Patients with Lemierre syndrome have a high risk of new thromboembolic complications, clinical sequelae and death: an analysis of 712 cases.

BACKGROUND: Lemierre syndrome is characterized by head/neck vein thrombosis and septic embolism usually complicating an acute oropharyngeal bacterial infection in adolescents and young adults. We described the course of Lemierre syndrome in the contemporary era. METHODS: In our individual-level analysis of 712 patients (2000-2017), we included cases described as Lemierre syndrome if these criteria were met: (i) primary site of bacterial infection in the head/neck; (ii) objectively confirmed local thrombotic complications or septic embolism. The study outcomes were new or recurrent venous thromboembolism or peripheral septic lesions, major bleeding, all-cause death and clinical sequelae. RESULTS: The median age was 21 (Q1-Q3: 17-33) years, and 295 (41%) were female. At diagnosis, acute thrombosis of head/neck veins was detected in 597 (84%) patients, septic embolism in 582 (82%) and both in 468 (80%). After diagnosis and during in-hospital follow-up, new venous thromboembolism occurred in 34 (5.2%, 95% CI 3.8-7.2%) patients, new peripheral septic lesions became evident in 76 (11.7%; 9.4-14.3%). The rate of either was lower in patients who received anticoagulation (OR: 0.59; 0.36-0.94), higher in those with initial intracranial involvement (OR: 2.35; 1.45-3.80). Major bleeding occurred in 19 patients (2.9%; 1.9-4.5%), and 26 died (4.0%; 2.7-5.8%). Clinical sequelae were reported in 65 (10.4%, 8.2-13.0%) individuals, often consisting of cranial nerve palsy (n = 24) and orthopaedic limitations (n = 19). CONCLUSIONS: Patients with Lemierre syndrome were characterized by a substantial risk of new thromboembolic complications and death. This risk was higher in the presence of initial intracranial involvement. One-tenth of survivors suffered major clinical sequelae.

Diagnosis and treatment of inherited thrombocytopenias.

Knowledge in the field of inherited thrombocytopenias (ITs) has greatly improved over the last 15 years. Several new genes responsible for thrombocytopenia have been identified leading to the definition of novel nosographic entities and to a much better characterization of the phenotypes of these diseases. To date, ITs encompass 22 disorders caused by mutations in 24 genes and characterized by different degrees of complexity and great variability in prognosis. Making a definite diagnosis is important for setting an appropriate follow-up, choosing the best treatments and providing proper counseling. Despite the abovementioned progress, diagnosis of ITs remains difficult and these disorders are still underdiagnosed. The purpose of this review is to provide an updated guide to the diagnosis of ITs based on simple procedures. Moreover, the currently available therapeutic options for these conditions are recapitulated and discussed.

R705H mutation of MYH9 is associated with MYH9-related disease and not only with non-syndromic deafness DFNA17.

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disease caused by mutation of MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (NMMHC-IIA). MYH9-RD patients have macrothrombocytopenia and granulocyte inclusions (pathognomonic sign of the disease) containing wild-type and mutant NMMHC-IIA. During life they might develop sensorineural hearing loss, cataract, glomerulonephritis, and elevation of liver enzymes. One of the MYH9 mutations, p.R705H, was previously reported to be associated with DFNA17, an autosomal dominant non-syndromic sensorineural hearing loss without any other features associated. We identified the same mutation in two unrelated families, whose four affected individuals had not only hearing impairment but also thrombocytopenia, giant platelets, leukocyte inclusions, as well as mild to moderate elevation of some liver enzymes. Our data suggest that DFNA17 should not be a separate genetic entity but part of the wide phenotypic spectrum of MYH9-RD characterized by congenital hematological manifestations and variable penetrance and expressivity of the extra-hematological features.

Thrombopoietin receptor agonists in hereditary thrombocytopenias.

Hereditary thrombocytopenias (HTPs) constitute a heterogeneous group of diseases characterized by a reduction in platelet count and a potential bleeding risk. As a result of advances in diagnostic methods, HTPs are increasingly being identified, and appear to be less rare than previously thought. Most HTPs do not have effective treatments, except for platelet transfusion when bleeding occurs and in preparation for procedures associated with a risk of bleeding. Preliminary clinical evidence suggests that thrombopoietin receptor agonists (TPO-RAs) with an established use in the treatment of certain acquired thrombocytopenias are well tolerated and provide clinical benefits in patients with some forms of HTP. These drugs may therefore be considered for the treatment of HTPs in clinical practice. However, caution and close monitoring are recommended, owing to the absence of long-term safety data and the potential risks posed by prolonged bone marrow stimulation in certain HTPs. In this review, we summarize the available clinical data on TPO-RAs in the treatment of HTPs, and discuss their use in patients with these disorders. We believe that TPO-RAs will play a major role in the treatment of HTPs, particularly myosin heavy chain 9-related disease, Wiskott-Aldrich syndrome, X-linked thrombocytopenia, and thrombocytopenia caused by THPO mutations.

