Mechanisms of neutrophil death in human immunodeficiency virus-infected patients: role of reactive oxygen species, caspases and map kinase pathways.

Neutrophils from human immunodeficiency virus-positive (HIV+) patients have an increased susceptibility to undergo programmed cell death (PCD), which could explain neutropenia during advanced disease. In this work, key steps of PCD have been evaluated in neutrophils from HIV+ patients. The role of caspase-3, caspase-8, mitogen activated protein kinase (MAPK) and reactive oxygen species (ROS) was analysed. Spontaneous neutrophil death is dependent upon caspase-3 but independent of caspase-8, suggesting that the intrinsic pathway is involved as a pathogenic mechanism of PCD. Inhibition of ROS decreased spontaneous PCD and caspase-3 hydrolysis, connecting oxidative stress and caspase-3 activation with neutrophil PCD in HIV-infected patients. Additionally, an increased neutrophil death was observed in HIV+ patients, following inhibition of p38 MAPK, suggesting a role for p38 MAPK in cell survival during the disease. We conclude that oxidative stress secondary to HIV infection can accelerate neutrophil death.

Profiles of NK, NKT cell activation and cytokine production following vaccination against hepatitis B.

Human natural killer (NK) cells (CD56+ CD3-) represent crucial components of the innate immune system especially against viral infections and because their activation can modulate the outcome of the adaptive immune response. NKT cells (CD56+CD3+), a lymphocyte T population characterized by expression of surface markers of NK cells, are known to be abundant in the liver and their activation could be associated with hepatic injury. Using three-color flow cytometry to measure surface receptors and intracellular cytokines, we have explored early activation signals and cytokine production in NK and NKT cells within a group of hepatitis B vaccinated and non-vaccinated individuals. A specific increase of the CD56bright cell population, the activation receptor CD69 and IFN-gamma, was observed in NK cells following incubation with recombinant HBsAg in responders to vaccination. Comparable results were observed in NKT cells showing an increment of CD69, CD25, IL-2 and IFN-gamma expression in responder subjects. These parameters were statistically diminished in non-responder individuals (p<0.05) in both groups of cells. These results demonstrate a diminished activation of these cells in non-responders to the vaccine, suggesting that NK and NKT cells play an important role in the immune response following hepatitis B vaccination.

Long-term care for the elderly in Japan.

This article, using data from the first author's research, presents selected issues in long-term care (LTC) of the elderly in Japan. A brief discussion of historical and cultural factors frame the current realities of LTC. These realities include the vast numbers of elderly people in Japan, changing definitions of the relationship of the individual to the group, and enactment of the new Care Insurance Law for the Elderly to be implemented in the year 2000. Some of the work underway for this implementation is detailed.

[Role of iron in immunity and its relation with infections]. / Participación del hierro en la inmunidad y su relación con las infecciones.

Experimental evidence in the last decades show that iron is a fundamental element for normal development of the immune system. Its deficiency affects the capacity to have an adequate immune response. The role of iron in immunity is necessary for immune cells proliferation and maturation, particularly lymphocytes, associated with the generation of a specific response to infection. The body has the capacity to reduce the iron availability to be consumed by infectious elements by proteins such as transferrin and lactoferrin. Also, iron is essential for the proliferation of bacteria, parasites, and neoplastic cells. Thus excess iron could potentially facilitate the development of infections and the invasion of tumoral cells. The immune system has bacteriostatic mechanisms that reduce the availability of the metal, interfering with bacterial growth. Additionally the system uses iron as the intermediary in the production of bacteriostatic cells.

Effects of Ajoene on lymphocyte and macrophage membrane-dependent functions.

Ajoene, (E, Z) -4, 5, 9-trithiadeca-1, 6, 11-triene 9 oxide, is a compound originally isolated from ethanolic extracts of garlic that impairs platelet aggregation by inhibiting the functional exposure of platelet integrins GPIIb/IIIa. In vitro, Ajoene is toxic for several tumoral cell lines, and exert an antiproliferative effect on T. cruzi and murine malaria parasites. Here we show that Ajoene strongly inhibited the proliferation induced in human lymphocytes by the mitogens phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and anti-CD3, and the capping formation induced in B lymphocytes by anti-IgM antibodies. On macrophages, Ajoene was also found to partially inhibit the lypopolysaccharide-induced production of Tumor Necrosis Factor (TNF), and to decrease the phagocytic activity of thioglycolate-elicited mouse peritoneal macrophages for IgG-opsonized, human erythrocytes. Ajoene also partially prevented the lytic effect of human and rabbit TNF on Actinomycin D-treated WEHI 164 cells. These results strongly suggest that Ajoene is a potent modulator of membrane-dependent functions of immune cells.

