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Biomedical subjects

L Larrad

Publications and source records attributed to L Larrad.

At least 19 recordsLinked to original sources
PubMed2006

Generation of rabbit antibodies against death ligands by cDNA immunization.

Specificity problems, especially in immunoblot analysis, have been shown for several commercial antibodies raised against the death ligand Fas ligand (FasL) using conventional protein and/or peptide immunizations. In this work, we have optimized the development of rabbit antisera and isolated pAb against the death ligands FasL, Apo2 ligand/TRAIL and Apo3 ligand/TWEAK by cDNA intramuscular immunization. This alternative approach has generated specific pAb in all three cases, which are useful for immunoblot purposes. The present data suggest that for the production of antibodies against certain glycosylated membrane proteins, cDNA immunization could be the method of choice.

Animals↗
PubMed2004

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) cells develop resistance to nucleoside analogs over time. This chemoresistance may be caused by selection for B-CLL cells with defects in the particular apoptosis pathway triggered by these drugs. Therefore, anticancer agents that induce apoptosis through alternative pathways might be useful in treating chemoresistant B-CLL. Farnesyltransferase inhibitors (FTIs) are a class of synthetic drugs with definite molecular targets, which have demonstrated cytotoxicity against leukemic cell lines. We have studied the ex vivo effect of the FTI BMS-214662 on cells from 18 patients with B-CLL. Low concentrations (<1 microM) of BMS-214662 prevented farnesylation of the chaperone marker HDJ-2 and had no effect on Akt activation. BMS-214662 induced apoptosis in B-CLL cells from all patients studied, including those showing resistance to cladribine and fludarabine ex vivo and in vivo. Treatment with BMS-214662 induced loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, proapoptotic conformational changes of Bax and Bak, reduction in Mcl-1 levels and activation of caspases 9 and 3. The general caspase inhibitor Z-VAD-fmk did not prevent BMS-214662-induced cell death. These results indicate that BMS-214662 may be a useful drug for treating B-CLL and, in particular, an alternative for the therapy of purine analog-resistant or relapsed B-CLL.

Alkyl and Aryl Transferases↗
PubMed2002

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia.

We have evaluated the role of caspases and the mitochondrial apoptosis inducing-factor (AIF) in apoptosis induced by cladribine (2CdA), in vitro, in cells from patients of B-CLL and in peripheral blood lymphocytes from normal donors. In sensitive B-CLL cells, apoptosis was characterized by cell shrinking, loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, activation of caspases 3, 7, 8 and 9, reduction of Mcl-1 levels, translocation of AIF from mitochondria to nucleus and chromatin condensation. No significant variations in the levels of Bcl-2, Bax and Bak proteins were noticed upon treatment with 2CdA. Co-treatment of cells with the pan-caspase inhibitor Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis and delayed 2CdA-induced DeltaPsi(m) loss, but did not prevent cell death. Z-VAD-fmk did not prevent 2CdA-induced AIF translocation but in this case apoptotic cells displayed only peripheral chromatin condensation, characteristic of AIF action. Reduced or negligible caspase 3 expression did not prevent 2CdA toxicity in cells from four patients. Cells from three patients that responded poorly to 2CdA lacked expression of caspases 9 or 3. Cells from another patient resistant to 2CdA expressed caspases 3, 7, 8 and 9 but they were not activated by treatment. These results indicate that execution of apoptosis is carried out independently by AIF and caspases, which are responsible for the development of apoptotic phenotype in response to 2CdA. Although caspases can also collaborate in DeltaPsi(m) loss, proapoptotic proteins from the Bcl-2 superfamily may be the key inducers of DeltaPsi(m) loss and apoptosis in B-CLL cells sensitive to 2CdA.

Aged↗
PubMed2001

Differential secretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand-carrying microvesicles during activation-induced death of human T cells.

Preformed Fas ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL) are stored in the cytoplasm of the human Jurkat T cell line and of normal human T cell blasts. The rapid release of these molecules in their bioactive form is involved in activation-induced cell death. In this study, we show by confocal microscopy that FasL and APO2L/TRAIL are mainly localized in lysosomal-like compartments in these cells. We show also by immunoelectron microscopy that FasL and APO2L/TRAIL are stored inside cytoplasmic compartments approximately 500 nm in diameter, with characteristics of multivesicular bodies. Most of these compartments share FasL and APO2L/TRAIL, although exclusive APO2L/TRAIL labeling can be also observed in separate compartments. Upon PHA activation, the mobilization of these compartments toward the plasma membrane is evident, resulting in the secretion of the internal microvesicles loaded with FasL and APO2L/TRAIL. In the case of activation with anti-CD59 mAb, the secretion of microvesicles labeled preferentially with APO2L/TRAIL predominates. These data provide the basis of a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation and could modify the interpretation of the role of FasL and APO2L/TRAIL as effector mechanisms in physiological and pathological situations.

