Aquatic pollutants are associated with reproductive alterations and genotoxicity in estuarine fish (Sciades herzbergii - Bloch, 1794) from the Amazon Equatorial Coast.

Estuaries are constantly subject to continuous environmental impacts of human activities, such as fisheries, port or industry, and domestic sewage, with fish being one of the most affected aquatic animals, reflecting the impacts directly on their bodies. Thus, the aim of this study was to carry out the biomonitoring of an estuary located on the Amazonian Equatorial Coast through analysis of PAHs (Polycyclic Aromatic Hydrocarbons) in the water, in addition to trace metals, histopathological alterations and analysis of erythrocyte micronuclei in Sciades herzbergii. S. herzbergii was used as a model species, due to its estuarine-resident behavior. Gonad and gill samples were subjected to histopathological evaluations. The quantification of trace metals was performed in samples of skeletal muscles of the animals collected, where concentrations of Lead (Pb), Copper (Cu), Zinc (Zn), Cadmium (Cd), Magnesium (Mg), Iron (Fe) and Aluminum (Al) were found. Except for Cadmium (Cd), all the concentrations were above the recommended limits. The PAHs analysis revealed the presence of Naphthalene and Acenaphthene in the water samples Histopathological and genotoxic analyses revealed of lesions in 100% of the study specimens. Thus, the histological and genotoxic alterations found in 100% of S. herzbergii specimens captured in São José Bay-MA are potentially associated with PAH concentrations present in the water. These results are potentially associated with the presence of PAH and trace metals, both in water and in animal tissues, inferring a general scenario of environmental contamination which directly implies a risk to the health and survival of the local biota. This study shows the relevance of continuous biomonitoring of estuarine ecosystems, in order to guide authorities regarding sewage management and ensure the evolutionary development of estuarine species, especially fishes of importance in the local cuisine, therefore related to human food security.

CR1 on erythrocytes of Brazilian systemic lupus erythematosus patients: the influence of disease activity on expression and ability of this receptor to bind immune complexes opsonized with complement from normal human serum.

Hypocomplementaemia and low expression of CR1 on erythrocytes (E) of patients with systemic lupus erythematosus (SLE) are associated with defective clearance of circulating immune complexes (IC) and so they may have pathogenic significance. Here, we investigated whether the reduced CR1/E in SLE patients per se might affect the binding of IC to CR1/E. First, we analysed the expression of CR1 on E of active (n=30) and inactive (n=34) SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry. Both groups of patients had a significantly reduced CR1/E expression compared with healthy controls (n=40). It was also observed that the number of E bearing CR1 was reduced in both groups of SLE patients studied. Second, we determined the functional activity of CR1/E by measuring the binding to E of FITC-bovine serum albumin (BSA)/rabbit anti-BSA complexes, formed at equivalence, which were opsonized with complement from normal human serum (NHS). On the other hand, we did not find differences between the patient and control groups in the ability of E to bind IC/NHS. There was also a positive correlation between the CR1/E expression and the number of E bearing CR1 in control and inactive SLE groups, which was not observed in the group of active SLE patients. Considering the involvement of low levels of complement and CR1/E expression on complex processing, in this in vitro model the results show that an effective coating of the complexes with complement is sufficient to bind them preferentially to CR1 over normal levels of receptor expression.

Application of the chemiluminescence systems to evaluate the role of Fcgamma and complement receptors in stimulating the oxidative burst in neutrophils.

We have established a luminol- and a lucigenin-dependent CL methods to investigate the role of the receptors for Fc portion of IgG (FcgammaR) and/or complement receptors (CR) in mediating the oxidative burst in neutrophils from systemic lupus erythematosus (SLE) patients compared with healthy controls. In the luminol-CL system, all the reactive oxygen species (ROS) are responsible for light production, whereas in the lucigenin-CL system, only the first ROS generated, converts the lucigenin into an unstable intermediate molecule, which also emits light. First, neutrophils from healthy controls and SLE patients were stimulated with different IC opsonized or not with complement from normal human serum (NHS) or SLE serum, in presence of 10(-4) M luminol. This method was able to differentiate the role of the FcgammaR, CR and FcgammaR/CR co-operation in mediating the oxidative burst, as well as show that the oxidative burst mediated by these receptors was reduced in neutrophils from SLE patients. Second, neutrophils from healthy controls and SLE patients were stimulated with different IC, opsonized or not with NHS, in presence of 10(-3) M lucigenin. In this case, the lucigenin-CL system was also able to differentiate the role of FcgammaR and FcgammaR/CR co-operation, as well as show differences among healthy controls and two different groups of SLE patients according to their clinical manifestations. In conclusion, we have established two sensitive CL systems to study the role of FcgammaR and/or CR in stimulating the oxidative burst of neutrophils, which can be applied in monitoring the involvement of these receptors in the immunopathogenesis of SLE.