T lymphocytes subsets in experimental iron overload.

Several abnormalities of the immune system have been reported in association with clinical and experimental iron overload. To dissect further such abnormalities, changes in lymphocyte subsets were evaluated in iron-loaded male Sprague-Dawley rats. The iron-loading protocol consisted of a total dose of irondextran (1.5 mg/Kg body weight) divided in daily intramuscular injections over twenty consecutive days. At days 0, 20, and 50 after initiation of iron injections lymphocyte subsets in blood, spleen and mesenteric lymph nodes were estimated by indirect immunofluorescence using monoclonal antibodies recognizing T cells (W3.13), the subset of helper T cells in (W3.25), and the subset of cytotoxic T cells (OX.8). By day 20, there was no change in the number of W3.25+ T cells in the blood of iron-loaded animals as compared to the controls, but the OX.8+ T cells were significantly elevated. At this time, the ratio W3.25 +/OX.8+ cells was significantly decreased (0.5 in experimental rats vs 2.0 in controls). Similar results were obtained at day 50. In the spleen, there was a decrease in the proportion of W3.25 +T cells and an increase in OX.8+ T cells at day 20. However, these values returned to normal by day 50. A negative correlation between W3.25 +/OX.8+ ratio and serum ferritin was observed in blood and spleen during iron administration. These changes were associated with abnormalities in lymphocyte proliferative response. No changes in W3.25 +/OX.8+ ratio were observed in mesenteric lymph nodes. These results demonstrate that iron overload alters the distribution of T lymphocytes in various compartments of the immune system.

Lipid peroxidation and changes in T lymphocyte subsets and lymphocyte proliferative response in experimental iron overload.

This study was undertaken to investigate the hypothesis that lipid peroxidation might be associated with immunological abnormalities in experimental hemosiderosis. The correlation between the degree of plasma and spleen lipid peroxidation with lymphocyte proliferative response and with the proportion of T lymphocyte subsets was studied in normal and iron overloaded male Sprague Dawley rats. The iron-loading protocol consisted of a total dose of iron-dextran (1.5 mg/Kg body weight) divided in daily i.m. injections over twenty consecutive days. Lipid peroxidation was measured by the thiobarbituric acid assay in plasma and in homogenates of spleen. Plasma lipid peroxide level increased rapidly after i.m. administration of iron-dextran and decreased sharply at 48 h after the last injection. Conversely, a progressive increase of lipid peroxidation in homogenates of spleen was observed in the course of the iron overload protocol, remaining high even at 50 days after initiation of iron-dextran injections. The increase of spleen lipid peroxide levels was associated with decreased lymphocyte proliferative response to Con A in iron overloaded rats. The addition of superoxide dismutase and catalase to lymphocyte cultures reversed the inhibition of the proliferative response, implicating reactive species of oxygen as the causative agents of these alterations. These effects may be related with the enhanced membrane and DNA damage occurring during intracellular and extracellular peroxidation. Negative correlations between helper/cytotoxic ratio and malondialdehyde levels were obtained in blood and spleen during iron administration. These results supports the hypothesis that lipid peroxidation plays a role in the immunological abnormalities observed in experimental hemosiderosis.

Effect of hepatic isoferritins from iron overloaded rats on lymphocyte proliferative response: role of ferritin iron content.

