Biomedical subjects
R De Maria
Publications and source records attributed to R De Maria.
Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1.
The production of red blood cells follows the sequential formation of proerythroblasts and basophilic, polychromatophilic and orthochromatic erythroblasts, and is promoted by the hormone erythropoietin (Epo) in response to tissue hypoxia. However, little is known about the negative regulation of this process. Death receptors are a family of surface molecules that trigger caspase activation and apoptosis in a variety of cell types. Here we show that immature erythroid cells express several death receptors whose ligands are produced by mature erythroblasts. Exposure of erythroid progenitors to mature erythroblasts or death-receptor ligands resulted in caspase-mediated degradation of the transcription factor GATA-1, which is associated with impaired erythroblast development. Expression of a caspase-resistant GATA-1 mutant, but not of the wild-type gene, completely restored erythroid expansion and differentiation following the triggering of death receptors, indicating that there is regulatory feedback between mature and immature erythroblasts through caspase-mediated cleavage of GATA-1. Similarly, erythropoiesis blockade following Epo deprivation was largely prevented by the expression of caspase-inhibitory proteins or caspase-resistant GATA-1 in erythroid progenitors. Caspase-mediated cleavage of GATA-1 may therefore represent an important negative control mechanism in erythropoiesis.
KDR receptor: a key marker defining hematopoietic stem cells.
Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.
Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis.
The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.
Fas/Fas ligand-driven T cell apoptosis as a consequence of ineffective thyroid immunoprivilege in Hashimoto's thyroiditis.
Hashimoto's thyroiditis (HT) is a chronic autoimmune disease resulting from Fas-mediated thyrocyte destruction. Although autocrine/paracrine Fas-Fas ligand (FasL) interaction is responsible for thyrocyte cell death during the active phases of HT, the role of infiltrating T lymphocytes (ITL) in this process is still unknown. Therefore, we investigated the expression and function of Fas and FasL in ITL. All ITL expressed high levels of Fas and CD69, an early marker of T cell activation associated with functional Fas expression in T cells in vivo. In contrast to thyrocytes that were found to produce high levels of FasL, ITL did not express significant amounts of FasL, suggesting that ITL are not directly involved in thyrocyte destruction. The analysis of ITL purified from HT thyroids showed that ITL were massively killed by Fas crosslinking and that a considerable number (24-36%) underwent spontaneous apoptosis within 36 h of culture. Accordingly, in situ TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining revealed that a significant number (10-15%) of ITL in proximity to FasL-producing thyroid follicles were apoptotic. Moreover, virtually all ITL in proximity to thyroid follicles were preapoptotic, as they expressed high levels of GD3 ganglioside, a killer glycolipid responsible for the generation of irreversible apoptotic signals that accumulate in hematopoietic cells shortly after Fas crosslinking. These data demonstrate that ITL are not directly involved in thyrocyte cell death during HT, suggesting that autocrine/paracrine Fas-FasL interaction is a major mechanism in autoimmune thyrocyte destruction.
Lipid and glycolipid mediators in CD95-induced apoptotic signaling.
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Caspase activation without death.
Since molecular cloning of the C. elegans ced-3 gene revealed its homology with mammalian IL-1beta-converting enzyme,1 14 members of the caspase family have been identified, which have often been involved as mediators of one or more phases of the apoptotic process. 2,3 However, an over-simplified role of these proteases may be insufficient to explain the usually constitutive expression of such a large and complex family of enzymes, many of which display overlapping specificity. In addition to the well-established role of caspase-1 in the production of active IL-1beta and IL-18 in inflammation,4 an increasing number of reports has recently suggested that caspases may have a function outside of apoptosis. In this review, the situations in which cells survive despite the presence of activated caspases in their cytoplasm will be examined and discussed, with the intent to gather all recent advances in this new field that promises to be a focus for caspase research in the near future.
Induction of Ca2+ flux by adhesion molecules in lymphocytes.
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Involvement of Fas/FasL system in the pathogenesis of autoimmune diseases and Wilson's disease.
The interaction of Fas with FasL has been demonstrated to be implicated in the pathogenesis of several autoimmune and liver diseases. Recently, attention has been focused on the hypothesis that thyrocytes and beta cells undergo massive Fas/FasL-mediated apoptosis during autoimmune response. Similarly, hepatocyte cell death occurring following copper accumulation points towards the same mechanism.
