Killer immunoglobulin-like receptors (KIR) and their HLA-ligands in Italian paroxysmal nocturnal haemoglobinuria (PNH) patients.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

Risk for the introduction of exotic ticks and pathogens into Italy through the illegal importation of tortoises, Testudo graeca.

In April 2008, 585 tortoises illegally imported into Italy from North Africa were examined for the presence of ticks. Of these, 221 tortoises (37.8%) were infested with a mean intensity of 3.9 +/- 3.1 ticks (range 1-17 ticks). A total of 798 ticks (672 males, 125 females and one nymph) were collected and identified as Hyalomma aegyptium (L.) (Acari: Ixodidae). The overall male : female ratio observed was 5 : 1. The prevalence and mean intensity [+/-standard deviation (SD)] of ticks were higher among male (67.4%, 4.0 +/- 3.2) than female (55.6%, 3.8 +/- 3.1) tortoises, although this difference was not significant. By contrast, the prevalence and mean intensity of ticks were significantly higher on tortoises weighing >100 g (61.5%, 4.0 +/- 3.2) compared with tortoises weighing <100 g (12.1%, 2.1 +/- 1.1). Of the infested tortoises, 89.8% had ticks on their hind limbs, 21.0% on forelimbs, 18.6% on the tail and pre-anal area, and 4.8% on the head; thus the hind limbs were evidently the preferred attachment site. The present report highlights the need to develop surveillance systems to prevent the introduction and spreading of exotic ticks and tick-borne pathogens in Italy and other European countries.

Large granular lymphocyte (LGL)-like clonal expansions in paroxysmal nocturnal hemoglobinuria (PNH) patients.

In paroxysmal nocturnal hemoglobinuria (PNH), clonal expansion of glycosylphosphatidylinositol-anchored proteins (GPI-AP)-deficient cells leads to a syndrome characterized by hemolytic anemia, marrow failure, and venous thrombosis. PNH is closely related to aplastic anemia and may share its immune pathophysiology. In vivo expansion of dominant T-cell clones can reflect an antigen-driven immune response but may also represent autonomous proliferation, such as in large granular lymphocytic (LGL)-leukemia. T-cell clonality can be assessed by a combination of T-cell receptor (TCR) flow cytometry and complementarity-determining-region-3 (CDR3) molecular analysis. We studied 24 PNH patients for evidence of in vivo dominant T-cell responses by flow cytometry; TCR-Vbeta-specific expansions were identified in all patients. In four cases, extreme expansions of one Vbeta-subset of CD8+/CD28-/CD56+ (effector) phenotype mimicked subclinical LGL-disease. The monoclonality of these expansions was inferred from unique CDR3-size peak distributions and sequencing of dominant clonotypes. We conclude that the molecular analysis of TCR-beta chain may demonstrate clonal LGL-like expansions at unexpected frequency in PNH patients. Our observations blur the classical boundaries between different bone marrow failure syndromes such as AA, PNH, and LGL, and support the hypothesis that in PNH, the mutant clone may expand as a result of an immune-escape from antigen-driven lymphocyte attack on hematopoietic progenitors.

Reinvestigation of the role of thiol groups of glyoxalase I purified from yeast (Saccharomyces cerevisiae).

Glyoxalase I has been purified to homogeneity from Saccharomyces cerevisiae and tested with two different thiol reagents, i.e., 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB). DTNB reacts with four thiol groups per molecule of enzyme and leads to a complete inhibition which is not reversed by addition of the disulfide-reducing agent dithiothreitol. On the other hand, CDNB slightly affects the glyoxalase-I activity and alkylates only one thiol residue/enzyme. In agreement, DTNB reacts with three thiol residues of the CDNB-reacted enzyme and no reactivation is observed after dithiothreitol treatment. The peptide containing the CDNB-reactive thiol group has been isolated and the sequence overlaps the segment 58-63 of the only known primary structure of glyoxalase I from Pseudomonas putida.

