Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

LDL oxidada circulante y anticuerpos contra LDL oxidada según niveles de ácido úrico en mujeres con exceso de peso / Oxidized LDL and anti-oxidized LDL antibodies according uric acid levels in overweight women

Objetivo: Establecer si el aumento de ácido úrico sérico se asocia a niveles más elevados de LDL oxidada (LDLox), anticuerpos contra LDLox (anti LDLox) e índices de oxidación de la LDL, en mujeres con exceso de peso. Método: Estudio transversal que incluyó 114 mujeres con índice de masa corporal > 25 kg/m². Se determinó peso, talla, circunferencia abdominal, presión arterial, glicemia, ácido úrico, perfil lipídico, creatinina, apolipoproteína B (ApoB), LDLox, anti LDLox e insulina. Se estimó resistencia a la insulina mediante HOMA. Se calcularon índice de masa corporal, ApoB asociada a LDL, índices de oxidación de la LDL y terciles de ácido úrico. Se diagnosticó síndrome metabólico según criterios del NCEP/ATP III. Resultados: De las mujeres estudiadas, 51.8% mostró sobrepeso y el resto fueron obesas; 66.7% presentó síndrome metabólico. En el grupo con sobrepeso y en el grupo total de mujeres, sólo el índice LDLox/HDLc fue significativamente mayor en el último tercil de ácido úrico. Las concentraciones séricas de LDLox y los índices LDLox/colesterol total, LDLox/HDLc, LDLox/ApoB y LDLox/ApoB asociada a LDL fueron significativamente mayores entre las obesas ubicadas en el tercil más elevado de ácido úrico. Las concentraciones de anti LDLox y el índice LDLox/Anti LDLox no se relacionaron con ácido úrico. Los niveles séricos de ácido úrico y ApoB predijeron la elevación de la LDLox. Conclusión: El aumento del ácido úrico sérico se asoció con mayor oxidación de la LDL entre mujeres obesas, sugiriendo la importancia que podría tener el control periódico de ácido úrico en mujeres con exceso de peso.

Objective: To establish whether increased serum uric acid is associated with higher levels of oxidized LDL (oxLDL), antibodies against human oxidized LDL (oxLDL Ab) and ratios of LDL oxidation in overweight women. Methods: Cross-sectional study that included 114 women with body mass index > 25 kg/m2. We determined weight, height, waist circumference, blood pressure, glycemia, uric acid, lipid profile, creatinine, Apolipoprotein B (ApoB), oxLDL, oxLDL Ab, insulin and insulin resistance was estimated using HOMA. Body mass index, LDL-associated ApoB, rates of LDL oxidation and tertiles of uric acid were calculated. Metabolic syndrome was defined using NCEP/ATP III criteria. Results: Of the women studied 51.8% were overweight and the rest was classified as obese; 66.7% had metabolic syndrome. In the total group and overweight group, only the oxLDL/HDL cholesterol ratio was significantly higher in the last tertile of uric acid. The serum levels of circulanting oxLDL and oxLDL/cholesterol, oxLDL/HDL cholesterol, oxLDL/ApoB and oxLDL/ LDL-associated ApoB ratios were significantly higher among obese women located in the highest tertile of uric acid. Concentrations of oxLDL Ab and oxLDL/oxLDL Ab were not related to the uric acid. Serum uric acid and ApoB predicted the elevation of oxLDL. Conclusion: Increased serum uric acid was associated with more oxidation of LDL among obese women. This suggests the importance of regular monitoring of uric acid in overweight women. Prospective research should be conducted.

Efeitos de autoanticorpos antilipoproteína lipase, antilipoproteína de baixa densidade e antilipoproteína de baixa densidade oxidada sobre o metabolismo lipídico e aterosclerose no lúpus eritematoso sistêmico / Effects of autoimmune antibodies anti-lipoprotein lipase, anti-low density lipoprotein, and anti-oxidized low density lipoprotein on lipid metabolism and atherosclerosis in systemic lupus erythematosus

