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Biomedical subjects

M de Sousa

Publications and source records attributed to M de Sousa.

At least 37 records · Page 2Linked to original sources
PubMed1998

HFE mutations in patients with hereditary haemochromatosis in Sweden.

OBJECTIVE: To determine the frequency of mutations (C282Y and H63D) in a newly identified gene HFE in patients with hereditary haemochromatosis (HH) in Sweden. DESIGN: Molecular genetic analyses of the HFE gene (polymerase chain reaction (PCR) followed by enzyme restriction) were performed in genomic DNA from unrelated patients with a clinical diagnosis of HH and in healthy subjects. SETTINGS: Patients with HH treated with phlebotomies at Karolinska Hospital and Huddinge Hospital were analyzed. SUBJECTS: Eighty-seven unrelated patients with HH and 117 healthy controls. RESULTS: It was found that the HFE C282Y mutation occurs in 94.2% of chromosomes from patients with HH. Eighty patients (92.0%) were homozygous for the C282Y mutation and one was heterozygous. Three patients were heterozygous for both C282Y and H63D mutations. One patient was homozygous and one was heterozygous for the H63D mutation. One patient carried normal alleles. In healthy controls, the C282Y mutation occurred in nine subjects (7.7%), all of which were heterozygous. The H63D mutation was found in 28 control subjects, one of which was homozygous. CONCLUSIONS: We found that the majority of patients with HH have the C282Y mutation in the HFE gene. The frequency of the H63D mutation was higher in controls than in patients with HH, although in chromosomes at risk the frequency of the H63D mutation was higher in patients.

Adult↗
PubMed1997

Restoration of lectin activity to a non-glycosylated ricin B chain mutant by the introduction of a novel N-glycosylation site.

Ricin B chain (RTB) is an N-glycosylated, galactose-specific lectin. Removal of the two native N-glycosylation sites at Asn95 and Asn135 by site-directed mutagenesis generated a recombinant protein devoid of lectin activity. Two novel N-glycosylation sites were introduced into RTB at Asn42 and Asn123, either singly or in combination. Microinjection of pre-RTB transcripts into Xenopus oocytes showed that these novel sites became glycosylated in vivo. The single oligosaccharide site chain at Asn42 restored lectin activity to RTB, whereas glycosylation at Asn123 or simultaneous glycosylation at Asn42 and Asn123 failed to do so.

Animals↗
PubMed1997

Relative impact of HLA phenotype and CD4-CD8 ratios on the clinical expression of hemochromatosis.

Hemochromatosis is a hereditary iron-overload disease linked to HLA. The clinical expression of hemochromatosis is influenced by sex and age. However, other factors must account for the notorious heterogeneity of expression of the disease independent of sex, age, and HLA phenotype. The present study attempts to clarify some of these additional factors based on exhaustive statistical analysis of data collected from 43 selected patients with hemochromatosis. The statistical analysis focused on three groups of variables: the first group included variables reflecting the clinical expression of the disease; the second group represented the biochemical and hematological values at the time of diagnosis; and the third group consisted of the independent variables sex, age, HLA phenotype, and T-cell subset profile, i.e., the percentages and total numbers of CD4+ and CD8+ cells and the CD4-CD8 ratios. The results show that the relative expansion of the two main T-cell subsets, in the context of the HLA phenotype, correlates significantly with the clinical expression of hemochromatosis and the severity of iron overload. The present findings substantiate further the postulate that T cells have a role in the regulation of iron metabolism.

Adult↗
PubMed1997

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28- T cells.

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8+ CD28- T cells with a corresponding reduction in the percentage of CD8+ CD28+ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4+ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8+ CD28+ T cell population 'to expand', coinciding with an 'expansion' of CD8+ CD28- T cells in peripheral blood of HLA-A3+ but not HLA-A3- HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR+ but not CD45RO+ cells was also found within the peripheral CD8+ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8+ CD28+ T cells both in controls and HH were able to expand in vitro; (ii) that CD8+ CD28- T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8+ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard 51Cr-release assays when compared with CD8+ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8+ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.

Adult↗
PubMed1997

Engineering protein mechanics: inhibition of concerted motions of the cellular retinol binding protein by site-directed mutagenesis.