Expression of the 67-kDa laminin receptor in acute myeloid leukemia cells mediates adhesion to laminin and is frequently associated with monocytic differentiation.

Lodgement, proliferation, and migration of leukemic cells within bone marrow (BM) microenvironment involves adhesion of these cells to the BM extracellular matrix molecules fibronectin and laminin. The 67-kDa laminin receptor (67LR) is a nonintegrin protein with high affinity for laminin, which plays a critical role in basement membrane invasion and metastasis of cancer cells. By Western blotting, we documented that 67LR was strongly expressed in myelomonocytic THP1 and histiocytic U937 cells and was weakly expressed in promyelocytic HL-60 cells. In HL-60 cells, 67LR expression almost disappeared after retinoic-induced granulocytic differentiation, whereas it strongly increased after phorbol ester-induced monocytic differentiation. We did not detect 67LR expression in normal BM hematopoietic cells, in precursor-B acute lymphoblastic leukemia, in chronic lymphocytic leukemia, or in chronic myeloid leukemia in chronic phase. By contrast, we detected enhanced 67LR expression in 40% of 53 de novo acute myeloid leukemias (AMLs), which frequently exhibited monocytic or myelomonocytic morphology and expressed CD14 and CD11a (P < 0.05). Using a colorimetric assay, we found that the expression pattern of this receptor corresponded to a higher adhesion to laminin; the adhesion was specific because in vitro addition to laminin-coated wells of recombinant 37-kDa laminin receptor precursor (37LRP), which is the cytoplasmic precursor containing both laminin-binding domains of cell surface 67LR, significantly reduced laminin binding of AML cells. The expression of 67LR on AML cell surface did not correlate with other differentiation and integrin antigens such as CD7, CD13, CD33, CD34, CD11b, CD11c, CD49d, CD49e, CD45RA, and CD45RO. In contrast with 67LR behavior in solid tumors, no statistically significant difference was found between 67LR expression and any hematological characteristic of the disease at diagnosis, nor between 67LR expression and outcome of the disease as measured by complete remission rate, disease-free survival, or overall survival. In conclusion, our results indicate that 67LR expression mediates specific adhesion to laminin and that the detection of this molecule may be a valuable addition to other lineage-associated antigens in identifying monocytic-oriented AML.

Long-lasting decrease of marrow and circulating long-term culture initiating cells after allogeneic bone marrow transplant.

We investigated bone marrow (BM) and circulating (PB) hematopoietic progenitor cells in 37 normal donors and in 25 patients 1 to 8 years after successful allogeneic bone marrow transplant. At the time of testing, transplanted patients had normal blood counts and bone marrow cellularity. By flow cytometry, BM CD34+ cells were found to be three- to four-fold decreased in transplanted patients compared to normal donors, while the number of PB CD34+ cells was the same as in normal donors. Using a methylcellulose colony assay, primary BM colony-forming cells (CFU-GM) were decreased 2.1-fold, whereas PB CFU-GM were only marginally decreased. In a long-term culture initiating cell (LTC-IC) assay, an eight-fold decrease of early progenitor cells was observed in the marrow of transplanted patients compared to normal donors, and a five-fold decrease was documented in peripheral blood. We found that the BM LTC-IC cell number correlated with concurrently determined BM CD34+ cells and committed progenitor cell number (measured as CFU-GM) and with PB LTC-IC number, but not with PB CFU-GM and CD34+ cells. We conclude that marrow and circulating early stem cell compartments, as measured by the LTC-IC assay, are greatly and permanently depressed following bone marrow transplant. The correlation between BM and PB LTC-IC indicates that the enumeration of circulating LTC-IC can be used as a measure of the stem cell compartment in the bone marrow after transplant. It seems that the deficiency of the most immature progenitor cells persists forever after successful bone marrow transplant; this means that a complete hematopoietic reconstitution can be sustained by a reduced stem cell pool.