Fcgamma and complement receptors: expression, role and co-operation in mediating the oxidative burst and degranulation of neutrophils of Brazilian systemic lupus erythematosus patients.

We have investigated the individual role of FcgammaR and CR, as well as their cooperation, in mediating the oxidative burst and degranulation of neutrophils of Brazilian systemic lupus erythematosus (SLE) patients. Neutrophils were stimulated with the immune complexes (IC)-IgG or -F(ab')2, opsonized or not with normal or SLE human serum. The oxidative burst was decreased in neutrophils of active SLE patients compared to healthy controls when this response was mediated by FcgammaR and/or CR, while the degranulation was unaffected. The SLE hypocomplementemia did not affect the oxidative burst mediated only by CR. FcgammaRII and CR1 expression on neutrophils of active SLE patients was reduced, while the expression of FcgammaRIII and CR3 was unaffected. These results suggest that the different FcgammaR and CR may be involved or cooperate in different ways in the mediation of the oxidative burst and the degranulation. Moreover, the decreased oxidative burst of neutrophils of active SLE patients may not depend only on SLE hypocomplementemia for IC opsonization. These observations are directed at the understanding of how each of these immune system components (FcgammaR, CR and complement) influences the precise biological neutrophil responses both in physiological and pathological conditions. Since the Brazilian population comprises many races, these results are important because they are directed at a specific population of SLE patients.

Association between EcoRI fragment-length polymorphism of the immunoglobulin lambda variable 8 (IGLV8) gene family with rheumatoid arthritis and systemic lupus erythematosus.

The human immunoglobulin lambda variable 8 (IGLV8) subgroup is a gene family containing three members, one of them included in a monomorphic 3.7-kb EcoRI genomic fragment located at the major lambda variable locus on chromosome 22q11.1 (gene IGLV8a, EMBL accession No. Z73650) at 100% frequency in the normal urban population. The second is a polymorphic RFLP allele included in a 6.0-kb EcoRI fragment at 10% frequency, and the third is located in a monomorphic 8.0-kb EcoRI fragment at 100% frequency, the last being translocated to chromosome 8q11.2 and considered to be an orphan gene. Our Southern blot-EcoRI-RFLP studies in normal individuals and in patients with rheumatoid arthritis (RA) or with systemic lupus erythematosus (SLE), using a specific probe for the IGLV8 gene family (probe pVL8, EMBL accession No. X75424), have revealed the two monomorphic genomic fragments containing the IGLV8 genes, i.e., the 3.7-kb fragment from chromosome 22q11.1 and the 8.0-kb fragment from 8q11.2, both occurring at 100% frequency (103 normal individuals, 48 RA and 28 SLE patients analyzed), but absence of the 6.0-kb IGLV8 polymorphic RFLP allele in all RA or SLE patients. As expected, the frequency of the 6.0-kb allele among the normal individuals was 10%. These findings suggest an association between the absence of the 6.0-kb EcoRI fragment and rheumatoid arthritis and systemic lupus erythematosus.

