1. Ann Biol Clin (Paris). 2012 Nov-Dec;70(6):695-701. doi: 10.1684/abc.2012.0744. Increased number of non-invariant NKT cells and low number of circulating CD1-expressing leukocytes in patients infected with hepatitis C virus. González LR(1), Conesa A, Blanca I, Machado I, Fernández S, Pujol FH, Toro F. Author information: (1)Instituto de inmunología-FCE center, Facultad de medicina, Universidad central de Venezuela, Caracas, Venezuela. Natural killer T (NKT) cells represent an heterogeneous T cell population involved in host immunity against several microorganisms. They also have important immunoregulatory functions. Studies on circulating levels of NKT cells during HCV infection have been focused on the invariant NKT (iNKT) subset which recognizes the non-classical Ag-presenting molecule CD1d, with little information about the non-invariant NKT (non-iNKT) cell subset. In the present study, we assessed the number of both NKT cells subsets and the surface expression of CD1a, b, c and d isoforms in peripheral blood of 31 HCV-infected patients and 31 ages matched healthy individuals. A significant increase of circulating non-iNKT cells was observed in HCV-infected patients as compared to controls (74 ± 57 cells/μL vs 42 ± 16 cells/μL respectively, p<0.0042) with no differences in the iNKT subset. In addition, the percentage of CD1a, CD1c and CD1d-expressing leukocytes was significantly low in patients as compared to controls. These findings suggest that both components, non-iNKT cells and CD1 molecules expression are involved in the control of natural immunity against HCV. PMID: 23207816 [PubMed - indexed for MEDLINE] 2. Ann Biol Clin (Paris). 2012 Mar-Apr;70(2):175-81. doi: 10.1684/abc.2012.0663. Allele and haplotype frequencies at human leukocyte antigen class I and II genes in Venezuela's population. Del Pilar Fortes M(1), Gill G, Paredes ME, Gamez LE, Palacios M, Blanca I, Tassinari P. Author information: (1)Institute of immunology, Universidad central de Venezuela, Caracas, Venezuela. Population studies represent an integral part and link in understanding the complex chain of host-pathogen interactions, disease pathogenesis, and MHC gene polymorphisms. Genes of Mongoloid, Caucasoid, and Negroid populations have created a distinctive HLA genetic profile in the Venezuelan population. Our objective was to determine the predominant HLA class I and II alleles and haplotype frequencies in the hybrid population of Venezuela. The study population consisted of 486 healthy unrelated native Venezuelans and 180 families. We examined the frequency of HLA A-B-C, HLA-DQ and HLA-DR genes by polymerase chain reaction and subsequent hybridization with sequence-specific oligonucleotide probes. Phenotypic, allelic and haplotype frequencies were estimated by direct counting and using the maximum-likelihood method. The predominant HLA class I alleles were A*02, A*24, A*68, B*35, B*44, B*51, B*07, B*15 and Cw*07. Regarding HLA class II, the most frequent alleles were DQB1*03 and DRB1*04, DRB1*15, DRB1*13, DRB1*07. The prevailing haplotype was HLA-A*02B*35 DQB1*03 DRB1*04. Some of these alleles and haplotype frequencies were predominantly present in Amerindians (A*02, A*24, B*35, Cw*07, DRB1*04, A*24 B*35). Previous reports have shown high incidence of A*02, B*44, B*51, DRB1*15, DRB1*13, DRB1*07 alleles in several European populations and A*68, B*07, B*15 alleles in African Americans, which could have contributed to the ethnic admixture of the Venezuelan population. We conclude that our results provide strong evidence that Venezuela's population represents an admixture of the primitive Mongoloid Aborigines, Caucasoid Europeans and Western African Negroid migrants. PMID: 22484528 [PubMed - indexed for MEDLINE] 3. Cell Immunol. 2010;264(1):86-92. doi: 10.1016/j.cellimm.2010.05.002. Epub 2010 May 10. CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells. Márquez ME(1), Millet C, Stekman H, Conesa A, Deglesne PA, Toro F, Sanctis JD, Blanca I. Author information: (1)Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. marmarqu@ivic.ve Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56(dim)CD16(bright) make up 90% circulating NK cells, whereas CD56(bright)CD16(-/dim) comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56(bright) NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-gamma and TNF-alpha and notably IL-12. 2010 Elsevier Inc. All rights reserved. PMID: 20553754 [PubMed - indexed for MEDLINE] 4. Tissue Antigens. 2010 Jun;75(6):724-9. doi: 10.1111/j.1399-0039.2010.01446.x. Epub 2010 Feb 24. Distribution of killer cell immunoglobulin-like receptor genes in the mestizo population from Venezuela. Conesa A(1), Fernández-Mestre M, Padrón D, Toro F, Silva N, Tassinari P, Blanca I, Martin MP, Carrington M, Layrisse Z. Author information: (1)Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, FOCIS Center of Excellence, Caracas, Venezuela. This study represents the first report on the distribution of KIR genes in 205 unrelated healthy mestizo Venezuelan individuals. Genotyping analysis showed that all KIR genes are present in this population. Frequency of inhibitory killer cell immunoglobulin-like receptors (KIRs) exceeded 0.69, except for KIR2DL2 (0.29) and 2DL5 (0.37). Activating KIRs showed low frequencies (0.11-0.29), except for KIR2DS4 (0.68). Forty-five different KIR genotypes were identified, with a predominance of three genotypes found in 50.7% of the population of which 25.9% were individuals homozygous for haplotype A. The frequencies of KIR genes reflect the ethnic admixture existing in the mestizo Venezuelan population. PMID: 20210918 [PubMed - indexed for MEDLINE] 5. J Immunol. 2008 Aug 1;181(3):1927-36. Modulation of T cell activation by stomatin-like protein 2. Kirchhof MG(1), Chau LA, Lemke CD, Vardhana S, Darlington PJ, Márquez ME, Taylor R, Rizkalla K, Blanca I, Dustin ML, Madrenas J. Author information: (1)FOCIS Centre for Clinical Immunology and Immunotherapeutics, Robarts Research Institute, and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada. T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation. PMCID: PMC2913160 PMID: 18641330 [PubMed - indexed for MEDLINE] 6. Mem Inst Oswaldo Cruz. 2004 Mar;99(2):179-84. Epub 2004 Jun 24. Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake. Contreras CE(1), Rivas MA, Domínguez J, Charris J, Palacios M, Bianco NE, Blanca I. Author information: (1)Instituto de Inmunología, Facultad de Medicina, Universidad Central, Apartado Postal 50109, Caracas 1050 A, Venezuela. contrerc@camelot.rect.ucv.ve 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. PMID: 15250472 [PubMed - indexed for MEDLINE] 7. Invest Clin. 2006 Dec;47(4):361-9. Immunophenotype characteristics of peripheral blood mononuclear leukocytes of chronic idiopathic urticaria patients. Garmendia JV(1), Zabaleta M, Aldrey O, Rivera H, De Sanctis JB, Bianco NE, Blanca I. Author information: (1)Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela. garmmenj@ucv.ve 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. PMID: 17176904 [PubMed - indexed for MEDLINE] 8. Allergy Asthma Proc. 2004 Mar-Apr;25(2):121-5. Total and biologically active serum-soluble CD154 in patients with chronic idiopathic urticaria. Garmendia JV(1), Zabaleta M, Blanca I, Bianco NE, De Sanctis JB. Author information: (1)Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracus. 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. PMID: 15176497 [PubMed - indexed for MEDLINE] 9. J Immunol. 2002 Jun 15;168(12):6090-8. IL-2 and IL-12 alter NK cell responsiveness to IFN-gamma-inducible protein 10 by down-regulating CXCR3 expression. Hodge DL(1), Schill WB, Wang JM, Blanca I, Reynolds DA, Ortaldo JR, Young HA. Author information: (1)Laboratories of. Experimental Immunology and Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA. Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-gamma-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression. PMID: 12055219 [PubMed - indexed for MEDLINE] 10. J Immunol. 2001 Dec 1;167(11):6132-9. Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells. Blanca IR(1), Bere EW, Young HA, Ortaldo JR. Author information: (1)Laboratory of Experimental Immunology, Division of Basic Sciences, National Cancer Institute, Frederick, MD 21702, USA. NK cells are a subpopulation of lymphocytes characterized primarily by their cytolytic activity. They are recognized as an important component of the immune response against virus infection and tumors. In addition to their cytolytic activity, NK cells also participate either directly or indirectly in the regulation of the ongoing Ab response. More recently, it has been suggested that NK cells have an important role in the outcome of autoimmune diseases. Here, we demonstrate that human NK cells can induce autologous resting B cells to synthesize Ig, including switching to IgG and IgA, reminiscent of a secondary Ab response. B cell activation by the NK cell is contact-dependent and rapid, suggesting an autocrine B cell-regulated process. This NK cell function is T cell-independent, requires an active cytoplasmic membrane, and is blocked by anti-CD40 ligand (anti-CD154) or CD40-mIg fusion protein, indicating a critical role for CD40-CD40 ligand interaction. Depletion studies also demonstrate that CD5+ B cells (autoreactive B-1 cells) and a heterogeneous population of CD27+ memory B cells play a critical role in the Ig response induced by NK cells. The existence of this novel mechanism of B cell activation has important implications in innate immunity, B cell-mediated autoimmunity, and B cell neoplasia. PMID: 11714772 [PubMed - indexed for MEDLINE] 11. Kidney Int. 1999 Feb;55(2):546-53. Effect of recombinant human erythropoietin on endothelial cell apoptosis. Carlini RG(1), Alonzo EJ, Dominguez J, Blanca I, Weisinger JR, Rothstein M, Bellorin-Font E. Author information: (1)Centro Nacional de Dialisis y Trasplante, Hospital Universitario de Caracas, Venezuela. BACKGROUND: Recombinant human erythropoietin (rHuEPO) induces endothelial cell growth and angiogenesis in vitro. The mechanisms are unknown. Because an increase in endothelial cell survival could play a role in this process, we examined the effect of rHuEPO on lipopolysaccharide (LPS)-induced apoptosis in bovine pulmonary artery endothelial cells (BPAECs). METHODS: Four groups of cells were studied. The first group was preincubated in serum-free medium followed by treatment with LPS. The second group was preincubated with rHuEPO followed by LPS. The third group was treated with only rHuEPO. Control cells were cultured in the absence of rHuEPO and LPS. Apoptosis was determined by flow cytometric DNA analysis, propidium iodide staining, cellular DNA fragmentation by ELISA, and gel electrophoresis. RESULTS: LPS-treated cells showed an increase in hypodiploid DNA (36.4 +/- 6.1%) compared with controls (9.8 +/- 3.3%, P < 0.001). Preincubation with rHuEPO decreased this effect to 14.7 +/- 5.1% (P < 0.001). Apoptosis determined by propidium iodide was observed in 33 +/- 8% of LPS-treated cells, but in only 9 +/- 3% of cells preincubated with rHuEPO cells (P < 0.001). Similarly, DNA fragmentation was decreased in rHuEPO pretreated cells compared with LPS alone (0.155 OD +/- 0.02 vs. 0.538 +/- 0.09 OD, P < 0.001). DNA breakdown was observed in only LPS-treated cells. CONCLUSIONS: These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium. PMID: 9987078 [PubMed - indexed for MEDLINE] 12. Clin Exp Immunol. 1998 Aug;113(2):206-12. Expression of low-density lipoprotein receptors in peripheral blood and tonsil B lymphocytes. De Sanctis JB(1), Blanca I, Rivera H, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Caracas. B lymphocytes, purified from peripheral leucocytes from young normolipaemic humans, expressed and internalized low-density lipoprotein receptors (LDLR). The expression was assessed by a monoclonal anti-LDLR. The internalization of LDL was assessed by LDL labelled with 125I (125I-LDL) and 1,1'-dioctadecyl-3,3,3',3' tetramethyl-indocarboxycyanine perchlorate (LDL-DiI). The expression of LDLR, assessed by anti-LDLR, was: 38 +/- 8% (n = 5) for fresh purified cells, 60 +/- 10% (n = 12) for non-stimulated cells, 79 +/- 5% (n = 10) for IL-2 (100 U/ml)-stimulated cells and 95 +/- 5% (n = 8) for pokeweed mitogen (PWM) (1:200 dilution)-stimulated cells. The optimal concentrations of agonist were 100 U/ml of IL-2, and 1:200 dilution of PWM. IL-2 and PWM increased the internalization of LDL-DiI by 1.5-fold. The internalization of LDL-DiI was maximal at 60 microg of protein/ml (48 +/- 8%). Scatchard analysis revealed a Kd of 3.2 +/- 0.22 x 10(-8) M and 2180 +/- 190 binding sites in non-stimulated cells, a Kd of 7.73 +/- 0.36 x 10(-9) M and 12,500 +/- 430 binding sites for IL-2 (100 U/ml)-stimulated cells, and a Kd of 7.2 +/- 0.43 x 10(-9) M and 13,250 +/- 450 binding sites for PWM (1:200 dilution)-stimulated cells. Lineweaver-Burk analysis of LDL binding (LDL-DiI) revealed that the apparent Kd for non-stimulated cells was 1.3 +/- 0.11 x 10(-8) M, and 9.2 +/- 0.2 x 10(-9) M and 7.5 +/- 0.25 x 10(-9) M for IL-2- and PWM-stimulated cells, respectively. B lymphocytes from tonsils also showed a high expression of LDLR assessed with anti-LDLR (70 +/- 6%). The high expression of LDLR and the avid internalization of LDL suggest that LDL may be important for B cell physiological responses. PMCID: PMC1905048 PMID: 9717969 [PubMed - indexed for MEDLINE] 13. Scand J Immunol. 