Sensibilización al maní en pacientes venezolanos con enfermedades atópicas y/o urticaria / Sensitization to peanut in Venezuelan patients with atopic diseases and / or uticaria

La sensibilización y las manifestaciones alérgicas al maní se han incrementado últimamente a nivel mundial, constituyendo el mismo la causa principal de anafilaxia por alimentos. Como la prevalencia de alergia al maní varía de acuerdo a las regiones nos propusimos evaluar, en una etapa preliminar, la sensibilización al maní por pruebas cutáneas (“skin prick test”) en pacientes venezolanos atópicos y/o con urticarias que acudieron a la Consulta ambulatoria de Alergía del Instituto de Inmunología. El 5,4 % de los pacientes manifestó algún tipo de manifestación cutánea o respiratoria al ingerir maní. Se demostró sensibilización al maní por pruebas cutánea en el 6,5 % de los pacientes. Sin embargo, un porcentaje pequeño (2 %) de ellos mostró, en conjunto, pruebas cutáneas positivas y síntomas a la ingesta del maní. Ningún paciente refirió síntomas severos tras la ingestión de maní. La mayoría de los pacientes con pruebas positivas al maní, también mostraron pruebas positivas a otros alimentos. Estos resultados concuerdan con la percepción de los médicos venezolanos de una baja frecuencia de reacciones adversas, especialmente graves, a la ingesta de maní en nuestro país

Peanut allergy and sensitization incidence has increased world wide to become the first cause of food anaphylaxis. Since the prevalence of peanut allergy changes according to geographical areas, the aim of the study was to assess, in a preliminary report, peanut allergy incidence by skin prick test in atopic Venezuelan patients with atopy and or urticaria from the outpatient allergy clinic of the Institute of Immunology. Cutaneous or respiratory manifestations after peanut ingestion was observed in 5.4 % of the patients studied. Cutaneous test was positive in 6.5 % of patients. In the other hand, a small group (2 %), showed positive skin test along with symptoms after peanut ingestion. None of the patients had severe reactions. Most of the patients with peanut positive skin test were positive to other food allergens. These results are in accordance with the general clinical perception of small frequency of adverse reaction, specially the most serious ones, to peanut ingestion in our country

Decreased transcription, expression and function of low-density lipoprotein receptor in leukocytes from patients with systemic lupus erythematosus.

The low-density lipoprotein receptor (LDLR) is directly involved in the metabolism of low-density lipoprotein (LDL) and its impairment causes accumulation of plasmatic LDL leading to atherosclerosis, a prevalent disease in patients with systemic lupus erythematosus (SLE). We studied LDLR transcription, expression and function in leukocytes patients with SLE and normal healthy donors (NHD). The ratio LDLR/glyceraldehyde-3-phosphate dehydrogenase (GADPH) mRNAs the expression of LDLR and the uptake of LDL-DiI were significantly lower (p < 0.001) in the patients with SLE. On the contrary, patients with SLE had significantly higher (p < 0.0001) levels of total cholesterol, LDL cholesterol and anti-oxLDL autoantibodies (AAb) as compared to NHD. No correlation between LDLR transcription, expression and function with the SLE disease activity index or with treatment was found. The decreased function of LDLR was independent of treatment. It seems dependent on the sterol regulatory binding protein and may be responsible for the increase of plasma LDL cholesterol and oxLDL AAb further increasing the risk of vascular diseases.

Serum adipokine levels in patients with systemic lupus erythematosus.

In patients with systemic lupus erythematosus (SLE) metabolic alterations are often observed, which may be due to either the disease, the genetic background or the treatment. We studied the serum levels of the adipokines leptin, adiponectin, resistin, visfatin and ghrelin in patients with SLE and controls. Leptin levels were lower and adiponectin, ghrelin and visfatin levels were higher in the patients. No significant differences were encountered for resistin. The values of adipokines were independent of treatment, even after correction for body mass index. Inverse correlations were found among leptin and adiponectin, ghrelin and visfatin. We conclude that adipokines are involved in the metabolic imbalance of patients with SLE.

Genetic contribution of major histocompatibility complex class II region to type 1 autoimmune hepatitis susceptibility in Venezuela.