Association between EcoRI fragment-length polymorphism of the immunoglobulin lambda variable 8 (IGLV8) gene family with rheumatoid arthritis and systemic lupus erythematosus

The human immunoglobulin lambda variable 8 (IGLV8) subgroup is a gene family containing three members, one of them included in a monomorphic 3.7-kb EcoRI genomic fragment located at the major lambda variable locus on chromosome 22q11.1 (gene IGLV8a, EMBL accession No. Z73650) at 100 percent frequency in the normal urban population. The second is a polymorphic RFLP allele included in a 6.0-kb EcoRI fragment at 10 percent frequency, and the third is located in a monomorphic 8.0-kb EcoRI fragment at 100 percent frequency, the last being translocated to chromosome 8q11.2 and considered to be an orphan gene. Our Southern blot-EcoRI-RFLP studies in normal individuals and in patients with rheumatoid arthritis (RA) or with systemic lupus erythematosus (SLE), using a specific probe for the IGLV8 gene family (probe pVL8, EMBL accession No. X75424), have revealed the two monomorphic genomic fragments containing the IGLV8 genes, i.e., the 3.7-kb fragment from chromosome 22q11.1 and the 8.0-kb fragment from 8q11.2, both occurring at 100 percent frequency (103 normal individuals, 48 RA and 28 SLE patients analyzed), but absence of the 6.0-kb IGLV8 polymorphic RFLP allele in all RA or SLE patients. As expected, the frequency of the 6.0-kb allele among the normal individuals was 10 percent. These findings suggest an association between the absence of the 6.0-kb EcoRI fragment and rheumatoid arthritis and systemic lupus erythematosus

Intravenous immunoglobulin (IVIG) attenuates antibody binding in acute haemorrhagic immunopneumonitis in a rat model of complement-dependent lung injury.

Administration of rabbit anti-rat lung serum (PNTS) to rats produces a fulminant haemorrhagic pneumonitis sensitive to the availability of complement. The present experiments were undertaken to assess whether a high dose of IVIG can affect the development of this kind of cytotoxic reaction. The experimental design included groups of Wistar rats pretreated intravenously with physiologic saline, IVIG or a preparation of human F(ab')2 fragments. One hour later the animals were challenged with either saline or PNTS. At 30 min after challenge, blood was collected and the lungs were removed. Pulmonary damage was evaluated by light microscopy; C3 deposits and the binding of immunoglobulins to the alveolar septa were assayed by immunofluorescence. The serum complement activity of the classical and alternative pathways was estimated by a kinetic technique. Pretreatment with IVIG decreased binding of rabbit anti-lung antibodies to alveolar septa and prevented the deposition of C3. These results indicate that pretreatment with IVIG inhibits the binding of the pathogenic antibody to lung tissue. Human IgG binding was not detected in any animal. The protection against lung injury afforded by pretreatment with IVIG, in contrast to the pneumotoxic effect of PNTS observed in control animals, was evident despite the administration of F(ab')2 to the rats. Since pretreatment with F(ab')2 failed to prevent the acute lung lesion, our results indicate that the attenuation afforded by IVIG in this model of complement-dependent tissue injury seems to be related to the integrity of the IgG molecule.

Complement haemolytic activity (classical and alternative pathways), C3, C4 and factor B titres in healthy children.

Values of complement lytic activity of classical and alternative pathways, assessed by measuring the time required to lyse 50% of target red blood cells, and the concentration of complement components C3, C4 and factor B were estimated in the sera of 103 healthy children aged 3 to 14 y. Age-dependent variations were seen in the C3 and factor B concentrations, but not in C4, with the highest values found among 5-6-y-old children. Variations in classical and alternative lytic activity were not detected in this group of children, although the values are significantly different from our previously published data on adults, using the same kinetic assay (1). We also evaluated the relationship between the lytic activity of the classical (CPT) and alternative pathways (APT) and the levels of complement components. There were significant correlations between: APT and factor B, APT and C3, C3 and C4, C3 and factor B, and C4 and factor B concentrations. The normal ranges measured here can be used in the initial screening of Brazilian children presenting diseases involving the complement system. This study also contributes to a better understanding of the complement system ontogeny.

Correlation of alternative pathway (AP) lytic activity and AP-dependent neutrophil phagocytosis with factor B levels and consumption in serum.