Antibodies, Monoclonal↗
PubMed2001

Determination of the immunoglobulin E postoperative variation as a measure of surgical injury.

The aim of this study was to ascertain postoperative changes in immunoglobulin E (IgE) in patients undergoing different types of surgery and the possible correlation with the duration and type of surgery. Evidence suggests that surgery induces a predominant activation pattern through the T-helper-2 (Th2) cell pathway, increasing interleukins (IL-4, IL-5, IL-10, IL-13), inhibiting Th1 cell activation, and promoting B and Th2 cell activation. IgE production may indicate predominant Th2 pathway activation and may be a more persistent and easily measurable postoperative marker than IL-6 for measuring surgical trauma. Altogether, 180 patients undergoing different types of surgery for nonneoplastic and nonparasitic diseases were studied. All patients received the same type of anesthesia. Before surgery and on the first (1PO) and 7th (7PO) postoperative days we determined in peripheral blood the CD3, CD4, CD8, CD16, and CD19 cell percentages; IL-1, IL-2, IL-4, IL-6, and tumor necrosis factor (TNF) levels; and the IgA, IgG, IgM, total IgE, C3, C4, and CIC levels. On 1PO, all variables decreased except IgE, IL-1, IL-2, IL-4, IL-6, CIC, and CD19. Only IgE, IL-6, and CD19 increases showed a significantly statistical (ss) difference regarding preoperative values (0.01, 0.05, 0.001, respectively). Relations between the IL-4 and IgE increases (p < 0.01) and between the IgG decrease and IgE increase (p < 0.001) were found. On 7PO, only IgE was increased (p < 0.001). The IgE increase correlated with surgical trauma intensity (p < 0.05). We concluded that IgE increases during the early postoperative period, correlating with surgical injury intensity. The increase in the IgE level may be detected 24 hours after surgery and during the first 7 postoperative days depending on the type of surgery.

Adult↗
PubMed2000

CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells.

Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.

Amino Acid Sequence↗
PubMed2000

Autologous blood transfusion as an immunomodulator in experimental sepsis.

BACKGROUND: Homologous blood transfusion is associated with immunosuppressive consequences. Some clinical and experimental studies have suggested an immunostimulating action of autologous blood transfusion. The aim of this paper is to ascertain the effects of either homologous blood transfusion or autologous blood transfusion on the lymphocyte subsets and cytokines in a model of intra-abdominal sepsis. MATERIALS AND METHODS: There were three study groups. Group A: 10 Wistar-Furth (WF) rats underwent cecal ligation and puncture (CLP) aimed at causing an intra-abdomial sepsis; Group B: 10 WF rats underwent CLP plus 1 ml homologous blood perioperative transfusion obtained from Fisher-344 rat while Group C: 10 WF rats underwent CLP plus 1 ml autologous blood perioperative transfusion. Changes of peripheral lymphocyte subsets, percentages of total T-lymphocytes (CD3), Helper T-lymphocytes (CD4), supressor/cytotoxic T-lymphocytes (CD8), CD4/CD8 ratio, Interleukin-2 receptor expression (IL-2R) and cytokines IL-1 and TNF-alpha were measured in peripheral blood on the preoperative, 1st, 3rd and 7th postsepsis (PO) days. RESULTS: Rats in homologous transfused group showed a decrease of %CD4 on the 3rd PO (from preoperative to 3rd PO;p < 0.01; and from 1st to 3rd PO; p < 0.05) and on the 7th PO (from preoperative to 7th PO; p < 0.05); %CD8 increased from preoperative to 3rd PO (p < 0.05), from 1st to 3rd PO (p < 0.01) and from 1st to 7th PO (p < 0.05). An initial decrease on day 1 (p < 0.01) followed by an increase on the 3rd PO (p < 0.01) with regard to IL-2R and a significant increase of IL-1 levels within the first 24h (p < 0.01). Rats in autologous transfused group showed an increase of %CD3 from preoperative to 7th PO (p < 0.05), and from 3rd to 7th PO (p < 0.01). CONCLUSIONS: We observed that homologous blood transfusions induce a greater alteration in the cellular immune response and of the cascade of cytokines than autologous transfusions. This modulates the variations of the immune response induced by sepsis.