INTRODUÇÃO: O desenvolvimento prematuro de aterosclerose em lúpus eritematoso sistêmico tem sido amplamente divulgado. Anticorpo antilipoproteína lipase pode ser uma das causas que contribuem para esta doença. OBJETIVO: Avaliar o grau de risco coronariano devido a autoanticorpos em termos de placa carotídea em pacientes com lúpus. PACIENTES E MÉTODOS: Comparamos 114 pacientes com lúpus documentado e 111 controles normais pareados por sexo e idade. Antilipoproteína lipase (A-LPL), antilipoproteínas de baixa densidade oxidada (A-OXLDL), e antilipoproteínas de baixa densidade (A-LDL) foram medidos pelo teste imunoenzimático - ELISA. LDL-triglicéride (LDL-Trig) e HDL-Trig também foram dosados. A placa foi medida por ultrassom bilateral de carótida. RESULTADOS: 45,6 por cento dos pacientes foram positivos para A-LDL e 34,4 por cento para A-OXLDL; 44 por cento dos controles foram positivos para A-LDL e 20 por cento para A-OXLDL. O risco aumentou acentuadamente nos subgrupos com níveis elevados de anticorpos. Pacientes com A-LDL e A-OXLDL > 0,40 (n = 12) mostraram correlações de risco coronariano de: ALDL vs LDL-Trig = 0,7008, P = 0,0111; ultrassom bilateral vs colesterol = 0,62205, P = 0,0308; LDL-Trig vs infarto do miocárdio (IM) = 0,76562, P =0,0037; triglicerídeos totais vs IM = 0,78191, P = 0.0027); LDL-Trig/LDL-colesterol vs IM = 0,80493, P = 0,0016; A-OXLDL vs USBL = 0,71930, P = 0,0084. Correlações do SLEDAI com as variáveis de risco foram altamente significativas somente nos subgrupos com níveis elevados de anticorpos (SLEDAI x A-OXLDL = 0,70366, P = 0,0107). CONCLUSÃO: A-LPL inicia o desenvolvimento de mutações de LDL, seguido pela produção de anticorpos, formação da placa e do risco coronariano em alguns pacientes com lúpus erimatoso sistêmico (LES).

INTRODUCTION: Premature development of atherosclerosis in systemic lupus erythematosus has been widely reported. Anti-lipoprotein lipase antibody may be one cause contributing to this disorder. OBJECTIVE: To assess the extent of coronary risk due to autoimmune antibodies in terms of carotid plaque in lupus patients. PATIENTS AND METHODS: We compared 114 documented lupus patients with 111 normal controls matched for sex and age. Anti-lipoprotein lipase (A-LPL), anti-oxidized low density lipoprotein (A-OXLDL), and anti-low density lipoprotein (A-LDL) were measured by enzme-linked immunoabsorbent assay. Low density lipoprotein-triglyceride (LDL-Trig) and high density lipoprotein-triglyceride (HDL-Trig) were also measured. Plaque was measured by bilateral carotid ultrasound. RESULTS: 45.6 percent of patients tested positive for A-LPL, and 34.4 percent for A-OXLDL. 44 percent of normal controls tested positive for A-LPL, and 20 percent for A-OXLDL. Risk increased sharply in subgroups with increased antibody levels. Patients with A-LPL and A-OXLDL > 0.40 (n = 12) showed coronary risk correlations of: A-LPL x LDL-Trig = 0.7008, P = 0.0111; bilateral ultrasound vs total cholesterol = 0.62205, P = 0.0308; LDL-Trig vs myocardial infarction (MI) = 0.76562, P = 0.0037; total triglycerides vs MI = 0.78191, P = 0.0027); LDL-Trig/LDL-cholesterol vs MI = 0.80493, P = 0.0016; A-OXLDL vs USBL = 0.71930, P = 0.0084. Correlations of SLEDAI with risk variables were highly significant only in subgroups of elevated antibody levels (SLEDAI x A-OXLDL = 0.70366, P = 0.0107). CONCLUSION: A-LPL initiates the development of LDL mutations, followed by antibody production, plaque formation and coronary risk in some SLE patients.