AIMS: Autoimmune hepatitis (AIH) is a progressive liver disease characterized by the presence of circulating autoantibodies, hypergammaglobulinaemia and a favourable response to immunosuppressive treatment. Although the pathogenesis of type 1 AIH is unknown, disease susceptibility is partially determined by genes linked to the class II region of the major histocompatibility complex. Type 1 AIH has been associated with DRB1*03, DRB1*04 and DRB3 alleles in European and North American Caucasians, with DRB1*0405 in Japanese, with DRB1*0404 in Mexican, and with DRB1*1301 in Argentinean populations. METHODS: To analyse the molecular basis of these associations in Venezuela (mestizo population), we examined the frequency of human leucocyte antigens (HLA)-A -B -C, HLA-DQ and HLA-DR genes by low- and high-resolution oligonucleotide typing in a population of 41 type 1 AIH patients and 111 ethnic- and aged-matched healthy subjects. RESULTS: The frequencies of both DRB1(*)1301 (P<0.0001) and DRB1*0301 (P<0.005) were significantly higher in patients than in controls. In addition, patients showed a strong association with the DRB3 allele (P<0.01). In contrast, the DQB1*04 allele was significantly decreased in the patient group (P<0.01). The frequencies of haplotypes A*01-B*08-DQB1*02-DRB1*03-DRB3, DQB1*05-DRB1*1301, DQB1*06-DRB1*1301 and A*02-DRB1*1301, B*45-DRB3 were significantly increased in type 1 AIH patients compared with the controls (P<0.01). CONCLUSIONS: In conclusion, our data indicate that type 1 AIH predisposition in a Venezuelan mestizo population of different ethnic backgrounds is associated with DRB1*1301 and DRB1*0301 alleles. In addition, our findings suggest that protection against disease might be conferred by the DQB1*04 allele, with distinct ethnic differences from other populations.

Immunophenotype characteristics of peripheral blood mononuclear leukocytes of chronic idiopathic urticaria patients.

The pathogenesis of chronic idiopathic urticaria (CIU) is not completely understood although autoimmunity has been proposed. The aim of the study was to assess the expression of different leukocyte antigens, by flow cytometry, assaying total blood of 29 patients with CIU and of 20 sex and age matched controls. Moreover, we assessed soluble CD154 a marker of immune cell activation, predominantly memory T cells. When patients were divided depending an their response to the autologous serum skin test (ASST), three different groups were encountered: group 1 (n=11): with negative ASST-, group 2 (n=11): positive ASST (ASST+) with normal lymphocyte counts and group 3 (n=7): ASST+ with low lymphocyte counts (< 1500 cells/mm3). A significant increase in CD19+ percentage and not in the absolute count (P < 0.05) was observed in group 1 as compared to controls and to the other groups. In contrast, CD30+, CD45RO+ and CD4+/CD45RO+ percentages and biologically active soluble CD154 levels were significantly higher (P < 0.05) in group 3 as compared to group 1 or to controls. In ASST positive groups, CD45RO+ and CD4+/CD45RO+ positiveness correlates with wheal diameter. In conclusion, memory cells may play a role in these different types of patients and in understanding CIU pathogenesis.

Secreción de interferon por los linfocitos T periféricos de los portadores crónicos del virus de hepatitis B / Interferon G secretion by the peripheral lymphocyts T of the chronic carries of the hepatitis B virus

Previamente demostramos que los linfocitos T periféricos de los portadores crónicos del VHB activados “in vitro” con fitohemaglutinina (PHA) o con anticuerpos monoclonales anti-CD3 secretan interleucina-2 (IL-2) y expresan receptores para IL-2, IL-6 y para el factor de necrosis tumoral. Investigamos la producción de interferón por células T altamente purificadas (98 por ciento CD3+) de 14 pacientes infectados crónicamente por el VHB. Los linfocitos T se activaron con PHA sola (0.5g/mL) y con PHA añadiendo 100 L de sobrenadantes de monocitos (SMo) obtenidos después de la activación de los monocitos autólogos y alogénicos con lipopolisacárido. Los linfocitos T estimulados con PHA de los pacientes infectados por el VHB secretaron interferón en forma similar a los controles (438+129 vs. 577+127 pg/mL). En presencia de PHA más SMo autólogos, los linfocitos T de los pacientes no sufrieron modificación en la secreción de interferon, siendo la misma significativamente menor a la demostrada por las células T de los controles cultivadas en iguales condiciones (589+124 vs. 922+121 pg/mL; p<0.05). Al adicionar SMo alogénicos (PHA + SMo de controles), los linfocitos T de los portadores crónicos del VHB incrementaron la producción de interferón a una concentración significativamente mayor a la obtenida en presencia de PHA sola (752+123 vs. 438+129 pg/mL; p<0.01). En conclusión, los linfocitos T periféricos de los portadores crónicos del VHB son capaces de secretar interferón y responden incrementando esta secreción cuando son expuestos a factores solubles de monocitos alogénicos lo que indica que esta población celular T preserva su habilidad funcional para mediar respuestas inmunes

