Cruzipain and Its Physiological Inhibitor, Chagasin, as a DNA-Based Therapeutic Vaccine Against Trypanosoma cruzi.

Chagas disease caused by the protozoan parasite Trypanosoma cruzi is endemic in 21 Latin American countries and the southern United States and now is spreading into several other countries due to migration. Despite the efforts to control the vector throughout the Americas, currently, there are almost seven million infected people worldwide, causing ~10,000 deaths per year, and 70 million people at risk to acquire the infection. Chagas disease treatment is restricted only to two parasiticidal drugs, benznidazole and nifurtimox, which are effective during the acute and early infections but have not been found to be as effective in chronic infection. No prophylactic or therapeutic vaccine for human use has been communicated at this moment. Here, we evaluate in a mouse model a therapeutic DNA vaccine combining Cruzipain (Cz), a T. cruzi cysteine protease that proved to be protective in several settings, and Chagasin (Chg), which is the natural Cz inhibitor. The DNAs of both antigens, as well as a plasmid encoding GM-CSF as adjuvant, were orally administrated and delivered by an attenuated Salmonella strain to treat mice during the acute phase of T. cruzi infection. The bicomponent vaccine based on Salmonella carrying Cz and Chg (SChg+SCz) was able to improve the protection obtained by each antigen as monocomponent therapeutic vaccine and significantly increased the titers of antigen- and parasite-specific antibodies. More importantly, the bicomponent vaccine triggered a robust cellular response with interferon gamma (IFN-γ) secretion that rapidly reduced the parasitemia during the acute phase and decreased the tissue damage in the chronic stage of the infection, suggesting it could be an effective tool to ameliorate the pathology associated to Chagas disease.

Use of cross-reactivity immunoassay to orient insulin replacement in diabetic patients with high levels of insulin antibodies.

The prevalence and high levels of anti-insulin antibodies (IA) have frequently been associated with brittle diabetes, lipodystrophy in the areas where the insulin is injected and/or poor metabolic control. When this happens the usual criterion adopted is the empirical change of insulin type and/or formulation intending to diminish the IA level and then to decrease the undesirable side-effects. Here, we present a rational two step radiometric method consisting in: A) a first-line radioligand binding assay (RBA) to assess IA in sera of these patients and detecting those with high levels. B) applying a displacement assay (RIA) to determine the in vitro cross-reactivity parameters (affinity constants and selectivity ratios) that quantify the relative degree of interaction between antibodies and alternative insulin analogs. From these results we conclude that conventional criteria for selection of insulin analogs, in terms of pharmacokinetic and pharmacodinamic parameters, should be complemented with an appropriate test to assess affinity parameters when high IA title is demonstrated. •This manuscript introduces a rational method to determine the appropriated insulin replacement when high insulin antibodies levels are present.•This protocol provides instructions and details in mathematical tools and laboratory processes for the analysis of serum samples.•This method proved to be successful in a single case and requires confirmation using a large group of patients.

A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin.

A new radioligand-binding assay (RBA) is described for the detection of insulin/proinsulin-specific antibodies using 35S-labeled proinsulin produced by a cell-free reticulocyte extract. Direct use of the crude expression product in the RBA was not feasible because the protein failed to fold properly (or had incorrectly paired disulphide bridges) and purification was hindered by interfering by-products. A refolding protocol and a chromatographic procedure were devised that readily allowed production of purified and immunochemically competent 35S-labeled proinsulin. The new RBA was compared with the reference test, in which the tracer was standard 125I-insulin. The analysis included sera from 41 diabetic patients and 25 healthy controls. Twenty-six (63.4%) and 29 (70.7%) patients scored positive by RBA using 35S-PI and 125I-insulin, respectively. The methods showed a satisfactory correlation with r(2)=0.77 and a slope not significantly different from unity (m=1.16+/-0.10; 95% confidence interval). Since the nuclide used in the assay is 35S, the procedure is compatible with standard assays for GADA and IA-2A, and thus may permit combined assays for the major early markers of autoimmune diabetes.