Anticorpos contra LDL-ox e síndrome coronariana aguda / Antibodies against OxLDL and acute coronary syndrome

FUNDAMENTO: A oxidação da lipoproteína de baixa densidade (LDL-ox) induz à formação de epítopos imunogênicos na molécula. A presença de autoanticorpos contra a LDL-ox tem sido demonstrada no soro de pacientes com doença arterial coronariana (DAC). Contudo, o papel desses autoanticorpos na fisiopatologia das síndromes coronarianas agudas (SCA) e o seu significado clínico permanecem indefinidos. OBJETIVO: Avaliar a associação entre autoanticorpos contra a LDL-ox e SCA. MÉTODOS: Os títulos de imunoglobulina G autoanticorpos contra a LDL-ox por cobre (antiLDL-ox) e contra o peptídeo sintético D derivado da apolipoproteína B (antipeptD) foram determinados por ensaio imunoenzimático (ELISA) em 90 pacientes, nas primeiras 12h de SCA (casos) e em 90 pacientes com DAC crônica (controles). RESULTADOS: Os resultados mostraram que os títulos de antiLDL-ox foram significativamente mais elevados (p = 0,017) nos casos (0,40 ± 0,22), do que nos controles (0,33 ± 0,23). Por outro lado, os títulos de antipeptD foram significativamente menores (p < 0,01) nos casos (0,28 ± 0,23) do que nos controles (0,45 ± 0,30). A diferença dos títulos de ambos anticorpos entre os dois grupos estudados foi independente de idade, sexo, hipertensão arterial, diabete melito, dislipidemia, índice de massa corporal, tabagismo, perfil lipídico, uso de estatinas e história familiar de DAC. CONCLUSÃO: Os resultados mostraram que os títulos de antiLDL-ox foram significativamente mais elevados nos pacientes com síndrome coronariana aguda quando comparados aos pacientes com doença arterial coronariana e podem estar associados à instabilidade da placa aterosclerótica.

BACKGROUND: The oxidation of low-density lipoprotein (oxLDL) induces the formation of immunogenic epitopes in molecules. The presence of autoantibodies against oxLDL has been demonstrated in the serum of patients with coronary artery disease (CAD). However, the role of these autoantibodies in the pathophysiology of acute coronary syndromes (ACS) and their clinical significance remain undefined. OBJECTIVE: To evaluate the association between antibodies against oxLDL and ACS. METHODS: Titers of IgG autoantibodies against oxLDL by copper (anti-oxLDL) and anti-D synthetic peptide derived from apolipoprotein B (antipeptD) were determined by Enzyme-linked immunosorbent assay (ELISA) in 90 patients, in the first 12 hours of ACS (cases) and in 90 patients with chronic CAD (controls). RESULTS: The results showed that the titers of anti-oxLDL were significantly higher (p = 0.017) in cases (0.40 ± 0.22) than in controls (0.33 ± 0.23). On the other hand, the titers of antipeptD were significantly lower (p < 0.01) in cases (0.28 ± 0.23) than in controls (0.45 ± 0.30). The difference in the titers of both antibodies between the two groups was independent of age, sex, hypertension, diabetes mellitus, dyslipidemia, body mass index, smoking, lipid profile, statin use and family history of CAD. CONCLUSION: The results showed that the titers of anti-oxLDL were significantly higher in patients with acute coronary syndrome as compared to patients with coronary artery disease and may be associated with atherosclerotic plaque instability.

Impacto da proteína-C reativa no risco cardiovascular de adolescentes / Impact of C-reactive protein on cardiovascular risk in adolescents

FUNDAMENTO: Vários estudos sugerem que a proteína-C reativa (PCR) se correlaciona com doença arterial coronariana em adultos. Entretanto, essa associação ainda é pouco explorada em adolescentes. OBJETIVO: Avaliar a associação entre a PCR e os fatores de risco cardiovascular em adolescentes obesos. MÉTODOS: Oitenta e quatro adolescentes (12,6 ± 1,3 anos), ambos os sexos, foram distribuídos nos grupos Eutrófico (n = 28), Sobrepeso (n = 28) e Obeso (n = 28), segundo o índice de massa corpórea (IMC). A concentração de PCR (ELISA ultrassensível), o perfil lipídico e o conteúdo de anticorpos anti-LDLox (ELISA) foram determinados após jejum de 12h. RESULTADOS: Os grupos foram semelhantes quanto a idade (p = 0,13) e sexo (p = 0,83). Colesterol total, HDL-C, CT/HDL-C e LDL-C/HDL-C apresentaram diferenças significativas entre os grupos Eutrófico e Obeso. Não houve variação significativa no conteúdo de anticorpos anti-LDLox. Os valores de PCR foram diferentes entre os três grupos (p < 0,01). PCR apresentou associação significativa com IMC (β = 2,533), CB (β = 2,645) e CC (β = 2,945), CT (β = 0,006), LDL-C (β = 0,006) e anticorpos anti-LDLox (β = 0,383) e negativa entre HDL-C (β = -0,017). CONCLUSÃO: Os resultados indicam que a PCR se associa significativamente com marcadores de risco cardiovascular em adolescentes.