Iron and ferritin impair a variety of immunological functions. To evaluate the effect of ferritin iron content on rat lymphocyte proliferative response, isoferritins that differ in their iron content and isoelectric point (pI) were isolated from iron overload rat livers by ultracentrifugation (isoferritins with high iron content and low pI) or crystallization (isoferritins with low iron content and high pI) methods. Additionally, commercial horse splenic ferritin (with a lower pI and higher iron content than rat isoferritins) was also tested. Proliferative response to Con A was decreased in a dose-dependent manner in all assays in which spleen cells were incubated with rat and horse isoferritins. However, isoferritins with higher iron contents (rat isoferritin obtained by ultracentrifugation and horse ferritin) caused a greater decrease of proliferative response at 5 and 25 micrograms/ml than the others. Rat and horse apoferritins showed no inhibitory effect on lymphocyte proliferative response, suggesting that the effect is due to iron probably through the damaging effect of reactive oxygen species generated by iron released by the isoferritins on lymphocyte functions. Additionally, the role of serum ferritin level on proliferative response was studied in an experimental model of iron overload in rats. An inverse relationship between the proliferative response and serum ferritin levels was observed. Our results suggest that the inhibitory effect of the isoferritins on lymphocyte proliferative response is due, at least partially, to the iron content of this protein and not exclusively to variation in pI as suggested by other authors. These results are in agreement with the possible immunosuppressor role of ferritin in vivo.

ABO-haemolytic disease of the newborn (ABO-HDN): factors influencing its severity and incidence in Venezuela.

The objective of this study was to determine the incidence of the Haemolytic Disease of the Newborn due to ABO blood group incompatibility (ABO-HDN) in the population of Caracas attending the Maternity Hospital 'Concepción Palacios'. The relationship between A and B antigens density of cord blood erythrocytes and the cytotoxic activity of antibodies in mothers' sera with the severity of the haemolytic disease, was also studied. From a sample of 245 blood group 'O' mothers, 68 gave birth to full term 'A' or 'B' blood group infants. The evolution of serum bilirubin and the routine haematological values, were followed in all the babies during 72 h after birth, allowing the diagnosis of ABO-HDN in 21 infants. Taking into account that in Venezuela the frequency of blood group 'O' is 59 per cent, it was concluded that in the general population of newborns, 16 per cent present foeto-maternal ABO incompatibility, and the incidence of ABO-HDN was near to 5 per cent. The density of the 'A' and 'B' antigens in cord red cells was studied using an immunoenzymatic assay. No statistically significant association between antigen maturity and severity of the ABO-HDN could be shown. A positive association was found between cytotoxic capacity of mothers' sera and development of ABO-HDN (P < 0.05). Twenty ABO incompatibles children presented moderate late anaemia at 3 weeks of age.

Treatment of ABO hemolytic disease with synthetic blood group trisaccharides.

Thirteen infants, 10 with A-O and 3 with B-O hemolytic disease of the newborn (ABO-HDN), were treated with synthetic A or B blood group trisaccharides (ATS, BTS) which cause dissociation of maternal antibody bound to infant red cells. The clinical outcome was compared with that of a control group of 21 infants treated with phototherapy during the preceding year. Exchange transfusion was required in 2 out of 13 infants in the experimental group and in 7 in the control group. A randomized prospective controlled study is necessary to confirm these results.

Iron polymers impair the function and maturation of macrophages.

An inhibitory effect of iron salts on various immune functions in vitro has been reported in several laboratories during the last years. This work extends such observations by showing that iron citrate, but not sodium citrate, inhibits the function and maturation of murine macrophages (MOs). However, such inhibition is only observed in the presence of ferric citrate with a metal-to-ligand molar ratio of 1:1, but not with ferric citrate with a metal-to-ligand molar ratio of 1:10 in which the hydrolyzation and polymerization of iron in physiological solutions is prevented. Accumulation of ferric iron on the cytoplasm of MOs was observed, but only in the group of MOs treated with ferric citrate 1:1. Increasing the concentration of serum in the culture medium diminished the inhibitory effect of ferric citrate 1:1. The inhibitory capacity of iron polymer was probably associated to its ability to both interact with the cell constituents of the cytoplasm and stimulate lipid peroxidation.

Evaluation of complement activity by an enzyme immunoassay.

