We identified different lipemic and metabolic responses after the ingestion of a standardized meal by healthy adults and related them to atherosclerotic markers. Samples from 60 normolipidemic adults were collected before and after a liquid meal (40 g fat/m² body surface) at 0, 2, 4, 6, and 8 h for measurements of lipids, free fatty acids (FFA), insulin, cholesteryl ester transfer protein (CETP), autoantibodies to epitopes of oxidized LDL (oxLDL Ab), lipolytic activities, and apolipoprotein E polymorphism. Mean carotid intima-media thickness (cIMT) was determined by Doppler ultrasound. The volunteers were classified into early (N = 39) and late (N = 31) triacylglycerol (TAG) responders to the test meal. Late responders showed lower HDL cholesterol concentration at fasting and in the TAG peak, lower insulin and higher FFA concentrations compared to early responders. Multivariate regression analyses showed that mean cIMT was associated with gender (male) and age in early responders and by cholesterol levels at the 6th hour in late responders. oxLDL Ab were explained by lipoprotein lipase and negatively by hepatic lipase and oxLDL Ab (fasting period) by CETP (negative) and FFA (positive). This study is the first to identify a postalimentary insulin resistance state, combined with a reduced CETP response exclusively among late responders, and the identification of the regulators of postalimentary atherogenicity. Further research is required to determine the metabolic mechanisms described in the different postalimentary phenotypes observed in this study, as well as in different pathological states, as currently investigated in our laboratory.

Abnormal surface expression of HLA-DR by leukocytes is associated with a poor prognosis in critical care patients. Critical care patients often receive total parenteral nutrition with lipid emulsion (LE). In this study we evaluated the influence of fish oil LE (FO) on human monocyte/macrophage (Mφ) expression of surface HLA-DR under distinct activation states. Mononuclear leukocytes from the peripheral blood of healthy volunteers (n=18) were cultured for 24 hours without LE (control) or with 3 different concentrations (0.1, 0.25, and 0.5%) of the follow LE: a) pure FO b) FO in association (1:1-v/v) with LE composed of 50% medium-chain trygliceride and 50% soybean oil (MCTSO), and c) pure MCTSO. The leukocytes were also submitted to different cell activation states, as determinate by addition time: no INF-γ addition, 18 hours before, or at the time of LE addition. HLA-DR expression on Mφ surface was evaluated by flow cytometry using specific monoclonal antibodies. In relation to controls (for 0.1%, 0.25%, and 0.5%: 100) FO decreased the expression of HLA-DR when added alone [in simultaneously-activated Mφ, for 0.1%: 70 (59 ± 73); for 0.25%: 51 (48 ± 56); and for 0.5%: 52.5 (50 ± 58)] or in association with MCTSO [in simultaneously-activated Mφ, for 0.1%: 50.5 (47 ± 61); for 25%: 49 (45 ± 52); and for 0.5%: 51 (44 ± 54) and in previously-activated Mf, for 1.0%: 63 (44 ± 88); for 0.25%: 70 (41 ± 88); and for 0.5%: 59.5 (39 ± 79)] in culture medium (Friedman p < 0.05). In relation to controls (for 0.1%, 0.25%, and 0.5%: 100), FO did not influence the expression of these molecules on non-activated Mφ [for 0.1%: 87.5 (75±93); for 0.25%: 111 (98 ± 118); and for 0.5%: 101.5 (84 ± 113)]. Results show that parenteral FO modulates the expression of HLA-DR on human Mφ surface accordingly to leukocyte activation state. Further clinical studies evaluating the ideal moment of fish oil LE infusion to modulate leukocyte functions may contribute to a better understanding of its immune modulatory properties.