Gene regulation in normal and leukaemic progenitor/stem cells.
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Acidic sphingomyelinase (ASM) is necessary for fas-induced GD3 ganglioside accumulation and efficient apoptosis of lymphoid cells.
Ceramides deriving from sphingomyelin hydrolysis are important mediators of apoptotic signals originating from Fas (APO-1/CD95). However, definitive evidence for the role played by individual sphingomyelinases is still lacking. We have analyzed lymphoblastoid cell lines derived from patients affected by Niemann Pick disease (NPD), an autosomal recessive disorder caused by loss-of-function mutations within the acidic sphingomyelinase (ASM) gene. NPD lymphoblasts, which display normal neutral sphingomyelinase activity, fail to activate ASM in response to Fas cross-linking, unlike normal lymphoblasts. NPD lymphoblasts also fail to accumulate GD3 ganglioside, a downstream mediator of ceramide-induced cell death (De Maria, R., L. Lenti, F. Malisan, F. D'Agostino, B. Tomassini, A. Zeuner, M.R. Rippo, R. Testi. 1997. Science. 277:1652-1655), and display a substantially inefficient apoptosis after Fas cross-linking. Inefficient apoptosis is due to lack of ASM activity, because proximal signaling from Fas in NPD lymphoblasts is not impaired and apoptosis can be efficiently triggered by passing the ASM defect with exogenous ceramides. Moreover, mannose receptor-mediated transfer of ASM into NPD lymphoblasts rescues their ability to transiently activate ASM, accumulate GD3, and rapidly undergo apoptosis after Fas cross-linking. These results provide definitive genetic evidence for the role of ASM in the progression of apoptotic signals originating from Fas.
Beta-galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliosidosis.
A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low beta-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus conmmunis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, beta-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total beta-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of GM1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of GM1-ganglioside band was 98.52% of all stained bands (GD1a, GD1b, GT1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat GM1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of beta-galactosidase deficiency do not completely fit with those of human type II GM1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline GM1-gangliosidosis.
Super-normal 201Tl retention in hibernating myocardium: an ex-vivo study using the failing human heart.
OBJECTIVE: Although the relationship between delayed 201Tl distribution and blood flow in acutely ischemic and infarcted myocardium has been widely explored in the experimental setting, its behaviour in chronically hypoperfused dysfunctioning human myocardium has not yet been evaluated. METHODS: In tissue samples of excised failing hearts taken from ischemic (IHD) patients and idiopathic dilated cardiomyopathy (IDC) controls, we evaluated the relationship between delayed 201Tl retention (4 h redistribution), blood flow (assessed by means of 99mTc-labelled human albumin microspheres injected during transplantation) and biochemically-assessed fibrosis. 201Tl activity was expressed as the percent of the activity in the region with highest flow and the least fibrosis. RESULTS: Fibrosis and 201Tl activity were inversely related (r = -0.62, P = 0.0001). In IDC controls, low flows corresponded to uniformly preserved 201Tl retention. In IHD, 46 segments with flows < or = 0.60 ml.min-1.g-1 and 20 segments with flows > 0.60 ml.min-1.g1 showed matching delayed 201Tl retention and flow values; in the remaining 27, there was a disproportionately high tracer accumulation in comparison with flow (flow/201Tl mismatch). Despite significantly less fibrosis and lower flows, the mismatch segments showed significantly greater. 201Tl activity than the segments with concordantly high tracer retention and flow values. Conversely, at equivalent flow rates, the mismatch regions had less fibrosis than the areas with concordantly depressed 201Tl activity and perfusion. CONCLUSIONS: This super-normal 201Tl retention in hibernating myocardium may indicate a mechanism of cell adaptation to chronic hypoperfusion.
Tumour grading and the one-year post-surgical prognosis in feline mammary carcinomas.