BACKGROUND: Several studies suggest that C-reactive protein (CRP) is associated with coronary artery disease in adults. However, this association has not been thoroughly explored in cases of adolescents. OBJECTIVE: To evaluate the association between CRP and cardiovascular risk factors in obese adolescents. METHODS: Eighty-four adolescents (12.6 ± 1.3 years) of both genders were divided into the following groups: Normal weight (n = 28), Overweight (n = 28), and Obese (n = 28), according to body mass index (BMI). CRP levels (ultrasensitive ELISA), the lipid profile, and anti-oxLDL antibody levels (ELISA) were determined after a 12-hour fast. RESULTS: The groups were similar in age (p = 0.13) and gender (p = 0.83). Total cholesterol, HDL-C, TC/HDL-C, and LDL-C/HDL-C showed significant differences between Normal weight and Obese groups. There was no significant variation in anti-oxLDL levels. CRP values were different among the three groups (p < 0.01). CRP levels showed a significant association with BMI (β = 2.533), AC (β = 2.645), WC (β = 2.945), TC (β = 0.006), LDL-C (β = 0.006), and anti-oxLDL antibodies (β = 0.383), and a negative association with HDL-C (β = -0.017). CONCLUSION: The results indicate that CRP is significantly associated with markers of cardiovascular risk in adolescents.

Antibodies against electronegative LDL inhibit atherosclerosis in LDLr-/- mice

In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25 percent cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

Auto-anticorpos anti-LDLox e sua correlação com o perfil lipídico e o estado nutricional de adolescentes / Anti-oxLDL autoantibodies and their correlation with lipid profile and nutritional status in adolescents

OBJETIVO: Avaliar se o conteúdo de auto-anticorpos anti-LDL oxidada (anti-LDLox) no plasma de adolescentes correlaciona-se com suas medidas antropométricas e com o perfil lipídico. MÉTODOS: O estudo incluiu 150 adolescentes com idade entre 10 e 15 anos, recrutados do ambulatório de obesidade da Universidade Federal de São Paulo (SP) e de escolas públicas de Piracicaba (SP). Foram avaliadas medidas antropométricas, como índice de massa corporal, circunferência de cintura e do braço, classificando os adolescentes em eutrófico, sobrepeso e obeso. Para as análises bioquímicas, foi realizado o perfil lipídico através de métodos enzimáticos colorimétricos, e para detecção do conteúdo de auto-anticorpos anti-LDLox, utilizou-se o método de ELISA. RESULTADOS: Segundo análises das variáveis antropométricas, o grupo obeso apresentou perfil alterado em relação aos grupos eutrófico e sobrepeso (p < 0,01), indicando risco cardiovascular. Quando o perfil lipídico foi avaliado, observaram-se diferenças estatisticamente significativas para as concentrações de colesterol total (p = 0,011), HDL-colesterol (p = 0,001) e LDL-colesterol (p < 0,042) nos grupos eutrófico e obeso. Para as análises de auto-anticorpos anti-LDLox plasmática, os grupos sobrepeso (p = 0,012) e obeso (p < 0,001) apresentaram valores superiores ao grupo eutrófico. Também houve correlações entre os auto-anticorpos anti-LDLox e variáveis antropométricas. CONCLUSÃO: A presença de auto-anticorpos anti-LDLox em adolescentes e as alterações metabólicas no perfil lipídico variaram de modo proporcional com parâmetros antropométricos, o que torna o conteúdo de anti-LDLox um potencial indicador bioquímico de risco para síndrome metabólica.