Marcadores inmunológicos y genéticos en diabetes mellitus tipo 1 en Argentina / Immunologic and genetic markers in type 1 diabetes mellitus in Argentina

El objetivo del trabajo fue evaluar la prevalencia y asociación de los marcadores inmunológicos (anticuerpo anti-islote pancreático: ICA, autoanticuerpo anti-insulina: IAA, anticuerpo antidecarboxilasa del ácido glutámico: GADA y anticuerpo anti ICA512) y con el genotipo HLA DQBl en pacientes con diabetes tipo 1 de reciente debut, hermanos de diabéticos y personas sin historia de enfermedad autoinmune en población argentina. Se estudiaron 79 niños con diabetes tipo 1 de reciente debut, 79 niños controles y 68 hermanos sanos de niños con diabetes 1. En todos ellos se determinó IAA, GADA, ICA, ICA512 y alelos HLA DQB1. La sensibilidad para ICA fue de 67.1 por ciento; para IAA de 36,7 por ciento; para GADA de 74,6 por ciento, y para ICA512 de 63,4 por ciento. Ninguno de los niños control presentó marcadores inmunológicos positivos. La sensibilidad combinada de ICA-IAA-GADA fue de 89,8 por ciento, similar a la de ICA512-GADA (87.3 por ciento) o la combinación de ICA512-GADA-IAA (91.1 por ciento). El valor de GADA presentó correlación positiva con el de ICA, no encontrándose correlación alguna entre los valores de IAA, ICA512 e ICA. El valor de IAA presentó correlación negativa y el de GADA positiva con la edad de los pacientes. La presencia de IAA se asoció con DQB1 *0201, mientras que la de ICA e ICA512 con DQB1 *0302. Entre los hermanos, 3/68 (4,4 por ciento) fueron positivos para IAA, uno (1,5 por ciento) lo fue para GADA y otro (1.5 por ciento) para ICA512. Nuestros resultados muestran que la combinación de múltiples marcadores incrementa la sensibilidad predictiva, siendo la asociación ICA512-GADA altamente sensible y equivalente a otras combinaciones propuestas como ICA-IAA-GADA

Immunologic and genetic markers in insulin-dependent diabetes mellitus (Type 1) in a argentine population

The objective was to evaluate the prevalence and association of several markers (islet cell antibodies: ICA, ainsulin autoantibodies: IAA, glutamic acid decarboxylase antibodies: GADA and ICA512 antibodies: ICA512A) along with HLA DQB1 genotype in type 1 diabetes mellitus of recent onset, including siblings and individuals without any history of this disease, in an Argentine population. A total of 79 children with type 1 diabetes mellitus of recent onset were studied, as well as 79 control children, and 68 healthy siblings of type 1 diabetic cases. IAA, ICA, GADA, ICA512A and HLA DQB1 alleles were determined. Sensitivity was 67.1 por ciento for ICA, 36.7 percent for IAA, 74.6 por ciento for GADA and 63.4 por ciento ICA512A. None of the control subjects was positive for the immunological markers. Combined sensitivity of ICA-IAA-GADA was 89.8 por ciento, similar to the ICA512A- GADA (87.3 percent) or ICA512A-GADA-IAA combination (91.1 por ciento ). GADA correlated positively with ICA, but no such correlation was found between IAA, ICA512A and ICA. IAA correlated negatively and GADA positively with age. IAA was associated to DQB1*0201, whereas ICA and ICA512A associated to DQB1*0302. Among siblings, 3/68 (4.4 percent) were positive for IAA and a single case (1.5 percent) was positive for GADA and one for ICA512A. Our findings show that the combination of multiple tests increases the sensitivity for prediction, with the ICA512A-GADA combination proving highly sensitive and equivalent to other proposed combinations, such as ICA-IAA-GADA.