Alternative immunosuppression in patients failing immunosuppression with ATG who are not transplant candidates: Campath (Alemtuzumab).

Antithymocyte globulin (ATG)-based immunosuppression remains the standard immunosuppressive therapy (IST) for aplastic anemia (AA) patients lacking a sibling donor; however, treatment failures are relatively frequent, including about one-quarter to one-third of patients who do not show any response to initial IST, and about half of the initial responders who may experience subsequent relapses or require continuous maintenance IST. For these patients, there is the option of further IST, which may include additional courses of ATG-based IST, or attempts with alternative IST regimens. Alemtuzumab is a monoclonal anti-CD52 Ab, which has been recently investigated as novel IS agent for the treatment of AA patients. Recent data from different groups have clearly demonstrated the biological efficacy of Alemtuzumab in AA patients, ruling out the initial concerns about possible unacceptable infectious risks secondary to its extremely powerful lympholytic effect. Preliminary data demonstrate a remarkable efficacy, especially in the context of relapsed and, to less extent, refractory patients, whereas data in naïve patients are still limited. On the basis of these results, Alemtuzumab-based immunosuppression is a worthy option for AA and other marrow failure patients requiring a second-line IST. Here we describe a consensus regimen that the European Group for Blood and Marrow Transplantation Severe Aplastic Anemia Working Party suggests for AA patients failing initial IST who are not indicated for SCT.

Supportive care in severe and very severe aplastic anemia.

Optimal management of aplastic anemia (AA) is not confined to immediate diagnosis, early decision making and timely initiation of major treatment strategies (immunosuppression or SCT) but also involves supportive treatment as a crucial part of patient care. Patients are threatened by complications of cytopenia. Here, we summarize current recommendations for prevention and early treatment of fungal, bacterial and viral infections, transfusion strategy and iron chelation and assess the evidence basis. In fact, many recommendations for patients with AA are not based on randomized studies in AA itself, but they are deduced from other conditions with similar severity of cytopenia. Prevention and treatment of complications like hemorrhage, bacterial and fungal infections and of secondary events like alloimmunization to blood products and iron overload have a significant impact on the prognosis of AA patients and need to be carefully observed in daily practice. More controlled studies on supportive care should be performed in this rare disease.

Novel immunosuppressive strategies for bone marrow failure syndromes: a focus on alemtuzumab.

Acquired bone marrow failure syndromes (BMFS) are a heterogeneous group of hematological disorders characterized by impaired bone marrow function and subsequent cytopenia of one or more blood cell lineages [1,2]. The well-accepted pathogenic mechanism of the typical bone marrow failure - aplastic anemia (AA)- is a T cell mediated immune attack targeting the hematopoietic tissue [3]. This pathogenic mechanism is at least partially shared by other bone marrow failure syndromes, such as lineage-restricted aplasias and some myelodysplastic syndromes. Thus, for these disorders immunosuppression (IS) is the pivotal etiologic treatment. While the standard IS regimen include the heterologous anti-thymocyte globulin [4], here we review the recent data on the anti-CD52 monoclonal antibody alemtuzumab as a novel IS agent for marrow failures. Alemtuzumab led to objective responses in aplastic anemia patients in 3 recent prospective studies, with overall response rates ranging between 37% and 72%. Adverse events were irrelevant, ruling out even the concerns about the risk of infectious complications. Alemtuzumab was effective even for the treatment of lineage-restricted marrow failure, with very acceptable toxicity and excellent response rates (as high as 80%). More recently, even patients suffering from myelodysplastic syndromes showed a remarkable hematological response to alemtuzumab-based IS treatment. Thus, alemtuzumab is a novel IS agent representing an excellent alternative to ATG for all immune-mediated marrow failure syndromes. Even if the dose and the schedule may still require further refining, the available data support the need of large prospective trials comparing alemtuzumab to current standard IS regimens.