Recently we reported on the dynamic properties of the cellular retinol binding protein, a member of the fatty acid binding protein family. A few conserved glycines were identified as important for producing the conformational changes necessary for the uptake and release of retinol. Here, we describe a multidisciplinary analysis of a genetically engineered mutation of one of these glycines (Gly67), designed to inhibit an observed hinge bending motion. The correctly folded mutant protein is unable to bind retinol. Analysis of the molecular dynamics simulations of the mutant and wild type protein using the essential dynamics method shows that the mutation indeed inhibits the hinge bending motions which are important for retinol binding.

Binding Sites↗
PubMed1997

In vivo mucosal uptake, mucosal transfer and retention of iron in mice.

An improved and sensitive method for studying iron absorption in mice with alterations in body iron stores is described. Mice with varying iron status were given a double isotope-labelled test dose containing 59Fe and 51Cr as a non-absorbable indicator, via an oroesophageal needle. Using a whole-body counter it was possible to measure in vivo the initial mucosal iron uptake and long-term iron retention and to calculate mucosal iron transfer. A significant difference was demonstrated between normal and both anaemic and dietary iron-loaded mice with regard to the various steps of iron absorption. When mice were tested twice for iron absorption, the results were highly reproducible. In conjunction with other parameters, the method described is useful in studying the mechanism and the regulation of iron absorption in mice.

Absorption↗
PubMed1996

Defective iron homeostasis in beta 2-microglobulin knockout mice recapitulates hereditary hemochromatosis in man.

Previously, hepatic iron overload resembling that in hereditary hemachromatosis (HH) has been found in beta 2-microglobulin knockout (beta 2m-/-) mice. We have now characterized iron metabolism in beta 2m-/- mice. The mutant mice fail to limit the transfer of iron from mucosal cells into the plasma. Transferrin saturation is abnormally high. Pathologic iron depositions occur predominantly in liver parenchymal cells. Reconstitution with normal hematopoietic cells redistributes the iron from parenchymal to Kupffer cells, but does not correct the mucosal defect. We conclude that (a) iron metabolism is defective in the gut mucosa as well as the liver of beta 2m-/- mice; and (b) a beta 2m-dependent gene product is involved in iron homeostasis. Recently, a novel gene of the major histocompatibility complex class I family, HLA-H, has been found to be mutated in a large proportion of HH patients. Our data provide functional support for the proposed causative role of HLA-H mutations in HH.

Absorption↗
PubMed1996

Cobalt-chromium-molybdenum but not titanium-6aluminium-4vanadium alloy discs inhibit human T cell activation in vitro.

This study describes the effect of the presence of cobalt-chromium-molybdenum (CoCrMo) and titanium-6aluminium-4vanadium (Ti6AL4V) disc samples on the CD3-mediated in vitro response of human peripheral blood T lymphocytes. Lymphocyte proliferation in the presence and absence of these metal alloy discs was measured by [3H]thymidine incorporation. Inhibition of lymphocyte proliferation was observed in the presence of CoCrMo disc samples. In contrast, the presence of the Ti6AL4V metal alloy discs had no effect on T cell proliferation. Ultrastructural studies using scanning electron microscopy revealed that the differences in the number of blast cells on uncoated CoCrMo and Ti6AL4V discs from a 4 day culture were consistent with the results observed in the proliferation experiments, i.e. fewer blast cells were seen on the CoCrMo than on the Ti6AL4V discs. In addition, a quantitative analysis of trace elements using total reflection X-ray fluorescence spectrometry in supernatants from 68 h in vitro cultures containing Ti6AL4V or CoCrMo disc samples was performed, revealing differences in the relative metal concentrations in the culture conditions tested. These differences point to the presence of cobalt in the supernatants as a possible determining factor of the inhibition observed. Because cell viability did not appear to change, a more complex mechanism involving the interaction of metals with T lymphocytes may account for the results obtained.

Alloys↗
PubMed1995

Anti-TNF-alpha treatment down-regulates the expression of fibronectin and decreases cellular infiltration of cardiac allografts in rats.