Animals↗
PubMed2000

S-adenosylmethionine immunomodulator treatment in sepsis.

BACKGROUND: S-adenosylmethionine (SAMe) is a forerunner of glutathione. AIMS: The aim of the present study is to ascertain this drug effect on T-lymphocytes and cytokines in an experimental model of surgical sepsis. METHODS: Rats were allotted in two groups. In the control group, rats underwent anaesthesia and laparatomy with cecal ligation and puncture (CLP). In the second group, rats underwent the same CLP and received SAMe (14 mg/kg) i.m., on the 1st (1PO) and 2nd (2PO) postoperative days. A week before surgery (PRE), on the 1PO and on the 3PO: IL-1, IL-2, IL-4, IL-6, IL-10 and TNF levels (ELISA & MoAb), and CD3, CD4, CD8 cell and IL-2R percentages (%) (flow cytometry & MoAb) were determined in peripheral blood. RESULTS: Rats receiving SAMe do not show changes of CD3%, CD4% and IL-1 levels but show a significant increase of CD8% on the 3PO, showing a significant difference with regard to controls (p < 0.01). Both groups show a similar IL-2R variation pattern: increasing on 1PO (p < 0.05) and decreasing on 3PO (p < 0.05). CONCLUSION: In sepsis: SAMe inhibits the decrease of circulating immune-active cells and the IL-1 increase. This drug seems to have effects useful in avoiding immunological alterations in sepsis, that need to be tested in humans.

Adjuvants, Immunologic↗
PubMed1999

ITIH4 serum concentration increases during acute-phase processes in human patients and is up-regulated by interleukin-6 in hepatocarcinoma HepG2 cells.

The serum concentration of the inter-alpha trypsin inhibitor heavy chain 4 protein (ITIH4) increases (from 1.4-3 times) in male patients suffering of different acute-phase processes (myocardial infarction, unstable angina or programmed surgery). The concentration of C-reactive protein (CRP) in these samples ranged from 15 microg/ml to 133 microg/ml. Using the hepatocarcinoma HepG2 cell line we have observed up-regulation of ITIH4 mRNA expression upon dose-response treatments with interleukin-6 (IL-6). This effect correlates with the increase of radiolabeled ITIH4 in the cellular media of (35)S-labeled HepG2 cells treated with the cytokine. A similar effect was observed for haptoglobin mRNA, used as a control for acute-phase protein expression. IL-1beta, although up-regulating the expression of alpha(1)-acid glycoprotein in these cells, did not induce any effect in the expression of ITIH4. No changes were observed after TNF-alpha treatments. The results presented here indicate that ITIH4 is a type II acute-phase protein in humans.

Acute-Phase Reaction↗
PubMed1999

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (</=1 h) after PHA stimulation, well before the cell enters apoptosis. FasL- and APO2L-containing vesicles are also present in supernatants from PHA-activated fresh human PBMC. These observations provide the basis for a new and efficient mechanism for the rapid induction of autocrine or paracrine cell death during immune regulation.

Amino Acid Sequence↗
PubMed1999

Study of HLA as a predisposing factor and its possible influence on the outcome of multiple sclerosis in the sanitary district of Calatayud, northern Spain.

The relationship between multiple sclerosis (MS) and the HLA antigens DR2 and DQ1 is well recognised, but, in Spain, it has not been clearly defined. The aim of our study was to investigate the relationship between MS and HLA antigens in the sanitary district of Calatayud, northern Spain, and to correlate these antigens with the progression of the disease. Thirty-four patients were selected from a long-term (October 1990 to July 1996) prospective survey in the region where there was a prevalence rate of 58 per 100,000 population. The HLA antigens were determined in 31 patients. A control group of 895 people of Caucasian race was recruited from the same population. We performed serologic tests on all participants. Nucleotide typing was carried out in DR2-positive patients. The most frequent antigens in excess in MS were: A19 (odds ratio, OR: 2.29, p = 0.04), B5 (OR: 2.85, p = 0.02), B41 (OR: 7.65, p = 0.04), CW7 (OR: 3.4, p = 0.004), DR6 (OR: 6.18, p = 0.0001) and DR10 (OR: 3.4, p = 0. 004). The DR2 antigen was also more frequent in MS patients (39%) than in controls (19%; OR: 2.69, p = 0.01). All positive DR2 patients showed the DR15(2) split but not the DR16(2) split. The frequency of antigens CW4 and DR1 was lower in MS patients than in controls. The CW4 antigen was detected in 12% of the patients and in 33% of the controls (OR: 0.28, p = 0.04). The DR1 antigen was found in 20% of the controls and in none of the MS patients (OR: undefined, p = 0.01). The DQ1 antigen was observed in 68% of the patients and in 50% of the controls (OR: 2.1, p = 0.07). We did not find any relationship between HLA antigens and progression of the disease. Although we found that DR2 antigen is linked to MS, we also found other antigens related to the disease. This suggests a genetic heterogeneity in our geographic area. We also concluded that the DR1 antigen may play a protective role, as it was detected in 20% of the controls and in none of the MS cases.

