Integrating Phenotypic and Chemoproteomic Approaches to Identify Covalent Targets of Dietary Electrophiles in Platelets.

A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.

"Sticki-ER": Functions of the Platelet Endoplasmic Reticulum.

Significance: The primary role of platelets is to generate a thrombus by platelet activation. Platelet activation relies on calcium mobilization from the endoplasmic reticulum (ER). ER resident proteins, which are externalized upon platelet activation, are essential for the function of platelet surface receptors and intercellular interactions. Recent Advances: The platelet ER is a conduit for changes in cellular function in response to the extracellular milieu. ER homeostasis is maintained by an appropriate redox balance, regulated calcium stores and normal protein folding. Alterations in ER function and ER stress results in ER proteins externalizing to the cell surface, including members of the protein disulfide isomerase family (PDIs) and chaperones. Critical Issues: The platelet ER is central to platelet function, but our understanding of its regulation is incomplete. Previous studies have focused on the function of PDIs in the extracellular space, and much less on their intracellular role. How platelets maintain ER homeostasis and how they direct ER chaperone proteins to facilitate intercellular signalling is unknown. Future Directions: An understanding of ER functions in the platelet is essential as these may determine critical platelet activities such as secretion and adhesion. Studies are necessary to understand the redox reactions of PDIs in the intracellular versus extracellular space, as these differentially affect platelet function. An unresolved question is how platelet ER proteins control calcium release. Regulation of protein folding in the platelet and downstream pathways of ER stress require further evaluation. Targeting the platelet ER may have therapeutic application in metabolic and neoplastic disease.

Focal Anticoagulation by Somatic Gene Transfer: Towards Preventing Cardioembolic Stroke.

Cardioembolic stroke (CS) has emerged as a leading cause of ischaemic stroke (IS); distinguished by thrombi embolising to the brain from cardiac origins; most often from the left atrial appendage (LAA). Contemporary therapeutic options are largely dependent on systemic anticoagulation as a blanket preventative strategy, yet this does not represent a nuanced or personalised solution. Contraindications to systemic anticoagulation create significant unmedicated and high-risk cohorts, leaving these patients at risk of significant morbidity and mortality. Atrial appendage occlusion devices are increasingly used to mitigate stroke risk from thrombi emerging from the LAA in patients ineligible for oral anticoagulants (OACs). Their use, however, is not without risk or significant cost, and does not address the underlying aetiology of thrombosis and CS. Viral vector-based gene therapy has emerged as a novel strategy to target a spectrum of haemostatic disorders, achieving success through the adeno-associated virus (AAV) based therapy of haemophilia. Yet, thrombotic disorders, such as CS, have had limited exploration within the realm of AAV gene therapy approaches-presenting a gap in the literature and an opportunity for further research. Gene therapy has the potential to directly address the cause of CS by localised targeting of the molecular remodelling that serves to promote thrombosis.

Multiple Electrode Aggregometry (Multiplate): Functional Assay for Vaccine-Induced (Immune) Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) was first described in 2021 and represents an adverse reaction to adenoviral vector COVID-19 vaccines AstraZeneca ChAdOx1 nCoV-19 (AZD1222) and Johnson & Johnson Ad26.COV2.S vaccine. VITT is a severe immune platelet activation syndrome with an incidence of 1-2 per 100,000 vaccinations. The features of VITT include thrombocytopenia and thrombosis within 4-42 days of first dose of vaccine. Affected individuals develop platelet-activating antibodies against platelet factor 4 (PF4). The International Society on Thrombosis and Haemostasis recommends both an antigen-binding assay (enzyme-linked immunosorbent assay, ELISA) and a functional platelet activation assay for the diagnostic workup of VITT. Here, the application of multiple electrode aggregometry (Multiplate) is presented as a functional assay for VITT.

Mechano-Redox Control of Mac-1 De-Adhesion by PDI Promotes Directional Movement Under Flow.