To investigate whether the degree of differentiation in feline mammary carcinoma (FMC) can indicate the post-surgical survival time (PST), tumours were surgically resected from 55 cats and histologically graded according to a method derived from human breast cancer studies. One year after the resection, 26 cats (47.3%) were alive while 29 (52.7%) had died as a consequence of FMC. Formalin-fixed, paraffin wax-embedded sections stained with haematoxylin and eosin were used to classify the FMCs initially, according to the WHO system. Histological grading was then performed on the basis of three main features: degree of tubule formation, nuclear and cellular pleomorphism, and accurate mitotic count obtained from a defined area. Immunohistochemical examination with an anti-actin antibody was used for the accurate detection of "in situ" carcinomas. Age and histological type were not significantly correlated with the PST. Seven tumours (12.7%) were graded as well-differentiated carcinoma (WDC; grade I), 33 (60%) as moderately differentiated carcinoma (MDC; grade II), and 15 (27.3%) as poorly differentiated carcinoma (PDC; grade III). The tumour-related death rates after the first post-surgical year were 0 in cats with WDC, 14 (42.4%) in those with MDC, and 15 (100%) in those with PDC. Six cats with tumours showing extensive myoepithelial differentiation were all alive after 1 post-surgical year. The grading system seemed to have a good predictive value in respect of grades I and III of FMC but not for grade II. Myoepithelial differentiation may be relevant to clinical prognosis in FMC.
Fas-FasL interactions: a common pathogenetic mechanism in organ-specific autoimmunity.
Organ-specific autoimmunity is characterized by the accelerated loss of selected cell types, resulting in specific tissue destruction and disease. The role of different genetic or environmental factors in initiating the autoimmune reactivity is still unclear. However, novel mechanisms responsible for tissue destruction have recently been revealed. Here, Ruggero De Maria and Roberto Testi propose that Fas ligand may represent a common weapon during the destructive phase of organ-specific autoimmunity.
Long-term survival effect of metoprolol in dilated cardiomyopathy. The SPIC (Italian Multicentre Cardiomyopathy Study) Group.
OBJECTIVE: To evaluate the additive effect of metoprolol treatment on long-term incidence of fatal and non-fatal cardiac events in idiopathic dilated cardiomyopathy. DESIGN: 586 patients with idiopathic dilated cardiomyopathy were prospectively enrolled in a multicentre registry and followed up for a mean (SD) of 52 (32) months. Metoprolol, carefully titrated to the maximum tolerated dose, was added to conventional heart failure treatment in 175 patients. RESULTS: Survival and transplant-free survival at seven years were significantly higher in the 175 metoprolol treated patients than in the remaining 411 on standard treatment (81% v 60%, p < 0.001, and 69% v 49%, p < 0.001, respectively). By multivariate analysis, metoprolol independently predicted survival and transplant-free survival (relative risk reduction values for all cause mortality and combined mortality or transplantation 51% (95% confidence interval 21% to 69%), p = 0.002, and 34% (5% to 53%), p = 0.01, respectively). New York Heart Association class, left ventricular end diastolic diameter, and pulmonary wedge pressure were also predictive. Seven year survival (80% v 62%, p = 0.004) and transplant-free survival (68% v 51%, p = 0.005) were significantly higher in 127 metoprolol treated cases than in 127 controls selected from the entire control cohort and appropriately matched. Metoprolol was associated with a 30% reduction in all cause mortality (7% to 48%, p = 0.015) and a 26% reduction in mortality or transplantation (7% to 41%, p = 0.009). CONCLUSIONS: The addition of metoprolol to standard heart failure treatment, including angiotensin converting enzyme inhibitors, was effective in the long-term, reducing both all cause mortality and transplantation in patients with idiopathic dilated cardiomyopathy.
In response to Milano A. Pratali S, De Carlo M, Borzoni G, Tartarini G, Bortolotti U. Ascending aorta dissection after aortic valve replacement. J Heart Valve Dis 1998; 7:75-80.
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Nitric oxide primes pancreatic beta cells for Fas-mediated destruction in insulin-dependent diabetes mellitus.
Fas is an apoptosis-inducing surface receptor involved in controlling tissue homeostasis and function at multiple sites. Here we show that beta cells from the pancreata of newly diagnosed insulin-dependent diabetes mellitus (IDDM) patients express Fas and show extensive apoptosis among those cells located in proximity to Fas ligand-expressing T lymphocytes infiltrating the IDDM islets. Normal human pancreatic beta cells that do not constitutively express Fas, become strongly Fas positive after interleuken (IL)-1beta exposure, and are then susceptible to Fas-mediated apoptosis. NG-monomethyl-L-arginine, an inhibitor of nitric oxide (NO) synthase, prevents IL-1beta-induced Fas expression, whereas the NO donors sodium nitroprusside and nitric oxide releasing compound (NOC)-18, induce functional Fas expression in normal pancreatic beta cells. These findings suggest that NO-mediated upregulation of Fas contributes to pancreatic beta cell damage in IDDM.