1998 May;47(5):496-501. Characterization of local memory cells in stage-classified pulmonary tuberculosis: preliminary observations. Urdaneta E(1), Feo-Figarella E, Montalvo C, Tálamo C, Castillo Y, Carrasco D, Rivera H, Blanca I, Machado I, Echeverría de Pérez G, De Sanctis JB, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University Hospital, Central University of Venezuela, Caracas. Immunophenotype analysis and proliferative responses were investigated in bronchoalveolar lavage (BAL) cells from 21 patients with stage-classified tuberculosis: six with localized pulmonary infiltrate (LPI); seven with diffuse pulmonary infiltrate (DPI); and eight with pleural effusions (PE). Bronchoalveolar lavage cells from these patients contained a high number of cells/ml. The macrophage number was significantly lower in the DPI group (P < 0.05) compared to the LPI or PE groups. Conversely, neutrophils were markedly increased in DPI patients compared to LPI (P < 0.01) and PE (P < 0.01) patients. Lymphocyte infiltration (97.7 +/- 2.3% CD3+, > 83% alphabeta+ and CD4+ > CD8+) was observed in the three groups. A significant increase in the number of total lymphocytes (P < 0.01) and CD4+ cells (P < 0.05) was observed in the LPI group compared to the PE group. In the LPI group CD4+CD45RO+ cell infiltration was higher than CD4+CD45RA+ cells (P < 0.001), contrasting to similar numbers of these subpopulations in the DPI group. Lymphocytes from three out of three LPI patients (alphabeta+CD4+CD45RO+) responded against tuberculin purified protein derivative contrasting to the unresponsiveness of five patients with either DPI or PE. This impaired response was reverted in two out of five patients by using peripheral blood monocytes instead of alveolar macrophages. It is suggested that, in humans, alphabetaCD4+CD45RO cells are the main lymphocyte type involved in the initial local cell-mediated immune response against Mycobacterium tuberculosis. PMID: 9627135 [PubMed - indexed for MEDLINE] 14. Clin Sci (Lond). 1997 Nov;93(5):413-21. Nitric oxide in different types of hypertension during pregnancy. Garmendia JV(1), Gutiérrez Y, Blanca I, Bianco NE, De Sanctis JB. Author information: (1)Internal Medicine Department, Maternidad Concepción Palacios Hospital, San Martín, Caracas, Venezuela. 1. Serum nitric oxide (NO) levels (determined by its products of oxidation) were assessed in non-pregnant women, normal pregnant women and patients suffering from mild pre-eclampsia (MPE), severe pre-eclampsia (SPE), chronic hypertension (CHT) and CHT with pre-eclampsia (CHT + PE). The levels of NO products were significantly reduced during pregnancy in MPE (P < 0.001), CHT + PE (P < 0.01) and SPE (P < 0.05). Significant reductions of NO products were also observed in puerperium (P < 0.001) in all groups except CHT + PE (P < 0.05). 2. In normal pregnancy, three events were related to NO levels: (1) negative correlations were found between the levels of nitrite (r = -0.73, P = 0.0003), nitrate (r = -0.53, P = 0.017) and the number of weeks of gestation; (2) in the caesarean section group, the levels of NO at puerperium were significantly lower (P < 0.05) than those during pregnancy; and (3) there was a significant reduction in NO levels in the pregnant women carrying male fetuses as compared with female fetuses (P < 0.05). 3. In SPE, the patients with a family history of hypertension had lower levels of NO compared with the patients without such a history (P < 0.05). 4. A negative correlation was observed between systolic blood pressure, diastolic blood pressure and NO levels in MPE (r = -0.62, P = 0.013 and r = -0.68, P = 0.0049 respectively) and SPE (r = -0.72, P = 0.004 and r = -0.53, P = 0.037 respectively). 5. In SPE, positive correlations were observed between platelet count and nitrite (r = 0.67, P = 0.006) and nitrate levels (r = 0.56, P = 0.028). 6. In MPE, patients with anti-hypertensive treatment showed significantly (P < 0.05) higher levels of NO compared with the non-treated patients. 7. NO may be important in the physiopathology of hypertension during pregnancy, although several factors may affect its levels. PMID: 9486086 [PubMed - indexed for MEDLINE] 15. Immunology. 1997 Apr;90(4):526-33. Secretion of cytokines by natural killer cells primed with interleukin-2 and stimulated with different lipoproteins. De Sanctis JB(1), Blanca I, Bianco NE. Author information: (1)Institute of Immunology, Central University of Venezuela, Caracas, Venezuela. Natural killer (NK) cells were shown to secrete differentially interleukins (IL), IL-1 alpha, IL-1 beta, IL-2, IL-8, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukaemia inhibitory factor (LIF) upon stimulation with optimal concentrations of chylomicrons (CM), very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or acetyl-modified low-density lipoprotein (AcLDL). CM, VLDL, LDL and AcLDL induced LIF secretion which was absent in nonstimulated cells. CM, VLDL, and LDL did not affect IL-1 alpha secretion. CM stimulated IL-8 > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma, and decreased seventeen-fold GM-CSF secretion. VLDL stimulated IL-8 secretion > IL-1 alpha = IL-2 > IFN-gamma > TNF-alpha and decreased fivefold GM-CSF secretion. LDL stimulated IL-8 secretion > IL-1 alpha > IL-2 = IFN-gamma, it did not modify TNF-alpha and inhibited five hundred-fold GM-CSF secretion. HDL stimulated IL-2 secretion = IFN-gamma > IL-8, it decreased GM-CSF secretion > IL-1 alpha > IL-1 beta > TNF-alpha without affecting LIF. AcLDL stimulated IL-8 secretion > TNF-alpha > IL-1 alpha > IL-2 = IFN-gamma = IL-1 beta, and decreased GM-CSF secretion eightfold. When NK cells were primed with 10, 100 or 500 IU/ml of IL-2 before the addition of lipoproteins, a decrease in the secretion of cytokines was observed as compared with cells primed with IL-2 only. Differences in cytokine secretion were observed among the diverse type of lipoproteins used for cell stimulus. Thus, lipoproteins may condition NK cytokine secretion and cell activation. PMCID: PMC1456702 PMID: 9176105 [PubMed - indexed for MEDLINE] 16. Clin Exp Immunol. 