OBJECTIVE: To investigate whether levels of autoantibodies to oxidized LDL (anti-oxLDL) in the plasma of adolescents correlates with their anthropometric measurements and lipid profiles. METHODS: The study enrolled 150 adolescents aged between 10 and 15 years, recruited from the obesity clinic at Universidade Federal de São Paulo (SP) and from public schools in Piracicaba, SP, Brazil. Anthropometric measurements such as body mass index and waist and arm circumferences were used to classify the adolescents as having healthy weight, overweight or obesity. Colorimetric enzymatic methods were used for biochemical lipid profile analysis and ELISA was used to determine anti-oxLDL autoantibody levels. RESULTS: Analysis of anthropometric variables indicated that the obese group's profile was abnormal compared to the healthy weight and overweight groups (p < 0.01), indicating cardiovascular risk. Analysis of the lipid profiles demonstrated statistically significant differences in concentrations of total cholesterol (p = 0.011), HDL-cholesterol (p = 0.001) and LDL-cholesterol (p < 0.042) between the healthy weight group and the obese group. Analysis of plasma anti-oxLDL autoantibodies demonstrated that the overweight (p = 0.012) and obese groups (p < 0.001) had higher values than the healthy weight group. There were also correlations between anti-oxLDL autoantibody levels and anthropometric variables. CONCLUSIONS: In adolescents the presence of anti-oxLDL autoantibodies and metabolic changes to the lipid profile vary in proportion with anthropometric parameters, which makes anti-oxLDL concentration a potential biochemical indicator of risk of metabolic syndrome.

Connections: can the 20th century coronary heart disease epidemic reveal something about the 1918 influenza lethality?

This essay proposes that the ecologic association shown between the 20th century coronary heart disease epidemic and the 1918 influenza pandemic could shed light on the mechanism associated with the high lethality of the latter. It suggests that an autoimmune interference at the apoB-LDL interface could explain both hypercholesterolemia and inflammation (through interference with the cellular metabolism of arachidonic acid). Autoimmune inflammation, then, would explain the 1950s-60s acute coronary events (coronary thrombosis upon influenza re-infection) and the respiratory failure seen among young adults in 1918. This hypothesis also argues that the lethality of the 1918 pandemic may have not depended so much on the 1918 virus as on an immune vulnerability to it, possibly resulting from an earlier priming of cohorts born around 1890 by the 1890 influenza pandemic virus.

Efecto inmunosupresor in vitro de las lipoproteínas de baja densidad / In vitro inhibition of lymphocyte proliferation by low density lipoproteins

Background: immune cells participate in the formation of atheromatous plate, however little is known about the effects of native or oxidatively modified lipoproteins on these cells. Aim: To study the effects of lipoproteins on in vitro mononuclear cell proliferation. Material and methods: peripheral blood mononuclear cells were obtained from 10 patients with type 2 diabetes mellitus (aged 52 ñ 9 years old with a disease duration of 8.2 ñ 5.7 years and a mean glycosilated hemoglobin of 9.3 ñ 2.2 percent) and 10 non diabetic healthy controls (aged 50.3 ñ 7.1 years old). These were stimulated with phytohemagglutinin (PHA) alone or in the presence of native LDLS, malondialdehyde modified LDLs or glycated LDLs. Proliferation was measured as 3H-thymidine incorporation and expressed as Stimulation Index (SI). Results: SI of patients and healthy subjects, after PHA stimulation were similar: (57.5 ñ 29.8 and 61.1 ñ 23.5) respectively LDLs did not induce proliferation in neither group. Native LDLs produced a 98 percent inhibition of PHA induced proliferation. Malondialdehyde modified and glycated LDLs caused a 50 percent inhibition. The suppressive effect was maintained when lipoproteins were incorporated to culture media 60 min prior or after PHA stimulation. Conclusions: Lipoproteins inhibit in vitro PHA induced peripheral blood mononuclear cell proliferation both in diabetic and in non diabetic subjects

Production and characterization of monoclonal antibodies to oxidized LDL

Oxidized low density lipoprotein (LDL) seems to take a part in atherogenesis through direct interactions with macrophages, endothelial cells, and smooth muscle cells, and is thought to participate in renal glomerular injury. For the purpose of illustrating the role of oxidized LDL in the human diseases, monoclonal antibodies were developed and characterized, recognizing oxidized LDL-specific epitopes that do not exist on native LDL. LDL was oxidized by the incubation with CuSO4, and used as immunogen. Splenocytes from the immunized mouse and mouse myeloma cells were fused to produce hybridomas, which were screened for the secretion of oxidized LDL-specific antibodies. Immunoblot analysis and binding affinity assay showed that these monoclonal antibodies recognize malondialdehyde-conjugated peptide epitopes.