Defective platelet responsiveness to thrombin and protease-activated receptors agonists in a novel case of gray platelet syndrome: correlation between the platelet defect and the alpha-granule content in the patient and four relatives.

BACKGROUND: We report a novel case of gray platelet syndrome (GPS). A 14-year-old boy had bleeding diathesis, mild thrombocytopenia, giant platelets with severe defect of alpha-granule secretory proteins, myelofibrosis and splenomegaly. METHODS AND RESULTS: Platelet function studies showed a marked reduction of aggregation and Ca(2+) mobilization by thrombin, protease-activated receptor 1 (PAR1)-activating peptide (AP) and PAR4-AP, PAR1 expression at 55% of normal levels, and a more than two hundred fold reduction of in vitro whole-blood thromboxane B(2) (TXB(2)) production. Sequencing of coding regions of the PAR1 gene failed to show abnormalities. This patient was initially classified as a sporadic case of GPS, as electron microscopy failed to identify giant platelets and/or alpha-granule deficiency in his relatives. However, further studies on the father and three other relatives showed a relative lack of platelet alpha-granule proteins by immunofluorescence microscopy, a defective platelet response to PAR4-AP, and severely reduced in vitro whole-blood TXB(2) production. On this basis, we suggest that in this family, GPS was transmitted in a dominant fashion with highly variable penetrance. CONCLUSIONS: Our study suggests that current diagnostic criteria fail to identify some patients with a mild GPS phenotype and that such patients might be identified by the methods cited above. It also better characterizes the pathogenesis of defective platelet responses to thrombin, and raises interesting questions on the correlation between abnormal PAR function and the lack of alpha-granule content in GPS.

Inherited thrombocytopenia caused by ANKRD26 mutations misdiagnosed and treated as myelodysplastic syndrome: report on two cases.

Essentials Thrombocytopenia 2 (THC2) is an inherited thrombocytopenia (IT) with dysmegakaryopoiesis. Physicians often do not suspect the genetic origin of thrombocytopenia in patients with THC2. We report two THC2 patients misdiagnosed with myelodysplasia and treated with chemotherapy. IT should be always considered in patients with isolated thrombocytopenia and dysmegakaryopoiesis. SUMMARY: Thrombocytopenia 2 (THC2) is an autosomal-dominant disorder caused by point substitutions in the 5'UTR of the ANKRD26 gene. Patients have congenital thrombocytopenia, normal platelet morphology and function, and dysmegakaryopoiesis. Thrombocytopenia is frequently discovered only in adulthood and physicians often do not suspect its genetic origin. We describe two unrelated patients referred to two different institutions for investigation of thrombocytopenia. Based on the finding of dysmegakaryopoiesis at bone marrow examination, patients were diagnosed with myelodysplastic syndrome (MDS) (refractory thrombocytopenia) and treated with several courses of 5-azacytidine. Subsequently, demonstration of thrombocytopenia in their relatives eventually led to molecular diagnosis of THC2 in both families. These cases highlight that patients with THC2 are at risk of being misdiagnosed with MDS and receiving undue myelosuppressive treatments. Because dysmegakaryopoiesis is a feature also of other forms of inherited thrombocytopenia, a genetic disorder must always be considered when a patient presents with isolated thrombocytopenia and dysmegakaryopoiesis.

Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders.

Essentials There are many hereditary platelet disorders (HPD) but diagnosing these is challenging. We provide a method to diagnose several HPDs using standard blood smears requiring < 100 µL blood. By this approach, the underlying cause of HPD was characterized in ~25-30% of referred individuals. The method facilitates diagnosis of HPD for patients of all ages around the world. SUMMARY: Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 µL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.

Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.

BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Severe to profound deafness may be associated with MYH9-related disease: report of 4 patients.

MYH9-related disease (MYH9-RD) is a rare genetic syndromic disorder characterised by congenital thrombocytopenia and is associated with the risk of developing progressive sensorineural hearing loss, nephropathy and presenile cataracts during childhood or adult life. All consecutive patients enrolled in the Italian Registry for MYH9-RD with severe to profound deafness were included in a retrospective study. The study population involved 147 Italian patients with MYH9-RD: hearing loss was identified in 52% of cases and only 4 patients (6%) presented severe to profound deafness at a mean age of 33 years. Deafness was associated with mild spontaneous bleeding in all patients and with kidney involvement in 3 cases. Cochlear implantation was carried out in 3 cases with benefit, and no major complications were observed. Diagnosis was performed about 28 years after the first clinical manifestation of MYH9-RD, which was never suspected by an otolaryngologist. The clinical and diagnostic aspects of 4 patients with severe to profound deafness are discussed with a focus on therapeutic implications.