An ELISA-type assay useful for the evaluation of the complement activity in serum is described. Aggregated pooled human IgG (IgGn) prepared so as to exclude large and small aggregates, to resemble soluble circulating immune complexes, was used to coat polystyrene microwells to serve as initiator of complement activation. Fresh serum, at different dilutions, as the source of the complement to be evaluated was added and the plate incubated 90 min at 37 degrees C. Then, a peroxidase-labeled antihuman C3c antibody was added to react with the bound C fragments. This was followed by 2,2'-azino-di-3-ethyl benzothiazoline sulfonic acid (ABTS), as the color reagent used for detection of the enzyme activity. In this system, theoretically, the levels of activating and regulatory complement components are evaluated up to the level of C3 splitting. The assay was applied in healthy volunteers to set normal values and in 15 patients suffering from systemic lupus erythematosus making possible the differentiation of those with normal and low complement levels.

Spleen and thymus histology and proliferative response of splenic cells in rats fed raw and cooked Phaseolus vulgaris beans.

Histological studies of the spleen and thymus of rats fed raw black beans (Phaseolus vulgaris) show an atrophy of both lymphoid organs. Decrease in relative thymus weight was most marked. All histological organization of this organ appeared altered. An evident decrease in cell number was also observed in both organs. Proliferative response of splenic cells stimulated in vitro with Concanavalin A was increased as compared to that from animals fed the control diet. It is likely that histological changes observed in the spleen and the thymus are due mainly to a protein caloric deficiency, although the possibility that toxic factors present in the raw diet have an effect on the immune system of the rat can not be overruled.

Enzyme-linked immunosorbent assay for the detection of substances that carry blood group A specificity.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the amount of material carrying blood group A activity in biologic samples. A soluble synthetic form of the A antigenic determinant (A trisaccharide, ATS) conjugated to peroxidase competes with the blood group A substance present in a biologic sample for anti-A attached to a solid phase by a second antibody coating the plastic micro-wells. A reference curve is constructed by using known quantities of ATS to compete with a fixed amount of ATS-peroxidase conjugate. The A substance activity in a sample is obtained by extrapolating the degree of inhibition of the binding of the ATS-peroxidase conjugate to an equivalent amount of ATS in the reference curve. The assay is reproducible, specific, and sensitive. It has been used in pharmacologic studies to estimate the concentration of ATS in the blood and urine of rats, rabbits, and baboons and in a study with human samples, testing the potential clinical use of ATS to neutralize anti-A when therapeutically indicated. It is also useful for the detection of ABO natural products in secretions, thus allowing the accurate classification of secretor and nonsecretor individuals.

A modified colorimetric method for the measurement of phagocytosis and antibody-dependent cell cytotoxicity using 2,7-diaminofluorene.

A sensitive and rapid colorimetric method for the in vitro determination of phagocytic activity and antibody-dependent cell-mediated cytotoxicity (ADCC) is described. The assay uses red blood cells (RBC) as target cells and relies on the specific oxidation of 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of hemoglobin (Hb). Generation of fluorene blue (FB), the chromophore formed upon oxidation of DAF, was a linear function of erythrocyte concentration. The oxidation of DAF by peritoneal macrophages (M phi) containing myeloperoxidase was negligible, confirming that the development of color was exclusively due to the pseudoperoxidase activity of Hb. A positive correlation was observed between FB formation and increased phagocytosis of opsonized erythrocytes. Phagocytosis increased as a function of time, reaching a maximum at 90 min of incubation. The phagocytosis of IgG-opsonized erythrocytes was greater than non-opsonized erythrocytes and was inhibited by high concentrations of non-specific human or mouse IgG, showing that phagocytosis was mediated by the Fc gamma receptor of macrophages. The interaction between opsonized RBC and macrophages also evoked an antibody-dependent extracellular lysis, however this process was slower than ingestion. The DAF phagocytosis assay has shown to be very sensitive, simple, rapid and safe.

Interaction of iron polymers with blood mononuclear cells and its detection with the Prussian Blue reaction.

An inhibitory effect of iron salts on various immune functions in vitro has been reported in several laboratories during the last few years. This study confirms and extends those observations by showing that iron citrate inhibits the mitogen-induced (PHA, Con A and PWM) lymphocyte proliferation. Such inhibition is observed in the presence of ferric citrate with a metal-to-ligand molar ratio of 1:1 but not with ferric citrate with metal-to-ligand molar ratio 1:20 in which the formation of polynuclear iron complexes is prevented. Increasing the concentration of serum in the culture medium diminished the inhibitory effect of 1:1 ferric citrate. Using the Prussian Blue reaction the presence of ferric iron was observed on the cell surface. It is proposed that the deposition of polynuclear iron complexes on the lymphocytes membrane is one of the possible factors determining the iron inhibitory effect.