Producción de los reactivos primarios de un sistema inmunoenzimático ELISA para determinar lipoproteína (a) / Production of primary reactives of an ELISA immunoenzymatic system to determine lipoprotein (a)

El objetivo de nuestro trabajo fue desarrollar la metodología y la producción de los reactivos primarios para realizar un método inmunoenzimático (ELISA) tipo sandwich para determinar lipoproteina (a) en suero humano. La concentración final del antisuero policlonal antiapolipoproteína (a) (anti-apo [a]) obtenida fue de 1,3 mg/dL, purificado, por cromatografía de afinidad y de intercambio iónico. El antisuero antilipoproteína de baja densidad (anti LDL) de carnero purificado por cromatografía de afinidad fue utilizado en la obtención del conjugado policlonal perxodasa-IgG anti-LDL. La concentración óptima de recubrimiento con anti-apo (a) fue de 2 µg/mL y la dilución óptima del conjugado fue 1/7000, con lo cual obtuvimos una sensibilidad alta del ensayo. Poder producir en nuestro laboratorio reactivos primarios para el desarrollo de un sistema inmunoenzimático de determinación de Lp (a) hace posible la accesibilidad de la determinación con fines asistenciales e investigativos, así como un ahorro en moneda libremente convertible, pues estos reactivos tienen un elevado costo en el mercado

Respuesta inmune contra lipoproteínas de baja densidad modificadas en pacientes con diabetes mellitus no-insulino dependiente / Immune response against modified low density lipoproteins in patients with non insulin dependent diabetes mellitus

LDLs obtainded from blood of healthy subjects, were glycated or altered with malondialbehyde and used as antigens. Serum autoantibodies against these LDLs were measured by ELISA in 22 patients with non insulin dependent diabetes mellitus aged 46 to 67 years old and 13 healthy controls aged 41 to 64 years old. Basal and LDL stimulated tumor necrosis factor production in vitro, by peripheral leukocytes of diabetics and controls was also measured. Results: The ratio of glycated LDL/native LDL antibodies was higher in diabetics than in controls (9.37 ñ 2.72 and 0.41 ñ 0.11 respectively p < 0.05) and the ratio of MDA modified LDL/native LDL antibodies was not significantly different (8.64 ñ 3.83 and 2.14 ñ 1.26 respectively, NS). Tumor necrosis or production by leukocytes was higher in diabetics than in controls in basal conditions (53.3 ñ 15.3 and 26.9 ñ 14.7 arbitrary units (a.u.) respectively), when stimulated withnative LDL (46.5 ñ 5 and 24.3 ñ 9.4 a.u. respectively), when stimulated with malondialdehyde modified LDL (50 ñ 16.2 and 24.4 ñ 7.7 a.u. respectively) or when stimulated with glycated LDL (38.3 ñ 8.8 and 14.4 ñ 7.5 a.u. respectively). Conclusions: Diabetic patients have an enhanced immune response against low density lipoproteins, factor that could contribute to the accelared atherogenesis of this disease

Monoclonal antibodies against low density lipoprotein with various degrees of oxidative modifications

Oxidative processes leading to the generation of oxidized low density lipoprotein (oxLDL) particles have been suggested to be an important factor in the pathogenesis of atherosclerosis. After initiation of the oxidative process, LDL undergoes a progressive protein and lipid fragmentation. To understand this process and the role of oxLDL in various diseases of inflammatory origin, we have generated mouse monoclonal antibodies against copper-oxidized human LDL. Mice were immunized intrasplenically and after one intravenous boost the spleen cells were fused with the Sp2/0 hybridoma fusion partner. The hybridoma clones obtained after selection and cloning were analyzed for reactivity against oxLDL with various degrees of copper-mediated oxidative modifications. Three hybridoma clones were purified and further characterized. The following observations were made: 1) the intrasplenic route of immunization, avoiding the use of mycobacterial adjuvants, yielded a high frequency of positive clones; 2) the individual hybridomas reacted against LDL with various degrees of oxidative modifications; 3) the monoclonal antibodies could be used in ELISA and to detect oxLDL in immunohistochemical tissue staining, and 4) the monoclonal antibodies also detected oxLDL from hamsters and rabbits. We conclude that these monoclonal antibodies could be useful to further investigate the role of oxLDL in inflammation and in the immune response.