Extracellular matrix (ECM) components provide costimulatory signals for T cell activation in vitro, and may be critical for lymphocyte migration and tissue positioning in vivo. We conducted a series of studies in rat recipients of cardiac allografts to evaluate intragraft expression of a prominent ECM protein, fibronectin (FN), and to analyze the effects of infusing a neutralizing anti-TNF-alpha serum on FN expression and lymphocyte migration into the transplants. LBNF1 cardiac allografts were rejected within 8 days in control LEW rats. A prominent immunohistochemical feature of this immune response was the dense deposition of FN at the graft site as early as 3 h, which then peaked at 4 to 6 days. The early 3-h FN deposition (likely plasma FN) was noted before cellular infiltration. Northern blot analysis established that a marked induction of FN mRNA expression occurred in rejecting cardiac allografts at day 4 after transplantation. To determine the source of FN mRNA, we conducted a series of in situ hybridization studies with probes for FN and lysozyme, a macrophage-specific marker. Indeed, the majority of graft-infiltrating cells expressed lysozyme mRNA and FN mRNA. Administration of anti-TNF-alpha serum into LEW hosts (0.5 ml i.v. at days 1 and 3 only) abrogated acute rejection and prolonged cardiac allograft survival to approximately 13 days. This was accompanied by depressed circulating and intragraft TNF-alpha levels, and markedly down-regulated FN mRNA/protein expression patterns, as compared with those in recipients given nonimmune rabbit serum. Anti-TNF-alpha treatment also markedly decreased graft infiltration by ED1+ monocytes/macrophages, OX-8+, and VLA-4+ cells, normally peaking at 4 days. Moreover, we found that the migration of 111In-labeled specifically sensitized lymph node lymphocytes to cardiac allografts in secondary rat recipients conditioned with anti-TNF-alpha serum was significantly decreased, as compared with that in controls. Thus, FN expression by intragraft macrophages occurs within the same interval as cellular infiltration, and may act as an ECM component "signal" for selective homing of recirculating lymphocytes in graft recipients. The results of this study support the notion that in vivo interactions between mononuclear cells and ECM may be vital for the ingress of alloreactive lymphocytes at the graft site, and offer potential novel sites for therapeutic intervention in the control of transplant rejection.

Animals↗
PubMed1995

Iron differentially modulates the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes.

Clinical and experimental studies performed in situations of iron overload have demonstrated that iron impairs several T-cell functions. We have examined the effect of iron in the form of ferric citrate on the CD4-lck and CD8-lck complexes in view of the key role played by the tyrosine kinase p56lck in regulating T-cell functions. Ferric citrate was seen to differentially modulate the CD4-lck and CD8-lck complexes in resting peripheral blood T-lymphocytes (PBLs) cultured in the presence of this metal salt for periods of 20 to 24 hr. Thus, whereas ferric citrate invariably induced a marked decrease in the in vitro activity of the CD4-associated lck by three- to fourfold at 100 microM (P < 3 x 10(-5)), it did not affect significantly the in vitro activity of the CD8-associated lck, although modest decreases were observed in some experiments. Immunoprecipitation and subsequent lck-immunoblotting revealed that the marked decrease in CD4-lck activity induced by 100 microM of ferric citrate was due to a decrease in the amount of p56lck on CD4 immunoprecipitates. Furthermore, flow cytometry analysis showed a decrease in the surface expression of the CD4 molecule in iron-treated PBLs, as judged by a decrease in the mean fluorescence intensity (MFI), that was accompanied by a decrease in the percentage of CD4+ T-lymphocytes. In marked contrast, whereas the surface expression of the CD8 molecule was slightly decreased, the percentage of CD8+ T-lymphocytes remained constant. This differential effect of ferric citrate on the CD4+ and CD8+ T-cell subsets led to a marked decrease in the CD4/CD8 ratios in iron-treated PBLs after the 20- to 24-hr period (P < 0.001). The present results indicate that iron in the form of ferric citrate can modulate key molecules involved in the process of T-cell activation and therefore influence T-cell-mediated functions.

CD4 Antigens↗
PubMed1994

In vitro modulation of T-cell surface molecules by iron.