La expresión anómala del HLA-DR sobre la superficie de los leucocitos se asocia con un pronóstico sombrío en pacientes críticos. A menudo, estos pacientes reciben nutrición parenteral total con una emulsión lipídica (EL). En este estudio, evaluamos la influencia de una EL de aceite de pescado (AP) en la expresión del HLA-DR en la superficie de monocitos/macrófagos humanos (MΦ) bajo diferentes estados de activación. Se cultivaron leucocitos mononucleares de sangre periférica de voluntarios sanos (n = 18) durante 24 horas sin la EL (control) o con 3 concentraciones distintas (0,1, 0,25 y 0,5%) de la siguiente EL: a) AP puro b) AP en asociación con EL (1:1 v/v) compuesta de 50% de triglicéridos de cadena media y 50% de aceite de soja (TCMAS), y c) TCMAS puro. También sometimos a los leucocitos a diferentes estados de activación celular, determinado por INF-γ tiempo de adición: no INF-γ adición 18 horas antes, o en el momento de añadir la EL. La expresión del HLA-DR sobre la superficie de los MΦ se evaluó mediante citometría de flujo utilizando anticuerpos monoclonales específicos. En relación con los controles (para 0,1%, 0,25% y 0,5%: 100) el AP disminuyó la expresión de HLA-DR cuando se añadía solo [en MΦ activados simultáneamente, para 0,1%: 70 (59 ± 73); para 0,25%: 51 (48 ± 56); y para 0,5%: 52.5 (50 ± 58)] o en asociación con TCMAS [en MΦ activados simultáneamente para 0,1%: 50,5 (47 ± 61); para 0,25%:(45 ± 52); y para 0,5%: 51 (44 ± 54) y en MΦ previamente activados para 0,1%: 63 (44 ± 88); para 0,25%: 70 (41 ± 88); y para 0,5%: 59,5 (39 ± 79)] en el medio de cultivo (Friedman p < 0,05). Con respecto a los controles, (para 0,1%, 0,25% y 0,5%: 100), el AP no influyó en la expresión de estas moléculas en MΦ no activados [para 0,1%: 87,5 (75 ± 93); para 0,25%: 111 (98 ± 118); y para 0,5%: 101,5 (84 ± 113)]. Los resultados muestran que el AP parenteral modula la expresión del HLA-DR sobre la superficie de los MΦ humanos en función del estado de activación leucocitaria. Estudios clínicos adicionales que evalúen el momento idóneo de añadir la infusión de EL con aceite de pescado para modular las funciones leucocitarias podrían contribuir a entender mejor sus propiedades inmunomoduladoras.

Abnormal surface expression of HLA-DR by leukocytes is associated with a poor prognosis in critical care patients. Critical care patients often receive total parenteral nutrition with lipid emulsion (LE). In this study we evaluated the influence of fish oil LE (FO) on human monocyte/macrophage (Mphi) expression of surface HLA-DR under distinct activation states. Mononuclear leukocytes from the peripheral blood of healthy volunteers (n = 18) were cultured for 24 hours without LE (control) or with 3 different concentrations (0.1, 0.25, and 0.5%) of the follow LE: a) pure FO b) FO in association (1:1 - v/v) with LE composed of 50% mediumchain trygliceride and 50% soybean oil (MCTSO), and c) pure MCTSO. The leukocytes were also submitted to different cell activation states, as determinate by INF-γ addition time: no INF-γ addition, 18 hours before, or at the time of LE addition. HLA-DR expression on Mphi surface was evaluated by flow cytometry using specific monoclonal antibodies. In relation to controls (for 0.1%, 0.25%, and 0.5%: 100) FO decreased the expression of HLA-DR when added alone [in simultaneously-activated Mphi, for 0.1%: 70 (59 ± 73); for 0.25%: 51 (48 ± 56); and for 0.5%: 52.5 (50 ± 58)] or in association with MCTSO [in simultaneously-activated Mphi, for 0.1%: 50.5 (47 ± 61); for 25%: 49 (45 ± 52); and for 0.5%: 51 (44 ± 54) and in previously-activated Mphi, for 1.0%: 63 (44 ± 88); for 0.25%: 70 (41 ± 88); and for 0.5%: 59.5 (39 ± 79)] in culture medium (Friedman p < 0.05). In relation to controls (for 0.1%, 0.25%, and 0.5%: 100), FO did not influence the expression of these molecules on non-activated Mphi [for 0.1%: 87.5 (75 ± 93); for 0.25%: 111 (98 ± 118); and for 0.5%: 101.5 (84 ± 113)]. Results show that parenteral FO modulates the expression of HLA-DR on human Mphi surface accordingly to leukocyte activation state. Further clinical studies evaluating the ideal moment of fish oil LE infusion to modulate leukocyte functions may contribute to a better understanding of its immune modulatory properties.