Interaction of immune complexes isolated from hepatitis C virus-infected individuals with human cell lines.

We investigated the interaction of immune complexes (IC) isolated from hepatitis C virus (HCV)-infected individuals with several cell lines that differentially express Fc receptors, and analyzed viral infection by the presence of HCV RNA sequences. Monocytic (U937 and Monomac-6) and lymphocytic (MOLT-4 and Jurkat) cell lines were incubated with interferon- plus phorbol myristate acetate to stimulate the expression of Fc receptors before addition of IC. Cell interaction with IC was monitored by flow cytometry. Positive cell fluorescence was detected in U937 and Monomac-6 cells [mean fluorescence intensity (MFI) 10.56+/-0.8 and 11.60+/-0.8, respectively]. Incubation of cells with monoclonal antibodies against Fc receptors for IgG before addition of IC decreased MFI in both cell lines (U937 2.1+/-0.5, Monomac-6 4.4+/-0.8, P<0.001), indicating that cell-IC interaction through these receptors was inhibited. In particular, the blockage of FcgammaRII was responsible for this effect. No binding of IC with either MOLT-4 or Jurkat cell lines was detected, which correlated with a very low Fc receptor expression. HCV RNA sequences were identified in the cells up to 120 h of post incubation with IC. These results suggest that IC can mediate entry of HCV to both U-937 and Monomac-6 cell lines mainly through the FcgammaRII.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake.

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Total and biologically active serum-soluble CD154 in patients with chronic idiopathic urticaria.

The pathogenesis of chronic idiopathic urticaria (CIU) is not understood completely; however, autoimmunity has been implicated. Because membrane and soluble forms of CD154 have been reported to be increased, in several autoimmune diseases, we have quantified the soluble CD154 (sCD154) molecule by a sandwich enzyme-linked immunosorbent assay in serum samples of 32 patients with CIU (aged 32 +/- 12 years) and compared them with 32 age- and sex-matched nonallergic controls. A marked increase was observed in patients with CIU as compared with controls (4.8 +/- 2.6 ng/mL versus 2.9 +/- 0.9 ng/mL; p < 0.0005). No significant differences were found between groups of patients with positive or negative autologous serum skin test. A biological assay to determine sCD154 showed that patients with positive autologous serum skin test have the highest levels (4.9 +/- 1.2 ng/mL) of biologically active sCD154 as compared with their negative counterparts (2.2 +/- 1.3 ng/mL; p < .001) and controls (0.6 +/- 0.3 ng/mL; p < 0.001). Active sCD154 can be derived from mast cell activation or other leukocytes. It is concluded that active sCD154 may be involved in the immune activation observed in patients with CIU.

Lipoprotein lipase protects bovine endothelial cells from human NK cytotoxic activity.

Human lipoprotein lipase (LPL), in a dose dependent fashion, significantly inhibited spontaneous human natural killer (NK) cells, but not lymphokine-activated killer (LAK) cytotoxic activity against bovine pulmonary endothelial cells. The effect was dependent on endothelial heparan sulfate (HS) sites, since heparitinase reverted it. When HS is added before LPL, NK and LAK cytotoxicity are markedly reduced. Endothelial and NK cell priming, with LPL and HS+LPL, significantly induced CD40 and CD154 expression, respectively. Furthermore, CD40 expression was inversely proportional to lytic units (R2 = 0.9, P < 0.001). Treating endothelial cells simultaneously with indomethacin, CD154 fusion protein, and Wortmanin prevented the CD40 effect increasing xenograft rejection. LPL and HS+LPL protect bovine endothelial cells from NK cytotoxicity by inducing CD40, CD154 expression, and secretion of soluble factors. The high, non-modulated expression of adhesion receptors and the low number of HS sites account for the minor effect of CD40 in LAK cytotoxic responses against bovine endothelial cells.

Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

Expresión de receptores leucocitarios similares a las inmunoglobulinas (lirs) en eosinofilos humanos / Expresion leukocyte receptors similar the ammunoglobulins (lirs) in eosinophils human

Los eosinófilos son células efectoras de la respuesta inmune, implicadas principalmente en las enfermedades alérgicas y en las infecciones parasitarias. Los eosinófilos pueden ser activados in vitro por diversas clases de agonistas, como las inmunoglobulinas, los mediadores lipídicos y las citocinas. Los receptores leucocitarios tipo Ig (leukocyte Ig-like receptors, LIRs) constituyen una familia de receptores de superficie celular con funciones activadoras e inhibitorias. Los LIRs inhibitors regulan en forma negativa las respuestas celulares a través de secuencias de tirosina inmunoreguladoras de inhibición (ITIM) citoplasmáticas. Hay muy poca data sobre el mecanismo de acción de los LIRs, aunque se piensa que actúan a través de la cadena de receptor Fc, que contiene una secuencia de tirosina inmunoreguladora de activación. En este trabajo demostramos la espresión de los LIRs en eosinófilos hipo y normodensos de donantes sanos y pacientes con enfermedades alérgicas

Interleukin-2 induces peroxide production by primed normodense eosinophils of patients with asthma.

In this study we assessed, by flow cytometry, the effect of interleukin 2 (IL-2) on the oxidative burst of normodense eosinophils (Eos's) isolated from 15 patients with moderately severe extrinsic asthma and 17 controls. We found that IL-2 significantly induced peroxide (H2O2) production in normodense Eos's from patients with asthma on a time kinetics study. This rise was higher in patients with immunoglobulin E levels > 180 IU/mL versus normal immunoglobulin E values. The effect of IL-2 was partially blocked by using anti-Tac antibody. In contrast, IL-2 decreased H2O2 production in normodense Eos's from controls. Cell surface expression of CD25, CD122, CD132, and CD69 were also determined and no statistical differences were found between both groups. In conclusion, IL-2 is able to increase H2O2 production by normodense Eos's isolated from patients with asthma and it may contribute to bronchial epithelium damage and chronic inflammation.

Hypodense eosinophils: characterization of surface molecule expression.

Distinct eosinophil populations have been characterized on the basis of discontinuous Percoll density gradients. In peripheral blood, normal individuals show a low number of normodense and hypodense eosinophils, contrasting with the high amount of hypodense cells in patients who have allergies. To characterize these two eosinophil populations, we analyzed membrane expression of several antigens and cytokine receptors in normodense and hypodense eosinophils from patients who have allergies and controls. Hypodense eosinophils expressed higher levels of CD122, CD69, and CD4 in both patients with allergies and control individuals when compared to normodense eosinophils. The expression of CD125, CD124, CD25, CD132, and CD23 were similar in both cell types.

Expresión diferencial de moleculas de superficie en eosinofilos normodensos e hipodensos de pacientes alérgicos / The diferencial expression of surface molecules in normodense eosinophils and hypodense of allergic patients

Se han descrito poblaciones heterógeneas de eosinófilos, basado en gradientes de densidad. En sangre periférica de sujetos sanos se observan principalmente eosinófilos normodensos (fenotipo de reposo) mientras que un número aumentado de eosinófilos hipodensos (fenotipo activado) está presente en pacientes alérgicos. En el presente trabajo analizamos, por citometría de flujo, la expresión de moléculas de superficie en eosinófilos normodensos e hipodensos de sujetos sanos y pacientes alérgicos. Nuestros resultados mostraron que los eosinófilos hipodensos expresan altos niveles de CD122, CD69 y CD4 en ambos grupos de estudio respecto de los eosinófilos normodensos, mientras que la expresión de CD125, CD124, CD25, CD132 y CD23 fueron similares en ambos tipos celulares