BACKGROUND: Neutrophil migration is critical to the initiation and resolution of inflammation. Macrophage-1 antigen (Mac-1; CD11b/CD18, αMß2) is a leukocyte integrin essential for firm adhesion to endothelial ICAM-1 (intercellular adhesion molecule 1) and migration of neutrophils in the shear forces of the circulation. PDI (protein disulfide isomerase) has been reported to influence neutrophil adhesion and migration. We aimed to elucidate the molecular mechanism of PDI control of Mac-1 affinity for ICAM-1 during neutrophil migration under fluid shear. METHODS: Neutrophils isolated from whole blood were perfused over microfluidic chips coated with ICAM-1. Colocalization of Mac-1 and PDI on neutrophils was visualized by fluorescently labeled antibodies and confocal microscopy. The redox state of Mac-1 disulfide bonds was mapped by differential cysteine alkylation and mass spectrometry. Wild-type or disulfide mutant Mac-1 was expressed recombinantly in Baby Hamster Kidney cells to measure ligand affinity. Mac-1 conformations were measured by conformation-specific antibodies and molecular dynamics simulations. Neutrophils crawling on immobilized ICAM-1 were measured in presence of oxidized or reduced PDI, and the effect of PDI inhibition using isoquercetin on neutrophil crawling on inflamed endothelial cells was examined. Migration indices in the X- and Y-direction were determined and the crawling speed was calculated. RESULTS: PDI colocalized with high-affinity Mac-1 at the trailing edge of stimulated neutrophils when crawling on ICAM-1 under fluid shear. PDI cleaved 2 allosteric disulfide bonds, C169-C176 and C224-C264, in the ßI domain of the ß2 subunit, and cleavage of the C224-C264 disulfide bond selectively controls Mac-1 disengagement from ICAM-1 under fluid shear. Molecular dynamics simulations and conformation-specific antibodies reveal that cleavage of the C224-C264 bond induces conformational change and mechanical stress in the ßI domain. This allosterically alters the exposure of an αI domain epitope associated with a shift of Mac-1 to a lower-affinity state. These molecular events promote neutrophil motility in the direction of flow at high shear stress. Inhibition of PDI by isoquercetin reduces neutrophil migration in the direction of flow on endothelial cells during inflammation. CONCLUSIONS: Shear-dependent PDI cleavage of the neutrophil Mac-1 C224-C264 disulfide bond triggers Mac-1 de-adherence from ICAM-1 at the trailing edge of the cell and enables directional movement of neutrophils during inflammation.

Endothelial cell dysfunction: Implications for the pathogenesis of peripheral artery disease.

Peripheral artery disease (PAD) is caused by occluded or narrowed arteries that reduce blood flow to the lower limbs. The treatment focuses on lifestyle changes, management of modifiable risk factors and vascular surgery. In this review we focus on how Endothelial Cell (EC) dysfunction contributes to PAD pathophysiology and describe the largely untapped potential of correcting endothelial dysfunction. Moreover, we describe current treatments and clinical trials which improve EC dysfunction and offer insights into where future research efforts could be made. Endothelial dysfunction could represent a target for PAD therapy.

Assessment of immunological anti-platelet factor 4 antibodies for vaccine-induced thrombotic thrombocytopenia (VITT) in a large Australian cohort: A multicenter study comprising 1284 patients.

BACKGROUND: Vaccine-induced thrombotic thrombocytopenia (VITT) is a rare complication of adenovirus-based vaccines aimed to prevent and minimize COVID-19 and related pathophysiology. OBJECTIVES: To describe patterns of testing for anti-platelet factor 4 (PF4) antibodies using various ELISA assays in a large Australian cohort and comparative functional platelet activation assays in a subset. PATIENTS/METHODS: Asserachrom HPIA IgG ELISA was performed in 1284 patients over a period of 12 months, supplemented in select cohorts by comparative ELISA using three other methods (n = 78-179), three different functional assays (flow cytometry, serotonin release assay, and/or Multiplate; n = 476), and rapid immunological chemiluminescence anti-PF4 assay (n = 460), in a multicenter study. RESULTS: For first episode presentations, 190/1284 (14.8%) ELISA tests were positive. Conversely, most (445/460; 96.7%) chemiluminescence anti-PF4 test results were negative. All functional assays showed associations of higher median ELISA optical density with functional positivity and with high rates of ELISA positivity (64.0% to 85.2%). Data also identified functional positivity in 14.8%-36.0% of ELISA negative samples, suggesting false negative VITT by HPIA IgG ELISA in upward of one third of assessable cases. CONCLUSION: To our knowledge, this is the largest multicenter evaluation of anti-PF4 testing for investigation of VITT. Discrepancies in test results (ELISA vs. ELISA or ELISA vs. functional assay) in some patients highlighted limitations in relying on single methods (ELISA and functional) for PF4 antibody detection in VITT, and also highlights the variability in phenotypic test presentation and pathomechanism of VITT.

A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium.

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils' importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.

Allosteric disulphide bonds as reversible mechano-sensitive switches that control protein functions in the vasculature.