1997 Jan;107(1):205-12. Low density lipoprotein receptor expression and function in human polymorphonuclear leucocytes. Lara LL(1), Rivera H, Perez-P C, Blanca I, Bianco NE, De Sanctis JB. Author information: (1)Instituto de Immunología, Facultad de Medecina, Universidad Central deVenezuela, Caracas. Low density lipoprotein receptors (LDLR), capable of internalizing LDL, are expressed in polymorphonuclear neutrophils (PMN). The expression was assessed using anti-LDLR antibody by flow cytometry. The internalization of LDL was assessed by: (i) quantification of the uptake of labelled LDL with 1,1'-dioctadecyl-3,3,3',3' tetramethyl-indocarboxycyanine perchlorate (DiI) by flow cytometry; and (ii) the binding of LDL-125I. In fresh purified cells, Lineweaver Burk analysis of LDL binding (LDL-DiI) revealed that the calculated Kd (internalized LDL) for PMN (15.0 x 10(-9) M) is lower than the Kd for monocytes (1.1 x 10(-7) M) and the Kd for lymphocytes (3.2 x 10(-7) M). Scatchard analysis (LDL-125I) revealed 25,000 binding sites and a Kd of 9.6 x 10(-9) M for PMN. The interaction of LDL with its receptor caused a two-fold fast (peak at 1 min) and transient increase in the oxidative burst, measured by the formation of 2',7' dicholoflurescein (DCF) by flow cytometry. This effect was not observed in monocytes or lymphocytes, and it was blocked by anti-LDLR antibody. The stimulation of LDL was optimal at 10 microg of protein/ml. LDL was able to suppress DCF formation induced by phorbol myristate acetate (PMA) and PMA was unable to further stimulate LDL-treated cells, suggesting protein kinase-C (PKC) involvement in LDL effects. Using a PKC assay, LDL was shown to induce a two-fold increase in PKC translocation to the membrane. Thus, LDL increases PMN oxidative burst through a PKC-dependent pathway. PMCID: PMC1904546 PMID: 9010277 [PubMed - indexed for MEDLINE] 17. J Lipid Res. 1996 Sep;37(9):1987-2000. Regulatory effects of lipoprotein lipase on proliferative and cytotoxic activity of NK cells. De Sanctis JB(1), Blanca I, Bianco NE. Author information: (1)Instituto de Immunologia, Facultad de Medicina, Universidad Central de Venezuela, Caracas, Venezuela. Lipoprotein lipase (LPL) induced, in a dose-dependent fashion, a 2-fold and 11-fold increase in the proliferative response of peripheral blood lymphocytes (PBL) at 48 and 72 h, respectively; a 4- and 12-fold increase in natural killer (NK) cells, respectively; and a maximal 3-fold induction in interleukin-2 (IL-2)-treated NK cells at 72 h. T lymphocytes did not proliferate independently of the concentration of LPL used. LPL decreased the proliferative response of K562 and U937 cell lines. The effect on NK cells could be blocked by anti-LPL if it was added before LPL binding to the cell membrane. Contrary to its effects on NK proliferative response, LPL inhibited spontaneous cytotoxicity and lymphokine-activated killer activity (LAK). The effect was dose-dependent, target-dependent (U937 was more sensitive than K562 in LAK assays), but not LPL-binding time-dependent. Treatment of NK cells with heparinase overcame the inhibitory effect of LPL in spontaneous cytotoxicity. LPL binding to cell membranes, as assessed by flow cytometry, was as follows: K562 cells > monocytes > NK cells > LAK cells > U937 cells, absent in T lymphocytes and partially sensible to heparinase and IL-2 treatments. Protein kinase C translocation was observed upon treatment of NK cells with LPL. Three proteins in NK cell membrane (76, 57.2, and 27.2 kD), two in the cytosol (57.2 and 27.2 kD), and only one in ANA-1 cell membrane (76 kD) were precipitated with LPL-Sepharose. LPL receptors seem to be responsible for the proliferative and cytotoxic response observed in LPL-stimulated NK cells. PMID: 8895065 [PubMed - indexed for MEDLINE] 18. Cell Immunol. 1996 Jan 10;167(1):18-29. Expression and function of low-density lipoprotein receptors in CD3-CD16+CD56+ cells: effect of interleukin 2. De Sanctis JB(1), Blanca I, Radzioch D, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Sabana Grande, Caracas, Venezuela. Low-density lipoprotein receptors (LDLR) have been shown to be expressed, internalized, and transcribed in CD3-CD16+CD56+ cells. Only a low percentage (up to 12%) of NK cells express LDLR. Interleukin 2 (IL-2) (1000 IU/ml) induced a threefold increase in the expression of LDLR on the cell surface that results from, at least in part, augmentation of LDLR turnover from the cytosol to the membrane. Scatchard analysis revealed that IL-2 decreased the Kd of LDLR binding for LDL from 7.53 to 4.33 nM with an increment in the number of binding sites from 2500 up to 5000. Both the proliferative response and cytotoxic functions of these cells are affected by LDL. Low concentrations of LDL induce an increase in the proliferative response (up to eightfold) and in the cytotoxic response of NK cells (up to fivefold). High concentration (more than 60 micrograms/ml) of LDL hampers both proliferative response and cytotoxic activity of NK cells. LDL did not affect the cytotoxic functions of IL-2-activated NK cells. Overall, we have shown that LDLR is expressed on the surface of NK cells and can be augmented by IL-2. Furthermore, we propose some insights into the mechanism responsible for the enhanced expression of LDLR on NK cell surface. In addition, our data clearly delineate that LDLR plays an important role in the regulation of proliferative responses and cytotoxic activity of these cells. PMID: 8548841 [PubMed - indexed for MEDLINE] 19. Immunology. 1995 Nov;86(3):399-407. Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity. De Sanctis JB(1), Blanca I, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Caracas, Venezuela. Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed. PMCID: PMC1383943 PMID: 8550077 [PubMed - indexed for MEDLINE] 20. Clin Sci (Lond). 1995 Nov;89(5):511-9. Effects of different lipoproteins on the proliferative response of interleukin-2-activated T lymphocytes and large granular lymphocytes. De Sanctis JB(1), Blanca I, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Caracas. 1. T lymphocytes and large granular lymphocytes internalized chylomicrons, very low-density lipoprotein, low-density lipoprotein, high-density lipoprotein and acetyl modified low-density lipoprotein through different receptors as assessed by flow cytometry. The observed internalization ranged from 8% to 20%. 2. All lipoproteins induced proliferative responses in T lymphocytes and large granular lymphocytes at optimum concentrations (40 micrograms of protein/ml for all lipoproteins except high-density lipoprotein). Chylomicrons, very low-density lipoprotein and low-density lipoprotein increased T-lymphocyte proliferative response by fourfold while inducing respectively a seven-, nine- and sevenfold increment in large granular lymphocytes. Similarly, high-density lipoprotein and acetyl modified low-density lipoprotein respectively induced a nine- and sevenfold increment in T cells and a 17- and eightfold increment in large granular lymphocyte proliferative response. 