Disminución de la respuesta proliferativa de los linfocitos frente a antígenos ambientales, como indicador precoz / Precocious indicator of the lympocytary injuries induce by the immunodeficiency virus human

A fin de evaluar las células CD4+/CD29+ y sus respuestas frente a diferentes antígenos en estadíos de la infección por el Virus de Imunodeficiencia Humana (VIH), estudiamos 26 pacientes seropositivos asintomáticos (SP) y 15 pacientes con Síndrome de Inmunodeficiencia Adquirida (SIDA) simultaneamente con 20 individuos controles sanos (CS) y 10 hombres homo y bisexuales seronegativos (SN) y 15 pacientes con Síndrome de Inmunodeficiencia Adquirida (SIDA) simultáneamente con 20 individuos controles sanos (CS) y 10 hombres homo y bisexuales seronegativos (SN). Los fenotipos CD3, CD4, CD29 y CD45RA fueron analizados por citometría de flujo de dos colores. Encontramos depleción significativa de los linfocitos T CD4+ y de las subpoblaciones de células memoria (CD4+/CD29+) y virgen (CD45/CD45RA+) en los pacientes SP y SIDA, en comparación con los grupos CS y SN. Las respuestas proliferativas con dosis óptimas de Candida Albicans, Estreptokinasa y Toxoide Tetánico fueron exploradas en células mononucleares de sangre periférica (CMSP) y en poblaciones enriquecidas de células CD4+ y CD4/CD29+. En pacientes SP, la respuesta ante C. Albicans de CMSP estaba significativamente disminuída con respecto a la de CS (15.308 vs 35.951 cpm). La reducción de la proliferación frente a estreptokinasa fue evidente solamente con preparaciones enriquecidas de linfocitos Cd4+/cd29+ (3.084 vs 10.367 cpm). Una vez analizados los resultados, evidenciamos entre los pacientes SP, 7 individuos respondedores a por lo menos un antígeno y 7 no respondedores. En este último grupo, la ausencia de proliferación era independiente del número absoluto de células memoria y virgen. La respuesta frente a Toxoide Tetánico, aunque disminuida en SP, no era significativamente diferente de la CS. Nuestros resultados sugieren que la respuesta defectuosa frente a antígenos ambientales comunes, no relacionada con el número absoluto de linfocitos CD4+/CD29+, es probablemente un indicador temprano de lesión linfocitaria inducida por el VIH

The immunopathology of human schistosomiasis. III. Immunoglobulin isotype profiles and response to praziquantel

Immunoglobulin (Ig) isotype (IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD and IgE) levels were investigated, both pre- and post-treatment with praziquantel (PZQ), in 43 adults and children chronically infected with Schistosoma mansoni, by means of a two-site, isotype-specific immunoenzymometric assay. The patients were classified as responders (R) or non-responders (NR) on the basis of their circumoval precipitin test (COPT) results 12 months after treatment. In comparison with controls, pre-treatment R children showed significantly higher levels of IfG, IgG1, IgG4 (p<0.001) and IgE (p<0.01), and diminished IgG2 (p<0.05), while NR children showed significantly elevated levels only of IgE (p<0.05). Twelve months after therapy, R children maintained significantly lower levels of IgG2, but showed significantly decreased levels of IgG, IgG1, IgG4, and IgE, while the Ig isotype profile of NR children was unaltered. Adult R and NR showed similar isotype profiles before chemotherapy, with the exception of significantly elevated IgM levels in R. Twelve months after therapy, R adults showed significantly decreased levels of IgG, IgG1, and IgG4, while NR adults showed only diminshed IgG4 levels. These results reveal different Ig isotype profiles in untreated adults and children chronically infected with S. mansoni. The results further show that pre-treatment Ig isotype profile may be significantly modified after an effective R to chemotherapy, accounted for by down regulation of the IgG1 isotype in association with negative seroconversion of the COPT in R patients. The COPT reaction has been associated with the highly specific egg glycoprotein antigen omega 1, which shows a significant reduction in reactivity six months after treatment. IgG1 may thus play a main role in the response against the omega 1 antigen.