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.

Altered cytoskeleton organization in platelets from patients with MYH9-related disease.

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

Effects of staurosporine on ACTH-mediated stimulation of aldosterone production.

Incubation of rat adrenal glomerulosa cells with low concentrations (up to 50 nM) of the protein kinase (PKC) inhibitor staurosporine (ST) inhibited aldosterone (ALDO) and cyclic AMP (cAMP) production stimulated by adrenocorticotropic hormone (ACTH) and cholera toxin. Only higher concentrations (1.6 microM) of staurosporine inhibited dibutyryl-cAMP- and forskolin-induced stimulation of aldosterone production. cAMP levels were increased only with low concentrations of the PKC inhibitor. This latter increase was avoided by treatment with a maximal concentration of isobutylmethylxanthine (MIX). Our results suggest that: (1) second messengers other than cAMP are involved in ACTH action; (2) staurosporine inhibits different kinases involved in ACTH action in a dose-dependent manner; (3) the protein kinase inhibited by high concentrations of staurosporine appears to be the cAMP-dependent kinase, PKA; and (4) the protein kinase inhibited by low concentrations of staurosporine remains to be identified. This latter species is suggested as being involved in mediating ACTH-induced activation of Gs.

The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region.

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.

ß-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in platelet-associated genes partly explain inherent variability in platelet counts. Patients with monoallelic Bernard Soulier syndrome due to the Bolzano mutation (GPIBA A156V) have variable platelet counts despite a common mutation for unknown reasons. OBJECTIVES: We investigated the effect of the most common SNP (R307H) in the hematopoietic-specific tubulin isotype ß-1 in these Bernard Soulier patients and potential microtubule-based mechanisms of worsened thrombocytopenia. PATIENTS/METHODS: Ninety-four monoallelic Bolzano mutation patients were evaluated for the R307H ß-1 SNP and had platelet counts measured by three methods; the Q43P SNP was also evaluated. To investigate possible mechanisms underlying this association, we used molecular modeling of ß-1 tubulin with and without the R307H SNP. We transfected SNP or non-SNP ß-1 tubulin into MCF-7 and CMK cell lines and measured microtubule regrowth after nocodazole-induced depolymerization. RESULTS: We found that patients with at least one R307H SNP allele had significantly worse thrombocytopenia; manual platelet counting revealed a median platelet count of 124 in non-SNP patients and 76 in SNP patients (both ×10(9)  L(-1) ; P < 0.01). The Q43P SNP had no significant association with platelet count. Molecular modeling suggested a structural relationship between the R307H SNP and microtubule stability via alterations in the M-loop of ß tubulin; in vitro microtubule recovery assays revealed that cells transfected with R307H SNP ß-1 had significantly impaired microtubule recovery. CONCLUSIONS: Our data show that the R307H SNP is significantly associated with the degree of thrombocytopenia in congenital and acquired platelet disorders, and may affect platelets by altering microtubule behavior.

The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor.

The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol-acetyl transferase activity in cells transfected with a plasmid that possesses two canonical glucocorticoid-responsive elements. Unlike most glucocorticoids, deltaHOP does not induce the fragmentation of DNA in a regular pattern characteristic of apoptosis and it does not reduce thymus weight. This unusual dissociation of glucocorticoid parameters makes deltaHOP a useful tool to discriminate between mechanisms of action by which steroids can exert their biological effects.

Endothelin-1 stimulation of aldosterone and zona glomerulosa ouabain-sensitive sodium/potassium-ATPase.

Endothelin stimulates the cells of the zona glomerulosa of the adrenal gland and releases aldosterone. While it is a less potent aldosterone secretagogue than angiotensin II endothelin also potentiates the effects of angiotensin II on aldosterone biosynthesis. Two endothelin receptors have been cloned and are expressed in the adrenal zona glomerulosa. Intravenous infusion of endothelin at a rate of 80 ng/kg/min for 30 min into rats produced increases in blood pressure, adrenal content of aldosterone and stimulated the ouabain-sensitive sodium potassium ATPase in the zona glomerulosa, but not in the zona fasciculata, of the adrenal. The simultaneous infusion of the isopeptide specific endothelin receptor A (ETA) antagonist BQ-123 blocked the pressor effects of endothelin, but did not alter the increase in aldosterone content of the zona glomerulosa or the ouabain-sensitive sodium potassium ATPase activity. Infusion of Sarafotoxin 6b, an ETB agonist, also increased the aldosterone content of the adrenal and stimulated the ouabain-sensitive sodium potassium ATPase in the zona glomerulosa, further indicating that the effect of endothelin is probably mediated by ETB or isopeptide non-specific endothelin receptor. The mechanism by which endothelin stimulates the sodium potassium ATPase is unclear as is the relation between a stimulated sodium potassium ATPase and the potentiation of angiotensin II effect on the adrenal.