3. Both cell types internalized more lipoprotein when they were stimulated with interleukin 2. Chylomicrons and low-density lipoprotein internalization was increased threefold and very low-density lipoprotein internalization twofold, while high-density lipoprotein internalization was unchanged in both cell types. Acetyl modified low-density lipoprotein internalization was fourfold higher in large granular lymphocytes only. 4. The proliferative response of interleukin-2 stimulated cells was different from that of unstimulated cells. Chylomicrons and very low-density lipoprotein induced a sixfold increment in T-cell proliferative response but only a fourfold increment in large granular lymphocytes. Low-density lipoprotein and acetyl modified low-density lipoprotein induced respectively a sevenfold and eightfold increment in T cells and a eightfold and threefold increment in large granular lymphocyte proliferative response. High-density lipoprotein did not affect T-lymphocyte proliferative response while inducing a twofold increase in large granular lymphocytes. 5. Lipoproteins are important in the proliferative response of unstimulated and interleukin-2-stimulated cells. PMID: 8549066 [PubMed - indexed for MEDLINE] 21. Clin Diagn Lab Immunol. 1995 Jul;2(4):404-7. Decreased T-cell proliferative response to common environmental antigens could be an indicator of early human immunodeficiency virus-mediated lymphocyte lesions. Tassinari P(1), Deibis L, Blanca I, Bianco NE, Echeverría de Pérez G. Author information: (1)Instituto de Inmunología, Facultad de Medicina, Universidad Central de Venezuela, Caracas. To evaluate CD4+/CD29+ cells and their responses to different antigens in polar stages of human immunodeficiency virus (HIV) infection, we studied 26 HIV-seropositive carriers (SPCs) and 15 patients with AIDS simultaneously with 20 healthy volunteers (HVs) and 10 seronegative homosexual and bisexual men (SNH). CD3, CD4, CD29, and CD45RA phenotypes were analyzed by two-color flow cytometry. Significant depletion of CD4+ T cells and both memory (CD4+/CD29+) and naive (CD4+/CD45RA+) T-cell subsets was found among SPCs and AIDS patients compared with the numbers of such cells in the HV and SNH groups. Responses to optimal doses of Candida albicans, streptokinase, and tetanus toxoid were explored in peripheral blood mononuclear cells and CD4(+)- and CD4+/CD29(+)-enriched cell populations. In SPCs, the response to C. albicans in peripheral blood mononuclear cells showed a statistically significant diminution compared with the response of HVs (15,308 versus 35,951 cpm). In addition, a significantly reduced response to streptokinase was evident only when cell preparations were CD4+/CD29+ enriched (3,048 versus 10,367 cpm). Furthermore, the SPC group comprised seven responders to at least one antigen and seven nonresponders to any of the selected specific antigens. Absence of a response in these latter patients was independent of the absolute counts of memory and naive T-cell populations. The response to tetanus toxoid, although diminished in SPCs, was not significantly different from that in controls. Our results suggest that defective responses to common environmental antigens, unrelated to the absolute number of CD4+/CD29+ cells, is probably an early indicator of an HIV-induced lymphocyte lesion. PMCID: PMC170169 PMID: 7583914 [PubMed - indexed for MEDLINE] 22. Pharmazie. 1995 May;50(5):337-41. Synthesis of bicyclic ketones in prostaglandins and their antimitotic activity. Domínguez JN(1), Zapata AJ, Lobo GM, Blanca I. Author information: (1)Organic Synthesis Laboratory, Faculty of Pharmacy, Central University of Venezuela, Caracas. The synthesis of a second ring in the prostaglandin structure, located at positions C-11 and C-13, has been accomplished starting from prostaglandin A2. Also an efficient enantioselectivity was obtained through the conjugate addition of carbanions at position C-11, together with the stereospecificity of a Claisen rearrangement at position C-13. Evaluation of the inhibitory effects on the proliferation of the K-562 cell line, in vitro, is presented. A structure-activity relationship indicated that alterations of the functional groups incorporated in the second ring of the prostaglandin structure affected their hydrophobicity. The antimitotic activity for prostanoids 7b, 7c and 7e have shown substantial improvements in their activities according to their ID50 values (12.5, 9.0 and 1.12 micrograms/ml), respectively). Attention is called to the importance of derivative 7a in terms of its high potency, determined by its ID50 values (0.35 micrograms/ml). PMID: 7604067 [PubMed - indexed for MEDLINE] 23. Immunology. 1994 Oct;83(2):232-9. Lipoprotein lipase expression in natural killer cells and its role in their cytotoxic activity. de Sanctis JB(1), Blanca I, Radzioch D, Bianco NE. Author information: (1)Institute of Immunology, Faculty of Medicine, Central University of Venezuela, Caracas. Lipoprotein lipase (LPL) is the key enzyme in the metabolism of triglyceride-rich lipoproteins. The patterns of LPL mRNA expression and secretion of the enzyme have not yet been established in natural killer (NK) cells. We show in the present communication that CD3- CD16+ cells (NK cells) transcribe LPL mRNA, express LPL on the surface and secrete the enzyme. In contrast, there is no LPL expression on the surface of highly purified B and T lymphocytes. Stimulation of NK cells with interleukin-2 (IL-2) reduced the expression of LPL on their surface and augmented the secretion of LPL by the cells. The addition of anti-LPL antibodies to NK cells in culture led to a complete abrogation of cytotoxicity of NK cells against the K562 tumour cell line. Furthermore, IL-2 stimulation of effector cells reversed the anti-LPL antibody-induced inhibition of cytotoxic activity. Overall, these findings suggest that LPL plays a key role in the cytotoxic activity of NK cells. PMCID: PMC1414934 PMID: 7835940 [PubMed - indexed for MEDLINE] 24. J Pharm Sci. 1994 Apr;83(4):472-5. Synthesis of C(10)-halogenated prostaglandins. Dominguez JN(1), Taddei A, Cordero M, Blanca I. Author information: (1)Facultad de Farmacia, Universidad Central de Venezuela, Caracas. The synthesis of halogenated prostaglandins at position C(10), starting from prostaglandin A2, has been accomplished, as well as an efficient regioselective hydroxylation of the upper chain of the prostanoid structure. Evaluation of the inhibitory effects on the proliferation of the K-562 cell line in vitro is presented. When the prostaglandin was modified in the upper chain, the antimitotic activity for bromo derivatives 4b, c and iodo derivative 5b had shown substantial improvements in their activities according to their ID50 values (28, 25, and 22 micrograms/mL, respectively). Attention is called to the significance of chloro derivative 3a in terms of its high potency, determined by its ID50 value (0.06 micrograms/mL). PMID: 7913963 [PubMed - indexed for MEDLINE] 25. Farmaco. 