This work investigated the correlation between serum levels of factor B, AP-lytic activity, ratio of factor B activation by zymosan, and AP-dependent neutrophil phagocytosis in samples of normal human serum (NHS). In addition, since the antithyroid drug propylthiouracil (PTU) induces increased levels of AP lytic activity in rats, groups of these animals were treated with this drug in order to increase AP titers and to evaluate those parameters also in this condition. The results showed no correlation between factor B concentration and AP lytic activity in 18 samples of NHS or between factor B concentration and proportion of consumption by zymosan. Interestingly, this consumption was also not correlated with phagocytosis as measured by the chemiluminescence (CL) response of neutrophils to the opsonized particles. The two biological properties of phagocytosis and lytic activity, dependent of AP, were not correlated to each other in the NHS samples. In the samples of rat serum with increased AP lytic levels a different result was observed. A positive correlation between CL response and lytic activity occurred in serum of animals receiving a low PTU dose, but not in serum of animals receiving a high dose, where CL responses were lower than those of controls. The results are compared to literature data and discussed in terms of individual differences in resistance or susceptibility to infections and or diseases involving the complement system.

Augmented plasma and tissue kallikrein like activity in synovial fluid of patients with inflammatory articular diseases.

We studied some of the components of the kininogen-kallikrein-kinin system, simultaneously, in plasma and synovial effusions of patients with inflammatory articular diseases. Plasma and tissue kallikrein like activity and kininogen levels were evaluated. Active plasma and tissue kallikreins in plasma and synovial fluid were detected by their amidase activity upon specific chromogenic substrates. Kininogen levels were determined by a bioassay. Both specific amidase activity of plasma and tissue kallikreins were augmented in synovial effusions in relation to their own plasma activity. Kininogen levels in synovial fluid tended to be diminished in relation to plasma, however statistical significance was not reached. The consumption of kininogen is probably related to kinin production. This finding together with increased activities of plasma and tissue kallikreins reinforce the involvement of kinins in pathogenesis of inflammatory articular diseases.

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease

Numeros absolutos de leucocitos e linfocitos, de celulas T totais, indutoras/auxiliares, supresorras/citotoxicas e de celulas B estavam diminuidos no sangue periferico de pacientes com doenca de Chagas cronica. Como anticorpos antilinfocitarios estavam presentes em apenas uma minoria de pacientes, eles provavelmente nao sao responsaveis pelas anormalidades das subpopulacoes de linfocitos. Neutrofilos de pacientes estimulados por plasma autologo tratado por endotoxina mostravam atiividade quimiotatica diminuida que deve ser devida a um defeito celular intrinseco e nao a inibicao plasmatica. A migracao aleatoria dos neutrofilos estava normal. A reducao do corante "nitroblue tetrazolium" (NBT) por neutrofilos estimulados por endotoxina tambem estava diminuioda nos pacientes. Estes achados estendem a documentacao da imunossupressao na doenca de Chagas humana. Eles podem ser relevantes para autoimunidade e para defesa contra microorganismos e celulas tumorais, pelo menos em um subgrupo de pacientes com anormalidades mais pronunciadas

Lymphocyte subpopulations and neutrophil function in chronic human Chagas' disease.

The absolute numbers of total leukocytes, lymphocytes. T cells, helper/inducer, suppressor/cytotoxic and B cells were decreased in the peripheral blood of patients with chronic Chagas' disease. Since antilymphocyte antibodies were present only in a minority of patients they probably cannot account for the abnormalities in lymphocyte subsets. Patient neutrophils stimulated with endotoxin-treated autologous plasma showed depressed chemotactic activity and this seems to be an intrinsic cellular defect rather than plasma inhibition. Random migration of neutrophils was normal. Reduction of nitroblue tetrazolium by endotoxin-stimulated neutrophils was also decreased. These findings further document the presence of immunosuppression in human Chagas' disease. They may be relevant to autoimmunity, defense against microorganisms and against tumor cells at least in a subset of patients with more severe abnormalities.

Serum haemolytic classical and alternative pathways of complement in infancy: age-related changes.

The haemolytic activity of complement was evaluated in the serum of healthy children from birth to 2 years of age using the kinetic method for the determination of the time needed to lyse 50% of target red cells (t 1/2). No sex-linked differences were observed in any of the age groups studied and the lowest lytic activity levels for both complement pathways were detected in neonates. The two pathways, however, showed different maturation patterns, i.e., lytic activity levels similar to those of adults were reached between the 1st and 3rd month of life (classical pathway) and around the 13th month (alternative pathway). In the age group of 7 to 24 months, the lytic activity of the classical pathway was higher than in adults. The present data permitted us to establish normal ranges of t 1/2 values for the classical and alternative pathways in serum of healthy neonates and children aged 1 to 24 months.