In vivo stimulation of aldosterone biosynthesis by endothelin: loci of action and effects of doses and infusion rate.

Infusion of endothelin-1 (ET-1) into rats increased adrenal mitochondrial synthesis of aldosterone from deoxycorticosterone and the adrenal cytosolic content of aldosterone. The dose-response relationships for these last two effects of ET-1 were found to be biphasic with a maximum (corresponding to 80 to 200% increase) at 50 to 80 ng ET-1/kg/min, and were also dependent on the infusion rate. Plasma aldosterone levels were also increased in a similar ratio. Previous infusion of the converting enzyme inhibitor enalapril did not affect the ET-1-induced increase in steroidogenesis. Finally, pregnenolene production was also increased in incubations of mitochondria from treated rats. These results indicate that ET-1 augments aldosteronogenesis by increasing the early as well as the late pathway. These effects were independent of the formation of angiotensin II. Isolated glomerulosa cells responded to ET-1 increasing aldosterone production in a dose-related fashion. These results confirm a direct effect of ET-1 on the adrenal gland in vivo.

Expression of p53, bcl-2 and ras oncoproteins and apoptosis levels in acute leukaemias and myelodysplastic syndromes.

We analysed by immunocytochemistry the expression of p53, bcl-2 and ras proteins in bone marrow blasts from 59 patients with acute leukaemia (AL), 36 myeloid (AML) and 23 lymphoid (ALL), and from 22 patients with myelodysplastic syndrome (MDS); our aim was to examine if abnormalities in their expression were associated with peculiar biological and clinical findings, or with an altered apoptosis rate, as measured by TUNEL technique. The oncoproteins were expressed with extreme variability, without significant differences among the various morphological or immunological AL subtypes. The mean percentages of bcl-2+ blasts were significantly higher in AML than in MDS (p = 0.01), and in MDS with bone marrow blastosis than in the forms without excess of blasts (p = 0.007). The lowest percentages of apoptotic cells were observed in ALL (mean 1%, p = 0.006), whereas in MDS the apoptotic index was higher (16.7%) than in AML (8.6%) and than in the normal controls (10.8%). but the difference tended to be statistically significant only for cases of refractory anaemia. Whereas in AML and MDS the apoptotic rate was independent of the oncoprotein expression, in ALL there was a significant linear relationship between TUNEL and ras positivity (p = 0.01). Among AML patients treated with intensive polychemotherapy, no differences were observed in oncoprotein expression and apoptotic rate between responders and resistant cases. In conclusion, our data are in agreement with the hypothesis that decreased apoptosis and enhanced cell survival are associated with AL, whereas a high level of apoptosis may be responsible for the ineffective hematopoiesis in MDS; abnormal expression of oncoproteins, even if not strictly related to apoptosis level, may influence disease behaviour.

Mechanisms of action of endothelin-1 in rat adrenal.

Displacement curves of 125I-Endothelim-1 (ET-1) binding to rat adrenal cells with unlabeled ET-1, and the ET-1 receptor-related peptides sarafotoxin and BQ-123, show that rat adrenal cortex possess, as its bovine counterpart, two different receptors to ET-1 named ET-A and ET-B. Binding of ET-1 to its rat adrenal receptors stimulates i) aldosterone production, in vivo and in vitro ii) calcium influx, which is mediated through voltage dependent- and receptor operated- calcium channels, iii) cholesterol uptake, iv) stimulation of Na+/K+-ATPase and iv) diacylglycerol production. While the last effect is mediated through ET-A receptors the others involve binding of ET-1 to ET-B receptors. Finally, ouabain potentiates the ET-1-mediated stimulation of aldosterone production, suggesting that the effect of the peptidic hormone on Na+/K+-ATPase could act as a negative feedback mechanism.

Endothelin-1-induced incorporation of cholesterol into rat adrenals.

The effect of endothelin-1 (ET-1) on cholesterol uptake by adrenal cortex was evaluated through several experimental approaches: infusion of ET-1 followed by measurement of endogenous cholesterol in excised adrenals; infusion of ET-1 followed by tritiated cholesterol incorporation into adrenal quarters in vitro; coinfusion of ET-1 with tritiated cholesterol-enriched serum and determination of adrenal-associated radioactivity; and tritiated cholesterol incorporation in incubations of adrenal cells. In all cases ET-1 increased cholesterol uptake. Subcellular fractionation showed an ET-1-mediated augmentation in mitochondrial fraction. This increase was mediated by the subpopulation B of adrenal receptors for ET-1. In addition, ET-1 also increased cytochrome P450-SCC (side-chain cleavage) activity.