Adult↗
PubMed1998

Resistance to apoptosis correlates with a highly proliferative phenotype and loss of Fas and CPP32 (caspase-3) expression in human leukemia cells.

Apoptosis induced by effector cells of the immune system or by cytotoxic drugs is a main mechanism mediating the prevention or elimination of tumoral cells. For instance, the human T-cell leukemia Jurkat is sensitive to Fas-induced apoptosis and to activation-induced cell death (AICD), and the promonocytic leukemia U937 is sensitive to Fas- and TNF-induced apoptosis. In this work, we have analyzed the mechanisms of resistance to physiological or pharmacological apoptosis in human leukemia by generating highly proliferative (hp) sub-lines derived from Jurkat and U937 cells. These hp sub-lines were resistant to Fas- and TNF-induced apoptosis, as well as to AICD. This was due to the complete loss of Fas and TNFR surface expression and, in the case of Jurkat-derived sub-lines, also of CD3, CD2 and CD59 molecules. The sub-lines also completely lacked the expression of the apoptotic protease CPP32, present in parental cells. Moreover, these sub-lines were no longer sensitive to doxorubicin-induced apoptosis, which was efficiently blocked by the general caspase inhibitor Z-VAD-fmk in the parental cell lines. These data suggest a molecular mechanism for the development of resistance of leukemic cells to physiological and pharmacological apoptosis inducers, giving rise to highly proliferative tumoral phenotypes. These results also indicate that Fas and CPP32 could be useful prognostic markers for the progression and/or therapy outcome of human leukemias.

Antibiotics, Antineoplastic↗
PubMed1998

Immune response and cytokines in septic rats undergoing blood transfusion.

BACKGROUND: Intrabdominal sepsis and allogeneic blood transfusion have been associated with a depression of the immune response in patients undergoing surgery. Some authors have considered that an already immunocompromised host is probably primed for a potential detrimental effect of allogeneic blood. The aim of this paper is to ascertain the effects of allogeneic blood transfusion on the lymphocyte subsets and cytokines in septic rats. MATERIALS AND METHODS: Thirty rats were allotted into three groups: Sham-CLP, anesthesia and laparotomy; CLP, cecal ligation and puncture; CLP+BT, CLP and allogeneic blood transfusion. Preoperatively and on the 1st, 3rd, and 7th postoperative days, the cell percentages of lymphocyte subpopulations, the IL-2 receptor expression, and the IL-1, IL-2, TNF-alpha and IFN-gamma were measured in blood by flow cytometry and ELISA: RESULTS: CLP+BT rats showed on Day 3 a decrease of the CD4+%, an increase of the IL-2R expression directly correlated to the increase of the CD8+% phenotype, a steady increase of IL-1 levels, a decrease of the TNF-alpha levels on the 1st and 3rd days, and a decrease of the IL-2 and IFN-gamma on Day 1. CONCLUSIONS: An accumulative effect of the immunodepression induced by sepsis was observed when allogeneic blood transfusion is added. Blood transfusion + sepsis induces an extensive impairment on cellular immune response and an initial cytokine downregulation, except for IL-1.

Animals↗
PubMed1998

Lymphocyte T subset counts in children with hypercholesterolemia receiving dietary therapy.