Iron and zinc are known to have immunomodulatory functions. In the present work, iron (FeC6H5O7) and zinc (ZnCl2) were tested in comparison to nickel (NiCl2) and cobalt (CoCl2) for their effect on six different surface molecules known to be involved in recognition and activation processes, namely CD4, CD2, CD3, CD8, HLA-ABC, and HLA-DR. Iron was seen to down-modulate expression of the CD4 and the CD2 molecules on the surface of T-lymphocytes, as indicated by a decrease in the mean fluorescence intensity measured by FACS analysis. None of the other T-cell molecules tested were significantly affected. In addition, the iron-mediated CD4 down-modulation reached its lowest level by 12 hr, at which time a striking decrease in the percentage of CD4+, but not CD8+, cells was observed. This decrease was followed by a gradual recovery starting at 18 hr and reaching its highest level at 48 hr. When cells were treated with other metal salts, none of those effects were observed. The present results suggest that iron may play a regulatory role in processes of T-cell recognition and T-cell activation by selectively down-modulating T-cell molecules known to be involved in these processes.

CD4 Antigens↗
PubMed1994

The expression of extracellular matrix proteins during accelerated rejection of cardiac allografts in sensitized rats.

Extracellular matrix (ECM) proteins exert comitogenic effects on T cell activation in vitro, and may play a role in selective lymphocyte migration and tissue positioning in vivo. However, putative roles of ECM in host immune responses leading to graft rejection remain largely obscure. The present study evaluates intragraft expression of prominent ECM components, laminin (LN) and fibronectin (FN), and analyzes their interactions with host cellular repertoire in a well-defined cardiac allograft model in sensitized rats. (LEW x BN)F1 cardiac allografts are rejected within 24 hr in sensitized LEW rats. Immunohistochemical analysis has revealed that this brisk rejection response was associated with an early increase in the intermyocyte and endothelial deposition of LN and FN at the graft site, with their peak at 6 hr after transplantation. The upregulation of ECM preceded intragraft cellular infiltration, which at 6 hr consisted primarily of monocytes and macrophages. The infiltrating cells localized selectively in FN-rich cardiac interstitial and perivascular areas, as documented by two-color staining and laser scanning confocal microscopy. Marginal and transient increase in ECM expression was noted within control isografts. Next, we tested the role of ECM proteins in systemic lymphocyte recirculation. Specifically sensitized lymph node lymphocytes (LNL) were labelled in vitro with a DNA-binding fluorochrome, H33342, and injected i.v. into secondary engrafted recipients, which were sacrificed 6 hr later. These LNL were detected within host cervical lymph nodes in close association with FN deposits. Moreover, LNL labeled in vitro with a cationic membrane-binding fluorochrome, Di "I", were traced in hosts the lymph nodes of which were analyzed for simultaneous detection of LN and FN. Again, transferred LNL migrated selectively to FN-containing compartments, as shown by laser scanning confocal microscopy. Thus, ECM proteins should be regarded as active and important participants in host immune responses leading to graft rejection. FN may act as an ECM component "signal" for graft-infiltrating cells, and may play a key role in selective homing and specific tissue positioning of recirculating specifically sensitized lymphocytes in host peripheral lymphoid tissues.

Animals↗
PubMed1994

Iron overload in beta 2-microglobulin-deficient mice.

The present paper describes the results of a comparative histological and quantitative analysis of iron distribution in tissues of beta 2m-/- and beta 2m+/- mice of different ages. Progressive hepatic iron overload, indistinguishable from that observed in human hemochromatosis, was found only in mice homozygous for the mutated beta 2m gene. Total iron measurements done by flame atomic absorption showed statistically significant differences between liver samples from 5 beta 2m+/- heterozygotes (468 +/- 174 micrograms/g of dry weight) and 9 mice homozygous for the mutated beta 2m gene with average total hepatic iron levels of 1583 +/- 423 micrograms/g of dry weight.

Animals↗
PubMed1994

Lymphocyte traffic and positioning in vivo: an expanded role for the ECM, the VLA proteins and the cytokines.