1992 Dec;47(12):1487-94. Synthesis of modified prostaglandins and their inhibitory activity on natural killer cells. Dominguez JN(1), Flamerich J, Blanca I. Author information: (1)Facultad de Farmacia, Universidad Central de Venezuela, Caracas. PMID: 1294165 [PubMed - indexed for MEDLINE] 26. Clin Exp Immunol. 1992 Apr;88(1):143-8. Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2. Caldera LH(1), Leon-Ponte M, Acquatella G, Bianco NE, Blanca I. Author information: (1)Clinical Immunology Centre, University Hospital, Venezuela. Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma. PMCID: PMC1554352 PMID: 1373350 [PubMed - indexed for MEDLINE] 27. Am Rev Respir Dis. 1991 Mar;143(3):496-500. Cellular immunity in current active pulmonary tuberculosis. Andrade-Arzabe R(1), Machado IV, Fernandez B, Blanca I, Ramirez R, Bianco NE. Author information: (1)Clinical Immunology National Center, Ministry of Health, Caracas, Venezuela. A group of 10 patients with recently diagnosed pulmonary TB were studied and compared to 10 bacillus Calmette-Guérin (BCG) immunized healthy individuals. Cellular immune mechanisms were explored in vitro utilizing fresh and precultured peripheral blood mononuclear cells exposed to PHA, PPD, and recall antigens (SK/SD and CA). Proliferative assays were also carried out in the presence of either each patient's serum (autologous serum) or cocultured with CD3(+)-depleted adherent cells. Serum measurements of soluble interleukin-2 (IL-2) receptor and synthesis of IL-2 generated by mononuclear cells stimulated with PPD and SK/SD were also performed. Patient sera were able to inhibit autologous as well as allogeneic cell responses, and a significant adherent cell suppressive effect was observed. As a whole the group of patients showed decreased blast transformation to PPD, preserved proliferative responses to other recall antigens, and a low PPD-induced generation of IL-2. Furthermore, as possible evidence of preactivated T cells, these patients demonstrated high soluble IL-2 receptor serum levels. Early compromise of specific cell-mediated immunity, including IL-2 abnormalities, may be of significance in newly diagnosed pulmonary TB. PMID: 2001056 [PubMed - indexed for MEDLINE] 28. Bull Pan Am Health Organ. 1989;23(1-2):68-75. Immunopathogenic aspects of infection by the human immunodeficiency virus in Venezuela. Echeverría de Pérez G, Deibis L, Silvia García C, Olaria T, Márquez M, Blanca I, Bianco NE. A study of the immunopathogenic characteristics of HIV infection was begun in 1984 at the National Reference Center on Clinical Immunology (CNRIC) in Caracas, Venezuela, on 240 individuals with a variety of clinical manifestations. The most important findings were depletion of the CD4 cells in HIV-infected individuals, including asymptomatic carriers; significant reduction of the CD3-, CD16+ large granular lymphocytes (LGL) in AIDS cases; decrease in LGL cytotoxic activity in infected persons versus controls, along with increased lytic function induced by stimulation with recombinant interleukin-2 in both groups; and reduction of the CD4 population in AIDS patients, independent of the presence or absence of free serum antigen. Such research is helping to clarify the immunopathogenic mechanisms of HIV and possible geographic and demographic variations. PMID: 2785832 [PubMed - indexed for MEDLINE] 29. Rev Inst Med Trop Sao Paulo. 1988 Nov-Dec;30(6):400-5. Immunopathology of human schistosomiasis mansoni. II. NK activity and stimulation by specific antigen. Benarroch LK, Noya O, Noya B, Bianco NE, Blanca I. PMID: 3150866 [PubMed - indexed for MEDLINE] 30. Bol Oficina Sanit Panam. 1988 Nov-Dec;105(5-6):551-60. [Immunopathogenic aspects of infection by the human immunodeficiency virus in Venezuela]. [Article in Spanish] Echeverría de Pérez G, Deibis L, García CS, Olaria T, Marquez M, Blanca I, Bianco NE. PMID: 2977556 [PubMed - indexed for MEDLINE] 31. Cell Immunol. 1985 Oct 15;95(2):349-57. Cytotoxic activity of normal and Herpesvirus saimiri-transformed nonhuman primate cells. Ortaldo JR, Neubauer RH, Blanca I, Rabin H. Owl monkey mononuclear cells were separated from peripheral blood by centrifugation on Ficoll gradients, removal of adherent cells, and subsequent separation on discontinuous Percoll gradients. Lymphocytes recovered from the various fractions were tested for cytotoxic reactivity immediately after isolation. Low-density cells, enriched in large granular lymphocytes (LGL), demonstrated cytotoxic activity against the human natural killer-susceptible cell lines MOLT 4 and K562. In addition, IL-2-independent T-cell lines which had been obtained by immortalization with the primate herpesvirus Herpesvirus saimiri showed cytotoxicity, even after prolonged culture in vitro, similar to that demonstrated by fresh LGL. Cytotoxic activity of these lines was regulated by IL-2 in a fashion which appeared to be independent of the growth-promoting effects of this lymphokine. These results indicate a function for IL-2 beyond its role in supporting cellular proliferation. Cytotoxic activity could also be demonstrated in culture fluids from one of these cell lines (70N2). In addition, these results indicate the usefulness of immortalized cell lines (like 70N2) as a potential source for studies of the biochemical characterization and purification of supernatants containing cytotoxic factors. PMID: 2994888 [PubMed - indexed for MEDLINE] 32. J Clin Immunol. 1987 Sep;7(5):356-64. Helper activity by human large granular lymphocytes in in vitro immunoglobulin synthesis. Rodriguez MA(1), Blanca I, Baroja ML, Arama S, Leon-Ponte M, Abadi I, Bianco NE. Author information: (1)Centro Nacional de Enfermedades Reumaticas, Ministerio de Sanidad y Asistencia Social, Caracas, Venezuela. In the present study we have examined the effect of human large granular lymphocytes (LGL) from healthy donors on Ig synthesis by autologous B lymphocytes. The results showed that this cell population has a consistent helper activity in pokeweed mitogen-activated cultures even when added at very low numbers. LGL can mediate their effect by secreting soluble helper factors capable of modulating B-cell responses as evidenced by the enhancement of IgG and IgM production by supernatants obtained from LGL cultures. Preincubation with interferon gamma further potentiated the helper activity by LGL. PMID: 2958493 [PubMed - indexed for MEDLINE] 33. J Immunol Methods. 