation study indicate that D4 exposure caused no adverse effects on body weight, food consumption, or urinalysis parameters. In addition, there were no exposure related histopathological alterations at any site for any exposure group. A statistically significant increase in liver weight and the liver to body weight ratio was observed in both male (180-540 ppm) and female (20-540 ppm) rats, which was not observed in the 14-day recovery group animals. There were no other significant organ weight changes. Although statistically significant changes were observed in several hematological and serum chemistry parameters in both the terminal and 14-day recovery animals, the changes were marginal and within the normal range of values for the rat. Under these experimental conditions, there were no alterations noted in immune system function at any of the D4 exposure levels.

Assessment of cholinergic influences on a primary humoral immune response.

Cholinergic ligands can affect some lymphocyte functions, and binding of labelled cholinergic ligands to lymphocytes has been reported. However, the role of endogenous cholinergic stimulation in immunomodulation in vivo is unclear. It has been suggested that suppression of primary humoral immune responses in vivo by administration of organophosphorus compounds is caused by excessive cholinergic stimulation. If this is correct, it would demonstrate cholinergic immunomodulation in vivo and might serve as a useful model for the characterization of this phenomenon. In the present study, the organophosphorus insecticide parathion and its major metabolite, paraoxon, suppressed the primary IgM response to sheep red blood cells (SRBC) in vitro in Mishell-Dutton cultures at concentrations similar to those probably reached in vivo. In contrast, cholinergic agonists did not suppress the in vitro response, but tended to enhance it. However, antagonists also tended to enhance the response and the effects of agonists were not blocked by antagonists. Binding studies with a radiolabelled cholinergic antagonist ([3H-]QNB) did not indicate the presence of specific, saturable cholinergic receptors on lymphocytes. A membrane preparation from brain was used as a positive control, and specific, saturable binding was observed. These results suggest that suppression of primary immune responses in vivo by parathion is mediated at least in part by direct action of parathion and/or its major metabolite, paraoxon, on the immune system. The data provide no evidence that direct interaction of cholinergic ligands with the immune system contributes to parathion-induced immunosuppression. In fact, the absence of expected agonist-antagonist relationships in Mishell-Dutton cultures, the absence of saturable [3H]QNB binding, and puzzling inconsistencies in the literature on this subject cast doubt on the conclusion that lymphocytes express specific cholinergic receptors.

Assessment of carbamate pesticide immunotoxicity.

Adult female Swiss Webster and B6C3F1 mice received distilled water only or water containing 0.1, 1.0, 10, 100 or 1000 ppb of aldicarb daily for 34 days. The target concentration of aldicarb present in the 10 ppb dosing solution was analytically verified on a daily basis as was its stability over a 48-hr period. To develop an immune profile of this compound, functional parameters measured after exposure included resistance to infectious viral challenge; quantitation of splenic antibody-forming cells to sheep erythrocytes; splenic lymphocyte blastogenesis to B-cell mitogens; and T-cell mixed lymphocyte culture response. In addition, gross and histopathologic examinations of tissues relevant to the immune system were performed. The absence of significant effects on any of these parameters suggests that aldicarb at environmentally relevant exposure concentrations is not immunotoxic in rodents.

Assessment of the immune responsiveness of mice exposed to a 1.5-Tesla stationary magnetic field.

Humoral and cell-mediated immune responses were assayed following a 6-day exposure of LAF1/J mice to a 1.50 Tesla (1 T = 10(4) Gauss) stationary magnetic field. In tests of the immune response to sheep erythrocytes, the number of Jerne plaques formed by spleen lymphocytes and the level of serum IgM were not significantly different for the exposed mice in comparison with control animals. Tests for mitogen-induced lymphocyte proliferation also demonstrated no significant differences in the response of spleen lymphocytes from exposed and control groups of mice.

A plaque assay for assessment of immune responses to Streptococcus mutans cell wall carbohydrate.

A plaque-forming cell (PFC) assay has been developed for measurement of single cell responses to the serotype carbohydrate antigen of the Streptococcus mutans cell wall. Serotype g carbohydrate was purified from a mutanolysin (M1) enzyme digest (and designated M1g) of S. mutans 6715 cell walls by ion exchange and gel filtration chromatography. M1g carbohydrate was esterified by stearoylization (sM1g), and subsequently coated to sheep erythrocytes (sM1g-SRBC). This coating antigen was then used for enumeration of IgM anti-M1g PFC responses from spleens of either mice or rats immunized with S. mutans 6715 antigen. Good splenic IgM anti-M1g PFC responses were seen in either mice or rats given S. mutans whole cells or cell walls, while cell wall lysates or M1g were lowly immunogenic. Of interest was the finding that sM1g induced good IgM anti-M1g PFC responses in mice and in murine spleen cell cultures, in vitro. This study describes a method for assessment of individual antibody-producing cells to a major S. mutans cell wall determinant which should facilitate studies directed to determine mechanisms involved in the induction of immune responses to this important bacterium.

Assessment of antibody production in sex-linked and autosomal dwarf chickens.

The humoral immune response was examined in two dwarf and one control strain of Single Comb White Leghorn chicks. Circulating antibody levels in response to immunization with varying concentrations of sheep erythrocytes were determined following primary immunization and again following a secondary antigen challenge. The males of the sex-linked dwarf strain produced significantly less antibody to lower antigen dosages in both the primary and secondary response but as antigen dose was increased the humoral response was no longer depressed below that of the controls. The antibody response of females carrying the dw gene was not significantly impaired over the entire antigen dose range nor was that of either sex of the autosomal dwarf strain. Similar results were obtained when the numbers of splenic plaque-forming cells (PFC's) were examined. However, in this assay both the males and females of the sex-linked dwarf strain demonstrated significantly impaired plaque-forming activity. No differences were found between the control and autosomal dwarf strains in their ability to generate PFC's.

Immune response to Mycobacterium lepraemurium: assessment of the humoral immune response in mice.

Intraperitoneal infection of mice with Mycobacterium lepraemurium produced a primary immune response as represented by direct (IgM) plaque-forming cells at the splenic level within a short period after infection. This response was enhanced when animals were administered a second infection fifteen days after the first one. An identical situation was observed with the developed (IgG) plaques, although the magnitude of these plaques in animals given the second challenge was of a higher order than those given only one infection. In both instances, the number of plaques declined within a very short time after reaching a peak. Higher levels of both direct and developed plaques in animals given second infection was a consequence of the dual effect of antigenic stimulus offerred through the extracellular organisms in the second challenge and intracellular organisms from the previous challenge. The rapid decline, indicated a dampening of the immune response through restricted antigenic stimulus, not only due to all organisms becoming intracellular during the course of infection but also due to structural alterations in the spleens, caused by extensive proliferation of the infecting pathogen. Comparative analysis of the humoral immune response in animals infected with M. leprae, M. marinum and M. lepraemurium indicated similarities in terms of the latent period of induction and decline of the immune response and some very distinct differences in other respects. These differences could be attributed to various factors, such as the site and dose of infection, nature of the disease induced in the animal hose and the ability of the respective organisms to become intracellular. Based on these observations, it is suggested that neither the M. lepraemurium nor the M. marinum models can fully satisfy the requirements, at least immunologically, needed to consider these models as possible substitutes for evaluation of the host-parasite interactions in M. leprae infection of mice, although such a concept has been proposed previously, especially in regard to the M. marinum model.