Circulating immune complexes in sickle cell anaemia.

A polyethylene-glycol insoluble serum fraction was studied in patients with sickle cell anaemia during the steady state of the disease. The levels of C1q-precipitins were normal but increased amounts of proteins, IgM C3 and factor B were detected in this immune complex enriched serum fraction. These findings are a sign that circulating immune complexes can be detected even in the asymptomatic period of the disease.

Circulating immune complexes after splenectomy.

Circulating immune complexes were evaluated in 25 patients (age range 10 to 46 years) who had undergone splenectomy for non-malignant conditions by studying a polyethylene glycol insoluble serum fraction. Although the extent of binding to Clq was within normal limits, these patients had increased concentrations of factor B in the immune complex serum fraction. These findings indicate that an unusual type of circulating immune complex may be detected after splenectomy, suggesting a possible role for the spleen in the removal of circulating immune complexes.

Circulating immune complexes in sickle cell-beta zero thalassemia.

A serum fraction from patients with sickle cell-beta zero thalassemia prepared by treatment with polyethyleneglycol showed increased amounts of C1q-precipitable immune complexes, i.e., 216 micrograms/dl (range, 141-266 micrograms/dl) vs 181 micrograms/dl (range, 152-228 micrograms/dl) for controls (P less than 0.05), as well as increased amounts of protein. Levels of IgG, IgA, IgM, C3, C4 and factor B in the same fraction were within the normal range.

Circulating immune complexes in sickles cell-ß- thalassemia

A serum fraction from patients with sickle cell-betao thalassemia prepared by treatment with polyethyleneglycol showed increased amounts of C1q-precipitable immune complexes, i.e., 216 microng/dl (range, 141-266 microng/d) vs 181 microng/dl (range, 152-228 microng/dl) for controls (P<0.05), as well as increased amounts of protein. Levels of IgG, IgA, IgM, C3, C4 and factor B in the same fraction were within the normal range

Influence of antigen charge in the pathogenicity of immune complexes in rats.

Immune complexes (IC) formed in the presence of excess antigen with native human anionic (AA) or cationic (CA) albumin and anti-human albumin rabbit gamma globulin were administered to 51 female Wistar rats. In animals injected with IC formed with CA, IC deposition in the renal glomeruli (glomerular capillary walls and mesangium) occurred as early as 5 min after injection. These animals also showed slight alterations in renal structure and albuminuria, whereas in the animals injected with IC formed with AA there was no IC deposition in the renal glomeruli nor any alteration in renal structure or albuminuria. The serum complement levels of animals injected with IC formed with CA were significantly lower than those observed in animals treated with similar doses of IC formed with AA. In vitro experiments also showed that the IC formed with CA fixed more complement than those formed with AA.

Anticomplementary fraction from the poisonous secretion of the paratoid gland of the toad (Bufo marinus paracnemis Lutz).

Fractionation of the poisonous secretion of the toad Bufo marinus paracnemis Lutz, by dialysis and chromatography on QAE-Sephadex, led to the isolation of a fraction which was adsorbed to the ion exchanger. This fraction, when incubated with human serum, yielded an anticomplementary effect that was evaluated by measuring the kinetics of lytic activity on sensitized sheep red cells (classical pathway) and unsensitized rabbit cells (alternative pathway).

Experimental nephropathy induced by human polyclonal light chains.

We describe the nephrotoxic effects of a preparation of light chains (LC) obtained by reduction and alkylation of human IgG. The acute renal lesions in rats are dependent upon the dose and route of LC administration. The kidney alterations were not observed in animals treated with comparable amounts of human albumin or the F(ab')2 fragment of human IgG. Intravenous bolus injection of 120 mg LC into 80-100 g hydropenic rats induced extensive cast formation in distal and collecting renal tubules, which were similar to the human "myeloma kidney". In contrast, the slow infusion of the same amount of LC over 1 h produced degenerative changes in the proximal tubular cells without extensive cast formation. The morphological alterations of the kidney were investigated by classical histological methods, by immunofluorescence and in thin sections stained with toluidine blue. Urinary excretion and kidney content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase were increased only in the group of animals infused with LC. These findings may be relevant to the pathogenesis of human nephropathy occurring in multiple myeloma and in some autoimmune diseases.