[Biomolecules suppressing myelopoiesis]. / Biomoléculas supresoras de la mielopoyesis.

Hematopoietic stem cells are capable of self-replication and differentiation to lineage-committed progenitor cells. The progenitors proliferate and differentiate to lineage-specific, morphologically recognizable precursors and, finally, to terminal circulating blood cells. These homeostatic mechanisms are regulated by a complex set of interacting growth stimulatory and inhibitory factors that are produced by, or in collaboration with, the tissue's regulatory microenvironment. A number of well-characterized cytokines have been implicated in the negative regulation of hematopoiesis: ferritin H-subunit (HF), lactoferrin (Lf), prostaglandin E (PGE), tumor necrosis factor (TNF), interferon (IFN), transforming growth factor-beta (TGF beta), acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) or thymosin-beta 4, pyroGlu-Glu-Asp-Cys-Lys (pEEDCK), macrophage inflammatory protein-1 alpha (MIP-1 alpha), inhibin, superoxide dismutase (SOD), glutathione (GSH) and others not well-known yet. The role of inhibitors in restraining stem cells from entering the cell cycle and protecting them from the toxic side effects of chemotherapeutic drugs is opening an alternate strategy for the treatment of cancer patients.

A new hemolytic assay for bovine serum complement and its application during experimental bovine anaplasmosis.

A new hemolytic assay for bovine complement is presented. Using this assay we found a significant reduction in bovine serum complement activity during the acute phase of anaplasmosis, and an increase in the sensitivity of the red blood cells (RBC) to bovine complement lysis in vitro. The new hemolytic test is performed with bovine RBC, rabbit anti-bovine RBC serum and bovine serum complement. An isotonic sucrose Tris-buffered saline solution of ionic strength 0.094 and pH 7.2 was found to be adequate for this test. The titres obtained with this new assay, which uses autologous RBC, are comparable with those obtained using the guinea pig RBC assay. The finding of a reduction in bovine serum complement during anaplasmosis may be suggestive of a mechanism responsible for the pathology of this disease.

Functional characterization of mononuclear cells of normal and hypercholesterolemic rabbits.

Cholesterol-fed rabbits are more susceptible to respiratory and skin infections, and show a higher index of mortality than rabbits given a normal diet. Several tests were employed to estimate the proportion of T and B lymphocytes (L-T and L-B) and aspects of their physiology in rabbits with hypercholesterolemia. The proportion of L-T and L-B in hypercholesterolemic rabbits (HChR) was found to be similar to that of normal rabbits (NR). The proliferative response of mononuclear cells (MNC) from peripheral blood stimulated with mitogens in HChR decreased during the first ten days of the cholesterol rich-diet, being variable afterwards. The addition of HChR serum to the culture media decreased the proliferative response of MNC isolated from both HChR and NR and stimulated by Con A and PWM. A similar antibody response to sheep red blood cells (SRBC) and bovine serum albumin (BSA) antigens was found in HChR and NR.

Iron and transferrin uptake by phytohemagglutinin-stimulated human peripheral blood lymphocytes.

Iron (Fe) and transferrin (TF) uptake by human peripheral blood lymphocytes stimulated in vitro with phytohemagglutinin was measured. Pulses of 59FeTF or 125I-TF were added to the cultures either at time 0 or 8 hr before the end of a 72-hr incubation. In time-course experiments, peak iron and transferrin uptake coincided with the peak of tritiated thymidine uptake taken as a measure of cellular activation. Iron, but not transferrin, was accumulated by the cells. Non-linear relationships existed between both iron and transferrin uptake and the degree of activation. Both rose markedly above basal levels only at a level of activation at least 50% of the maximum observed. The results suggest that although iron utilization is related to cellular activity, the uptake mechanism is only activated when an increased iron metabolism has exhausted internal stores.