Disulphide bonds are covalent linkages of two cysteine residues (R-S-S-R') in proteins. Unlike peptide bonds, disulphide bonds are reversible in nature allowing cleaved bonds to reform. Disulphide bonds are important structural elements that stabilise protein conformation. They can be of catalytic function found in enzymes that facilitate redox reactions in the cleavage/formation of disulphide bonds in their substrates. Emerging evidence also indicates that disulphide bonds can be of regulatory function which alter protein activity when they are cleaved or formed. This class of regulatory disulphide bonds is known as allosteric disulphide bonds. Allosteric disulphide bonds are mechano-sensitive, and stretching or twisting the sulphur-sulphur bond by mechanical force can make it easier or harder to be cleaved. This makes allosteric disulphide bonds an ideal type of mechano-sensitive switches for regulating protein functions in the vasculature where cells are continuously subjected to fluid shear force. This review will discuss the chemistry and biophysical properties of allosteric disulphide bonds and how they emerge to be mechano-sensitive switches in regulating platelet function and clot formation.

Autoregulation of von Willebrand factor function by a disulfide bond switch.

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this "mechanopresentation" remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.

Protein Disulfide Isomerase in Thrombosis.

Protein disulfide isomerase (PDI) is a 57-kDa oxidoreductase that facilitates cysteine thiol reactions inside and outside the cell. It mediates reduction or oxidation of protein disulfide bonds, thiol/disulfide exchange reactions, and transfer of NO from one protein thiol to another. It also has chaperone properties. PDI is actively secreted by most, if not all, of the cell types involved in thrombosis, binds to integrins on the cell surface, and circulates as a soluble protein in blood. It plays a critical role in thrombosis in mice and presumably the same role in human thrombosis. Eight proteins involved in thrombosis have been identified as PDI substrates; however, the role of this oxidoreductase in this process is not fully understood. Novel small-molecule PDI inhibitors have been developed and are being evaluated as antithrombotics in clinical trials. This combination of ongoing laboratory and clinical studies will greatly accelerate the pace of discovery and targeting of PDI function in thrombosis.

Both platelet- and endothelial cell-derived ERp5 support thrombus formation in a laser-induced mouse model of thrombosis.

Protein disulfide isomerase (PDI) and endoplasmic reticulum protein 57 (ERp57) are emerging as important regulators of thrombus formation. Another thiol isomerase, endoplasmic reticulum protein 5 (ERp5), is involved in platelet activation. We show here the involvement of ERp5 in thrombus formation using the mouse laser-injury model of thrombosis and a specific antibody raised against recombinant ERp5. Anti-ERp5 antibody inhibited ERp5-dependent platelet and endothelial cell disulfide reductase activity in vitro. ERp5 release at the thrombus site was detected after infusion of Alexa Fluor 488-labeled anti-ERp5 antibody at 0.05 µg/g body weight, a dose that does not inhibit thrombus formation. Anti-ERp5 at 3 µg/g body weight inhibited laser-induced thrombus formation in vivo by causing a 70% decrease in the deposition of platelets and a 62% decrease in fibrin accumulation compared to infusion of control antibody (P < .01). ERp5 binds to ß3 integrin with an equilibrium dissociation constant (KD) of 21 µM, measured by surface plasmon resonance. The cysteine residues in the ERp5 active sites are not required for binding to ß3 integrin. These results provide evidence for a novel role of ERp5 in thrombus formation, a function that may be mediated through its association with αIIbß3.

Histological expression of angiogenic factors: VEGF, PDGFRalpha, and HIF-1alpha in Hodgkin lymphoma.

Angiogenesis is a prerequisite for solid tumor growth, but there is relatively limited data regarding Hodgkin lymphoma. The purpose of this study was to examine the immunohistochemical expression of angiogenic and proliferation markers in Hodgkin biopsies in relation to clinical parameters. Immunostaining was performed on 65 Hodgkin biopsies with vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 alpha (HIF-1alpha), platelet-derived growth factor receptor alpha (PDGFRalpha), Ki-67, and p53. Microvessel density (MVD) was determined by CD31 staining. In all cases, neoplastic cells and reactive background cells were evaluated. The neoplastic population expressed VEGF in 48% of the cases, HIF-1alpha in 54% of the cases, and PDGFRalpha in 95% of the cases. Both Ki-67 and p53 were positive in neoplastic cells in over 60% of the cases. The MVD had a median of 2.6/0.0625mm(2) which was not different from normal lymph nodes. VEGF in the non-neoplastic compartment showed increased staining in Ann Arbor stage I-II versus III-IV. In conclusion, VEGF, HIF-1alpha, and predominantly PDGFRalpha are expressed in neoplastic cells in the majority of Hodgkin lymphomas. As microvessel formation is not increased in Hodgkin, additional functions of these angiogenic molecules should be investigated.

Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: A methodological approach.

Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)V(H) unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgV(H) mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgV(H) mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p < 0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70's applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.

Thymoma with immunodeficiency (Good's syndrome): review of the literature apropos three cases.