BACKGROUND: In children with hypercholesterolemia, dietary therapy is indicated; however, we do not know if a low-fat diet can modify some organic functions, i.e. immune function. METHODS: 42 children with hypercholesterolemia received a low-fat, low-cholesterol diet during 6 months. At baseline and after the treatment period, we determined a lipoprotein profile and some immune characteristics: immunoglobulins G, A and M; complement components (C3, C4 and factor B), and blood lymphocyte subsets (CD3, CD4 and CD8). RESULTS: Total cholesterol serum concentrations showed a significant reduction after 6 months of dietary therapy (p = 0.008). After 6 months on a low-fat diet, lymphocyte T subset counts (CD3, CD4 and CD8) showed significant decreases (p < 0.01 to p < 0.003), but lymphocyte counts were always within normal ranges. There was also a significant correlation between changes in some lymphocyte T subset counts (CD3 and CD8) and changes in triglyceride serum concentrations (p < 0.05). CONCLUSIONS: A low-fat, low-cholesterol diet diminished CD3, CD4 and CD8 lymphocyte subset counts that are elevated in children with hypercholesterolemia. Dietary therapy, with emphasis on the intake of n-3 fatty acids, could be useful in the modulation of the immune response at the atheromatous plaque level.

Adolescent↗
PubMed1996

Relationship between immunoinflammatory proteins containing sialic acid and low-density lipoprotein serum concentrations.

The aim of this study was to investigate the relationship between sialoglycoproteins and the lipoprotein profile in a group of children with different levels of low-density lipoprotein cholesterol. We have studied 177 children of 132 families who were sent to our Pediatric Lipid Research Clinic because of serum cholesterol concentrations higher than 5.17 mmol/l. At the time of diagnosis, we analyzed the serum lipoprotein profile and the sialoglycoproteins: alpha 1-antitrypsin, acid alpha 1-glycoprotein, haptoglobin, alpha 2-macroglobulin, transferrin, IgA, IgG, IgM, complement C3 component and ceruloplasmin. At 7.0 to 10.9 years, alpha 1-glycoprotein serum concentrations were higher in the high risk group than in the moderate risk group (P < 0.05). At 2.0 to 6.9 years, IgA and IgM serum concentrations were higher in the moderate risk group than in the low risk group (P < 0.05 and P < 0.01, respectively), and IgG and IgM serum concentrations were also higher in the high risk group than in the low risk group (P < 0.05). Our results seems to reflect a general reaction to injury or inflammation which could be associated with the atherosclerotic process.

Adolescent↗
PubMed1996

Changes in blood lymphocyte populations in experimental bowel allograft rejection.

The aim of this study is to measure percentages of lymphocyte populations and IL-2R cellular expression in peripheral blood during the rejection of a small bowel allograft (SBA) in the rat. Thirty rats were allotted to three groups: A, control, no transplantation (Tx); B, rats receiving an orthotopic SBA; C, similar SBA but with thymostimulin (TP-1) administered before Tx, aimed at increasing the intensity of and accelerating rejection. The percentages of CD19, CD5, CD4 and CD8 cells and of IL-2R were determined when rejection was present. Rejection appeared in rats in group B between days 11 and 26 post-Tx and in group C between days 6 and 7 post-Tx (P < 0.001). In both B and C groups, CD5 and CD4 cells decreased (P < 0.005) and CD8 cells increased (P < 0.001). A correlation between CD8 and IL-2R content was found (P < 0.05). In group C, earliness of rejection correlated with the percentage of CD8 cells (P < 0.05) and the intensity of rejection with numbers of CD8 and CD19 cells (P < 0.05).

Animals↗
PubMed1996

Immune response and in vivo production of cytokines in patients with liver hydatidosis.

Cytokines play an important role in the human immunological response, but the exact role of cytokines in the human immune response against parasites, especially against Echinococcus granulosus, remains unclear. IL-1, IL-2, IL-4 and tumour necrosis factor (TNF) levels in peripheral blood of 21 patients with liver hydatidosis were evaluated before surgical treatment, and the levels of IgA, IgM, IgG, IgE, specific IgE against E. granulosus, C3, C4 and DF complement fractions and CD20, CD3, CD4, CD8 and CD16 cell percentages were also determined, as was the relationship between these variables and cytokine levels. Data from hydatid patients were compared with data obtained from 21 healthy volunteers. Hydatid patients showed increases of IgG, IgE, IgEs and IL-2 (P < 0.01), and decreases of IL-1 and TNF levels (P < 0.001), but these variables (respectively) increased in patients showing cysts in the central area of the liver or with a wide opening of cysts in the biliary tract. The increase of IL-1, IL-2 and IL-4 showed a close relationship with the number, characteristics and above all the location of cysts within the liver itself. IgG and IL-4 levels and also IgG and IgE levels showed a significant correlation (P < 0.05).

Adolescent↗