The continuous circulation of lymphocytes between blood and lymph, constitutes, with the specific recognition of foreign antigens and with memory, one of the hallmarks of the immunological system. In the routine process of recirculating between blood and lymph in adult life, lymphocytes display the unique capacity of recognizing "self within self". The physiological recognition of "self within self" is expressed at three levels: (1) an overall ability to distinguish between peripheral lymphoid and non-lymphoid organs, (2) the ability of lymphocyte subpopulations to discriminate between peripheral lymphoid organs, (3) a fine recognition of distinct microenvironments within the peripheral lymphoid organs. Historically, interest in regulation of entry into the lymph dominated this field, focusing primarily on the interaction of lymphocytes with post-capillary venules with high endothelium found in some higher vertebrate lymph nodes. Lymphocytes, however, enter the lymph in species without lymph nodes, and lymphocytes recirculation is well established in fetal life. Regardless of route of entry, lymphocytes in birds, fish, rodents, and humans enter the peripheral lymphoid organs and display the capacity to segregate and arrange themselves in distinct territories, a phenomenon called "ecotaxis". This paper reviews the evidence for the relative contribution of so called specialized lymphocyte endothelium interactions and other interactions, to the physiological regulation of lymphocyte traffic and positioning. Of the latter, interactions of lymphocytes with the ECM appear of some significance for two reasons: there is a selective distribution of some ECM components in peripheral lymphoid organ areas, as recently shown for tenascin in the thymus-dependent zones (Chilosi et al., Am J Pathol 143: 1348-1355, 1993). This selective distribution may serve as a basis for preferential T cell migration through those zones. The abnormal expression of tenascin in non-lymphoid organs with autoimmune lesions may serve as an identical basis for abnormal T lymphocyte migration in autoimmunity (Chilosi, personal communication). Additional evidence for abnormal cell-ECM interactions playing a role in autoimmune-like lesions comes from the recent observations in TGF-beta 1 knockout mice, in which mononuclear cell infiltration of the heart and lungs has been corrected by the systemic administration of synthetic FN peptides (Hines at al., PNAS 91:5187-5191, 1994). Changes in expression of ECM components have aso being described in rat heart allografts preceding lymphocyte accumulation in the process of allograft rejection (Coito et atl. Transplantation 57:599-605, 1994).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Decreased CD8-p56lck activity in peripheral blood T-lymphocytes from patients with hereditary haemochromatosis.

Hereditary haemochromatosis (HH) is an autosomal recessive disease linked to certain MHC class-I specificities. The disease is characterized by increased iron absorption and, in some patients, abnormally low numbers of CD8+ T cells in the periphery. We were interested in whether CD4- and CD8-associated p56lck kinase activities were altered in patients with HH. In a study of 18 patients with HH (with and without low numbers of CD8+ cells), the level of autophosphorylation of the CD8-associated p56lck as well as its phosphotransferase activity, as determined by phosphorylation of an exogenous substrate, was significantly reduced by two- to three-fold relative to a control population of 23 healthy blood donors (P < 6 x 10(-7). CD8-p56lck activity was decreased in 16 out of 18 patients (ranging from 1.5- to 10-fold decrease). By contrast, the level of CD4-p56lck activity did not show an overall decrease relative to controls. In addition to an occasional decrease in the amount of CD8-associated lck, HH patient-derived T cells showed a consistent decrease in the relative CD8-p56lck specific activity. Immunofluorescence staining showed further that the difference could not be accounted by a discrepancy in the expression of CD8 alpha alpha or CD8 alpha beta complexes or MHC class I molecules. Decreased CD8-p56lck activity was seen both in patients undergoing intensive phlebotomy treatment and in patients in maintenance therapy (i.e. patients who had reached normal levels of iron stores), indicating that this abnormality does not appear to be corrected by iron depletion. To our knowledge, this is the first demonstration of an abnormality in a src-like receptor associated kinase in a human disease state linked to MHC class-I antigens.

Adult↗
PubMed1994

Haemochromatosis as a window into the study of the immunological system: a novel correlation between CD8+ lymphocytes and iron overload.

In the present study we report a serial investigation of the numbers of the peripheral blood cells--erythrocytes, polymorphonuclear neutrophils, total lymphocytes, T-lymphocyte subpopulations (CD2, CD4, CD8), B lymphocytes and monocytes--in a group of 21 patients with haemochromatosis during the time of intensive phlebotomy treatment, i.e., from iron overload until the onset of iron deficiency. A remarkable individual stability of all blood cell populations studied was found in all patients. Patients differed in their relative proportions of CD4+ and CD8+. Each individual's CD4/CD8 ratio, as well as the absolute numbers, remained unaffected with time, confirming the existence of a strict homeostatic regulation of the relative numbers of the two major peripheral T lymphocytes. A significant positive correlation between CD4/CD8 ratios and the amount of iron mobilised by phlebotomy was found during this study. A novel correlation between the relative proportions of CD4+ and CD8+ cells and iron overload is confirmed by the follow-up of iron re-entry in the serum transferrin pool in the treated patients.

Adult↗