Good's syndrome is the association of thymoma with immunodeficiency, characterized by hypogammaglobulinaemia, depleted B-cells, diminished T-cells and inversion of the CD4/CD8 ratio. The initial clinical presentation is either with a mass lesion-thymoma that is usually benign, or with recurrent infections due to immunodeficiency. Thymectomy usually favourably affects associated conditions, such as pure red cell aplasia, but does not improve hypogammaglobulinaemia, thus the patient remains dependent on intravenous immune globulin and prone to infections. Infections usually affect the respiratory and/or the gastrointestinal tract. Common respiratory, opportunistic, and eventually life-threatening infections may occur. Moreover, patients with Good's syndrome may present other haematological conditions. We report 3 cases with long follow-up, sharing some common manifestations of the syndrome, but also showing unique features. The principal features of this rare syndrome are further discussed.

Autoimmune hepatitis type 1 and primary biliary cirrhosis have distinct bone marrow cytokine production.

We have recently reported differences in the hematopoiesis between autoimmune hepatitis type 1 (AIH-1) and primary biliary cirrhosis (PBC). In view of the notion that cytokines are regulators of hematopoiesis, we investigated in our tertiary center the cytokine production in the bone marrow (BM) of the same consecutive cohort of patients (13 AIH-1, 13 PBC, 10 healthy and 7 patients with cirrhosis due to chronic hepatitis B). Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) were determined in the supernatants of long-term BM cultures by ELISAs. IL-4, TNF-alpha and TGF-beta were found significantly increased in the BM of PBC patients compared to AIH-1 and both control groups. AIH-1 patients had significantly higher BM IL-10 compared to PBC patients and higher IL-10, IL-4 and TNF-alpha compared to controls. BM IFN-gamma was significantly higher in PBC and AIH-1 patients compared to controls. In AIH-1 patients, IL-10 was positively correlated with CD34+, CD34+/CD38- and CD34+/CD38+ cell proportions. In conclusion, the BM cytokine microenvironment of PBC and AIH-1 patients differs significantly compared to that of healthy individuals and cirrhotic patients of non-autoimmune etiology. Differences were also found between patients with PBC and AH-1. The implication of BM in the pathogenesis of autoimmune liver diseases is possible and needs further investigation.

Markers of cell activation and apoptosis in bone marrow mononuclear cells of patients with autoimmune hepatitis type 1 and primary biliary cirrhosis.

BACKGROUND/AIMS: We have reported quantitative and qualitative differences in bone marrow (BM) progenitor cells in autoimmune hepatitis type-1 (AIH-1) and primary biliary cirrhosis (PBC). This study investigated the apoptotic features and cytokine suppressors of haematopoiesis in long-term cultures of BM mononuclear cells (BMMCs) from AIH-1 and PBC patients. METHODS: Apoptotic markers and CD14 expression were evaluated in 13 AIH-1 patients, 13 PBC patients, 12 cirrhotic controls and 10 healthy subjects. TNF-alpha, TGF-beta and IFN-gamma were determined using ELISAs. RESULTS: All apoptotic markers and CD14 were increased in AIH-1 and PBC compared to controls (P<0.0001). Fas+ cells were positively correlated (P=0.0001) with apoptotic cells in AIH-1 and PBC. TNF-alpha and IFN-gamma were higher in AIH-1 (P=0.003 and P=0.001) and PBC (P=0.0001) compared to controls. No differences were found between the control groups. CONCLUSIONS: We demonstrate for the first time that the apoptotic process, macrophage activation and the production of cytokine suppressors of haematopoiesis in BMMCs from AIH-1 and PBC patients are higher compared to controls. The Fas-FasL pathway is likely to be involved in the apoptotic process; the increased levels of selected cytokines may contribute to Fas-FasL stimulation. Cirrhosis appears unlikely to be the cause of the above findings.

Relation between bone marrow angiogenesis and serum levels of angiogenin in patients with myelodysplastic syndromes.

Angiogenesis is implicated in the progression of myelodysplastic syndromes (MDS). Bone marrow microvascular density (MVD), serum angiogenin (ANG) and interleukin 6 (IL-6) were measured in 67 patients with untreated MDS. MVD, ANG and IL-6 were significantly higher in the patient group as a whole when compared to controls (P < 0.01). MVD and ANG were significantly higher in subtypes with a high-risk for leukemic transformation (RAEB, RAEB-t and CMML) than in low-risk subtypes (RA and RARS) (P < 0.01). In the MDS group, a positive correlation was found between ANG and IL-6 (P < 0.001) and also between MVD and IL-6 (P < 0.05). Using multivariate analysis, only IL-6 displayed independent prognostic value and was inversely related to MDS survival.