1985 Aug 2;81(2):207-14. The proliferation and function of human mononuclear leukocytes and natural killer cells in serum-free medium. Brown RL, Ortaldo JR, Griffith RL, Blanca I, Rabin H. We recently developed a serum-free (SF) culture medium that supports the growth of several established lymphoid cell lines. In an effort to develop a standardized medium for assay of human natural killer (NK) cell activity, we compared the cytotoxic activity of peripheral blood mononuclear leukocytes (PBL) and purified large granular lymphocytes (LGL) cultured in SF medium containing interleukin 2 (IL-2) or medium containing 10% fetal bovine serum (FBS) plus IL-2. The results indicated that PBL had a 30% increase in cumulative net cell growth and had as high or higher cytotoxic activity after growth in SF medium than in medium containing FBS. Purified LGL had a 50% increase in cumulative net cell growth and persisted approximately 2 weeks longer in culture in medium containing FBS than in SF medium. However, the cytotoxic activity of cells grown in SF medium persisted during the initial 3 weeks of culture. Purified LGL that were maintained and were subcultured at cell densities of 10(6) cells or greater per milliliter of either SF or FBS-containing medium had equivalent levels of cytotoxicity over a 44-day period in either medium compared with cells subcultured at a density of 5 X 10(5) cells per milliliter of medium. NK cells produced a cytotoxic factor (NKCF) in SF medium, and its cytotoxic activity was blocked by 10% FBS. We conclude that the SF medium supplemented with IL-2 can be used as an alternative to FBS-containing medium with IL-2 for the growth of NK cells and is advantageous for the production of NKCF. PMID: 3874913 [PubMed - indexed for MEDLINE] 34. Nat Immun Cell Growth Regul. 1985;4(1):48-59. Human natural killer cytotoxic factor. Studies on its production, specificity, and mechanism of interaction with target cells. Blanca I, Herberman RB, Ortaldo JR. Human peripheral blood lymphocytes cultured in vitro for 2 days in serum-free conditions produced a natural killer (NK) cytotoxic factor (NKCF) which selectively killed NK-susceptible targets. Optimal release of NKCF was achieved under serum-free conditions, while the presence of fetal calf serum inhibited both the production and activity of the factor. Mechanistic studies with NKCF demonstrated that the factor could be adsorbed by the target cells within 6 h, with no further exposure to NKCF required for maximal levels of lysis of the treated targets after additional 30-48 h of incubation, as assessed by a 111I release microcytotoxicity assay. NKCF adsorption to target cells and its cytotoxic activity were inhibited by some phosphorylated sugars (mannose-6PO4 and glucose-6PO4), but not by fructose-6PO4 or nonphosphorylated sugars (mannose, glucose, galactose). These results suggest a role of sugar-6PO4 at the level of interaction of NKCF with NK target cells. This was further supported by the finding that inhibition of target cell glycosylation by tunicamycin also inhibited absorption of NKCF to the target cells and direct killing by NKCF. Therefore, it appears that NKCF is a large granular lymphocyte produced factor which produces lysis as a result of the interaction with glycosylated structures on target cell membranes. Purification studies were performed to begin biochemical characterization of human NKCF. The results indicated that NKCF has an apparent molecular weight between 20,000 and 40,000 dalton. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action. The ability to elicit NKCF under serum-free conditions should facilitate its testing, purification, and biochemical characterization. PMID: 4033671 [PubMed - indexed for MEDLINE] 35. Adv Exp Med Biol. 1985;184:203-20. Studies of human natural killer cytotoxic factor (NKCF): characterization and analysis of its mode of action. Ortaldo JR, Blanca I, Herberman RB. Soluble natural killer cytotoxic factors (NKCF) have been detected in the supernatant of normal mouse, rat, and human lymphocytes stimulated in vitro for 1 to 3 days in serum-free medium. Stimulation of large granular lymphocytes (LGL) with NK-sensitive targets or mitogens has resulted in high levels of NKCF production. Previous studies in the mouse and human systems have analyzed the cells responsible for production, specificity, and general characteristics of NKCF. In the present study, using human NKCF as a model for cytolysis by LGL, we have analyzed a variety of agents previously demonstrated to inhibit NK activity. These have included: (i) phosphorylated sugars; (ii) protease inhibitors; (iii) antibodies to rat LGL granules; (iv) Ca++, and Mg++; (v) lipomodulin; (vi) nucleotides; (vii) prostaglandins; and (viii) inhibitors of lysosomal enzymes. All inhibitors were tested for their effects on production of NKCF after target cell interaction, binding of NKCF to target cells, and target cell lysis (after 6-hour NKCF absorption and washing of targets). Phosphorylated sugars and antibodies to rat LGL granules were found to inhibit the lysis of targets by NKCF, whereas the other agents tested had no detectable effect (ATP, cyclic AMP, protease inhibitors, prostaglandin E2). In regard to the production of NKCF, the data indicated that (i) the absence of calcium and magnesium, (ii) prostaglandin E2, and (iii) ATP inhibited production, whereas phosphorylated sugars did not. Studies with these types of agents will now enable us to dissect the sites at which these agents function within the lytic process. In addition to the above studies, purification studies were performed using tritiated arginine to label NKCF to begin biochemical characterization of human NKCF. The results indicated that radiolabeled NKCF has an apparent molecular weight between 20,000 and 40,000. This material demonstrated a pattern of binding to target cells which was similar to the pattern of lysis by NKCF. In addition, the binding of this material was competitively inhibited by unlabeled NKCF preparations. Such approaches with radiolabeled NKCF should be useful for the further study of the biochemical characteristics of human NKCF and of its mechanism of action. PMID: 3898750 [PubMed - indexed for MEDLINE] 36. J Natl Cancer Inst. 1984 Jul;73(1):1-6. Failure of cell-mediated effector mechanisms in lung cancer. Feo Figarella E, Morillo F, Blanca I, Bianco NE. The status of cell-mediated effector mechanisms was studied in 28 patients with lung cancer (25/28 in stage III). Patients' precultured peripheral blood mononuclear leukocytes, isolated by Ficoll-Hypaque, were tested for lymphocyte proliferation responses to alloantigens in mixed lymphocyte culture (MLC). The influence of autologous patients' sera was studied further on MLC responses from patients and controls. Cell-mediated lympholysis (CML), with the use of allogenic blast