La expresión anormal del HLA-DR en la superficie de los leucocitos se asocia con un pronóstico peor en los enfermos críticos. Estos enfermos a menudo reciben nutrición parenteral total con una emulsión lipídica (EL.) En este estudio evaluamos la influencia de la EL de aceite de pescado (AP) sobre la expresión del HLA-DR de superficie por los monocitos /macrófagos humanos (Mfi) en distintos estados de activación. Se cultivaron leucocitos mononucleares de sangre periférica de voluntarios sanos (n = 18) durante 24 horas sin EL (control) o con tres concentraciones diferentes (0,1, 0,25 y 0,5%) de la siguiente EL: a) AP puro b) AP en asociación (1:1 en v/v) con la EL compuesta de un 50% de triglicéridos de cadena media y 50% de aceite de soja (TCMAS), y c) TCMAS puro. Se sometió a los leucocitos a tres estados de activación diferentes, como venía determinado por el tiempo de adición de INF-γ: sin añadir INF-γ, 18 horas antes o en el momento de añadir la EL. La expresión de HLA-DR en la superficie de los Mfi se evaluó mediante citometría de flujo empleando anticuerpos monoclonales específicos. En relación con los controles (para 0,1%, 0,25% y 0,5%: 100) el AP disminuyó la expresión de HLA-DR cuando se añadía solo {en Mfi activados de forma simultánea, para 0,1%: 70 (59 ± 73); para 0,25%: 51 (48 ± 56) y para 0,5%: 52,5 (50 ± 58)} o en asociación con TCMAS [en Mfi activados de forma simultánea, para 0,1%: 50,5 (47 ± 61); para 25%: 49 (45 ± 52); y para 0,5%: 51 (44 ± 54) y en Mfiactivados previamente, para 1,0%: 63 (44 ± 88); para 0,25%: 70 (41 ± 88); y para 0,5%: 59,5 (39 ± 79)] en medio de cultivo (Friedman p < 0,05.) En relación con los controles (para 0,1%, 0,25% y 0,5%: 100), el AP no influyó en la expresión de estas moléculas en los Mfino activados [para 0,1%: 87,5 (75 ± Ó93); para 0,25%: 111 (98 ± 118); y para 0,5%: 101,5 (84 ± 113)}. Los resultados muestran que el AP parenteral modula la expresión del HLA-DR sobre la superficie de los Mfihumanos en función del estado de activación de los leucocitos. Estudios clínicos adicionales que evalúen el momento ideal de la infusión de la EL con aceite de pescado para modular las funciones leucocitarias podrían contribuir a un mejor conocimiento de sus propiedades inmunomoduladoras.

Background & aim: To compare the effect of fish oilbased (FO) lipid emulsions (LE) for parenteral administration with standard LE and a new FO containing LE composed of four different oils on the antigen presentation and inflammatory variables. Methods: Phytohemagglutinin (PHA) activated human mononuclear leukocytes were cultured with different LE - Control: without LE; SO: soybean oil; SO/FO: soybean and FO (4:1); MCT/SO: medium chain triglycerides and SO (1:1); MCT/SO/FO: MCT/SO and FO (4:1) and SMOF: a new LE containing FO. Cytokine production was evaluated by ELISA, the expression of antigen-presenting and co-stimulatory surface molecules were analyzed by flow cytometry and lymphocyte proliferation was assessed by H³-Thymidine incorporation, after tetanus toxoid-induced activation. Results: All LE decreased the HLA-DR and increased CD28 and CD152 expression on monocytes/macrophages and lymphocytes surface (p < 0.05). SO/FO and MCT/SO/FO decreased lymphocyte proliferation (p<0.05). All LE decreased IL-2 production, but this effect was enhanced with MCT/SO/FO and SMOF (p < 0.05). MCT/SO/FO decreased IL-6 and increased IL-10, whereas SO had the opposite effect (p < 0.05). Conclusion: FO LE inhibited lymphocyte proliferation and had an anti-inflammatory effect. These effects seem to be enhanced when FO is mixed with MCT/SO. SMOF had a neutral impact on lymphocyte proliferation and IL-6 and IL-10 production.