cells as targets, and antibody-dependent cell-mediated cytotoxicity (ADCC) against human red blood cells also were tested. Major differences between the cancer patients and controls were not demonstrated by MLC. Inhibition or enhancement of MLC responses by the autologous serum was shown; bimodal influence was significant; 72% of the sera caused inhibition and 28% caused enhancement. CML was depressed in 54% of the patients, and ADCC was depressed in 50%. The decrease in both cytotoxic responses was significant (P less than .005). Thirteen patients died after initiation of the investigative protocol; in 11 of 13, CML or ADCC was diminished. The altered cytotoxic capabilities were more prevalent among the epidermoid type, including the deceased patients. This study provides evidence that a severe impairment of cell-mediated effector mechanisms is frequent in advanced lung cancer and may be associated with poor clinical course and with the histologic type. PMID: 6330420 [PubMed - indexed for MEDLINE] 37. Am J Surg. 1984 Mar;147(3):334-8. Characterization of cell-mediated immunity in long-term survivors of gastric or colorectal cancer. Machado IV, Ruíz Diez C, Blanca I, Bianco NE. Cell-mediated immunity was assessed in 12 patients who were long-term survivors of gastric and colorectal adenocarcinomas. A slight decrease in the T-lymphocyte count was accompanied by preserved proliferative reactivity to mitogens (phytohemagglutinin) or alloantigens in 75 percent of the patients. The influence of autologous patient serum on in vitro lymphoproliferative test results was not significant. Selected sera from both study groups showed values of immune complexes that were within the normal range. The colorectal cancer group had antibody-dependent cellular cytotoxicity within the ranges already established for the normal control subjects. Cellular immune mechanisms seem to have been well preserved in long-term survivors of gastric or colorectal carcinoma. PMID: 6608279 [PubMed - indexed for MEDLINE] 38. Cancer. 1982 May 1;49(9):1810-6. Immunology of human gastric cancer: a preliminary report. Blanca I, Grases PJ, Matos M, Contreras CE, Ochoa M, Wright H, Bianco N. The immunological spectrum in fifteen patients with gastric cancer is presented. Patients were divided in three groups. Those with nonadvanced cancer, those with advanced but resectable lesions and those with advanced but nonresectable tumors. Preoperatively, elevated levels of circulating immune complexes (CIC) associated with hyporesponsiveness to phytohemagglutinin (PHA) and in mixed lymphocyte culture (MLC) as well as a positive leukocyte inhibitory serum factor (LIF-S) were found in nearly half of the patients. Inhibitory or enhancing autologous serum factors were detected. Postoperatively, immunologic parameters return to normal in patients with nonadvanced cancer, while in advanced cancer, antibody and cell-mediated immune response remained altered, with some changes associated with chemotherapy. These findings are probably related with the presence or absence of tumor and offer a distinct approach in evaluating the immunologic response of a tumor-bearing patient. PMID: 6210428 [PubMed - indexed for MEDLINE] 39. Int Arch Allergy Appl Immunol. 1982;69(1):7-11. Cell-mediated effector mechanisms in aging humans. Marcano NB, Rivas A, Figarella EF, Blanca I, Penchaszadeh GK, Pérez-Rojas G, Bianco NE. Specific and nonspecific cell-mediated effector mechanisms have been simultaneously assayed in 15 aged humans. 8 were female and 7 male, including a 114-year-old male in remarkably good health. Proliferative response to alloantigens, the generation of T killer cells and the ability to express cell-mediated lympholysis as well as the presence of natural cell-mediated cytotoxicity against K562 tumor cell line and the capacity to mount an ADCC response to RhD+ human red blood cell sensitized with anti-D antisera, revealed that in the human aged, while T function significantly declines, nonspecific cell-mediated effector mechanisms are operative. PMID: 6980843 [PubMed - indexed for MEDLINE] 40. J Natl Cancer Inst. 1979 Jan;62(1):83-8. Influence of inoculation site on development of the Lewis lung carcinoma and suppressor cell activity in syngeneic mice. Malavé I, Blanca I, Fuji H. The growth of the Lewis lung carcinoma (3LL) was studied in syngeneic C57BL/6 mice inoculated sc with similar numbers of tumor cells in either the flank or the hind footpad (fp). After injection of small numbers of 3LL cells, the incidence of tumors was lower in the flank than in the fp. However, after a successful 3LL transplant, tumors in the flank progressed faster than those in the fp, as evidenced by the early metastatic dissemination to the lungs and the shorter survival of the hosts. Local adoptive transfer tests demonstrated the early appearance of suppressor cell activity in spleens from mice bearing tumors in the flank. Adult thymectomy as well as treatement with antithymocyte serum after the tumor transplant inhibited the growth of a flank tumor but did not modify significantly that of an fp tumor. Thus variations in the site of a subcutaneous tumor implant resulted in differences in tumor development that appeared to depend on the characteristics of the immune response elicited by the inoculum. PMID: 281579 [PubMed - indexed for MEDLINE] 41. Clin Immunol Immunopathol. 1978 Aug;10(4):389-97. Changes in the development of the 3LL tumor in syngeneic mice in relation to protein calorie malnutrition. Blanca I, Malavé I. PMID: 99281 [PubMed - indexed for MEDLINE] 42. Int Arch Allergy Appl Immunol. 1978;56(2):128-35. Immune response in malnutrition. Effect of protein deficiency on the DNA synthetic response to alloantigens. Malavé I, Németh A, Blanca I. To evaluate the DNA synthetic response to alloantigens by lymphocytes from mice fed diets with normal (27%) and low (8%) protein content, we quantitated the uptake of 125IUDR in spleens of lethally irradiated adult F1 hybrids injected with cells with parental C57BL/6 donors kept on either diet. Thymocytes, spleen and lymph node cells from protein-restricted parental mice synthesized higher amounts of DNA than those from normally fed controls. Added thymocytes from either malnourished or normal F1 hybrids had similar inhibitory effects on the response of an active parental inoculum. The results indicate that cells from mice kept on a moderately protein-deficient diet for 3--5 weeks after weaning, have increased capacity to proliferate in response to alloantigens, suggesting a high proportion of reactive cells and/or changes in the ratio of the interacting cell populations in their lymphoid organs. PMID: 621113 [PubMed - indexed for MEDLINE]