Antecedentes & objetivo: Comparar el efecto de las emulsiones lipídicas (EL) basadas en aceite de pescado (AP) para la administración parenteral con las EL estándar y una nueva EL que contiene AP compuesta por cuatro aceites distintos sobre la presentación antigénica y las variables inflamatorias. Métodos: se cultivaron leucocitos mononucleares activados con fitohemaglutinina (PHA) con diferentes EL - Control: sin EL; AS: aceite de soja; AS/AP: soja y AP (4:1); TCM/AS: triglicéridos de cadena media y AS (1:1); TCM/AS/AP: TCM/AS y AP (4:1) y SMOF: una nueva EL que contiene AP. Se evaluó la producción de citocinas mediante ELISA, se analizó la expresión de moléculas de superficie de presentación de antígeno y co-estimuladoras mediante citometría de flujo y se evaluó la proliferación linfocitaria mediante la incorporación de timidina-H³ tras la activación inducida por el toxoide tetánico. Resultados: Todas las EL disminuyeron la expresión de HLA-DR y aumentaron la expresión de CD28 y CD152 sobre superficie de monocitos/macrófagos y linfocitos (p < 0,05). La AS/AP y la TCM/AS/AP disminuyeron la proliferación linfocitaria (p < 0,05). Todas las EL disminuyeron la producción de IL-2, pero su efecto se incrementó con las emulsiones TCM/AS/AP y SMOF (p < 0,05). La TCM/AS/AP disminuyó la IL-6 y aumentó la IL-10, mientras que el AS tuvo el efecto opuesto (p < 0,05). Conclusión: La EL AP inhibió la proliferación linfocitaria y tuvo un efecto antiinflamatorio. Estos efectos parecen estar potenciados cuando el AP se mezcla con TCM/AS. La SMOF tuvo un efecto neutro sobre la proliferación linfocitaria y la producción de IL-6 e IL-10.

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

The introduction of highly active antiretroviral therapy (HAART) for patients infected with HIV has significantly prolonged the life expectancy and to some extent has restored a functional immune response. However, the premature introduction of HAART has led to a significant and alarming increase in cardiovascular complications, including myocardial infarction and the appearance of abnormal distribution of body fat seen as lipodystrophy. One key element in the development of ischemic coronary artery disease is the presence of circulating and tissue-fixed modified low density lipoprotein (mLDL) that contributes to the initiation and progression of arterial lesions and to the formation of foam cells. Even though not completely elucidated, the most likely mechanism involves mLDL in the inflammatory response and the induction of a specific immune response against mLDL. Circulating antibodies against mLDL can serve as an indirect marker of the presence of circulating and vessel-fixed mLDL. In the present study, we measured antibodies to mLDL and correlated them with immune status (i.e., number of CD4+ T cells) in 59 HIV patients and with the clinical manifestation of lipodystrophy in 10 patients. We observed a significant reduction in anti-mLDL antibody levels related both to lipodystrophy and to an immunocompromised state in HIV patients. We speculate that these antibodies may explain in part the rapid development of ischemic coronary artery disease in some patients.

Hormone replacement therapy (HRT) reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp) and oxidized low density lipoprotein (LDL) have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11) and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02) (P<0.05 and P<0.001, respectively, ANOVA). The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

The uptake of lipids and lipoprotein particles by macrophages undergoes phagocytic activation and the formation of foam cells are key events in atherosclerosis. In this study we determined how intact high density lipoproteins (HDL) and apolipoproteins-HDL (removal of the lipid component from HDL, i.e., apoHDL) influence the phagocytosis of zymosan by mouse peritoneal macrophages. Zymosan particles preincubated together with lipoproteins or alone (control) were incubated with the macrophages. Phagocytosis activity was reported as the percent of macrophages that internalized three or more zymosan particles. HDL co-incubated with zymosan did not influence the over-all uptake of zymosan particles compared to apoHDL, which greatly enhanced the ability of the particle to be phagocytized (P<0.001). Part of this effect might be related to a greater binding of apoHDL to the particles compared to that of HDL (P<0.05). We conclude that this can be a useful method to study the ability of lipoproteins, including modified lipoproteins obtained from subjects with genetic forms of hyperlipidemia, to opsonize particles such as red blood cells and thus to investigate the processes that control the formation of foam cells and the mechanisms of atherogenesis.

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.