Achievements and limitations of complement inhibition by eculizumab in paroxysmal nocturnal hemoglobinuria: the role of complement component 3.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disorder characterized by complementmediated hemolytic anemia, thrombophilia and bone marrow failure. The clinical hallmark of PNH is evident chronic hemolysis due to the absence of the complement regulators CD55 and CD59 on PNH erythrocytes. Intravascular hemolysis drives the major clinical features of PNH, including anemia, hemoglobinuria, fatigue and other hemolysisrelated disabling symptoms, such as painful abdominal crises, dysphagia and erectile dysfunction. A peculiar thromboembolic risk has been associated with the hemolysis in PNH, but its pathophysiologic cause remains unclear. The treatment of PNH has remained supportive until a few years ago, when the first complement inhibitor, designated eculizumab, became available. Chronic treatment with eculizumab results in sustained control of intravascular hemolysis, leading to hemoglobin stabilization and transfusion independence in half of the patients. However, residual anemia may persist in a substantial fraction of patients. Recent observations by different groups, including our own, have demonstrated that residual hemolysis may be due to persistent activation of the early phases of the complement cascade, leading to progressive C3-deposition on PNH erythrocytes and possible subsequent extravascular hemolysis through the reticuloendothelial system. Here we critically review the available clinical results of eculizumab treatment for PNH patients, pointing out the recent insights into the pathophysiology of the disease. We discuss the role of the different components of the complement cascade leading to hemolysis, in both the absence and presence of the terminal effector pathway inhibition by eculizumab. Finally, we provide a theoretical rationale for the development of novel strategies of complement inhibition which could in the future further improve on the already substantial efficacy of eculizumab.

Fully processed lysyl oxidase catalyst translocates from the extracellular space into nuclei of aortic smooth-muscle cells.

Lysyl oxidase (LO), a secreted protein, was recently identified within the nuclei of vascular smooth-muscle cells (SMC) and 3T3 fibroblasts. A possible pathway by which LO can enter cell nuclei was explored in the present study. SMC were incubated with purified 32-kDa bovine aorta LO that had been fluorescently labeled with rhodamine (TRITC-LO). TRITC-LO entered the cytosol and then rapidly concentrated within the nuclei of preconfluent cultures of these cells, whereas carbonic anhydrase, a protein of similar molecular weight and similarly labeled, did not enter the cells under these conditions. LO that had been reductively methylated at lysine residues with [(14)C]HCHO was also taken up into the cytosolic and nuclear compartments. Intracellular uptake and intracellular distribution were not altered by inhibiting LO activity with beta-aminopropionitrile. An excess of native LO but not of carbonic anhydrase competitively inhibited the uptake of the isotopically labeled enzyme. Thus, once secreted and proteolytically processed, mature LO can enter the cells and concentrate within nuclei in a manner that appears to be specific and independent of its catalytic activity.

Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase.

Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.

Partial purification and properties of ornithine carbamoyltransferase from Citrus limonum leaves.

Ornithine carbamoyltransferase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3) has been partially purified from C.limonum leaves. The data indicate the presence of only the anabolic enzyme. The activity is strongly influenced by pH, ionic strength and ornithine concentration. Optimal activity for the enzyme dissolved in the tri-buffer: diethanolamine,2-(N-morpholino) ethanesulfonic acid, N-ethylenmorpholine (0.051 M/0.1 M/0.051 M) is at pH 9.0, when ornithine is 10 mM. The enzyme catalyses an ordered-sequential process in which carbamoyl phosphate binds first followed by L-ornithine and then L-citrulline leaves followed by phosphate. Support for this statement comes from product inhibition and evidence of abortive ternary complex formation.

T cell receptor VB repertoire diversity in patients with immune thrombocytopenia following splenectomy.

In recent years, a pathophysiological role for T cells in immune thrombocytopenia (ITP) has been established. We applied cDNA size distribution analysis of the T cell receptor (TCR) beta-variable (VB) complementarity-determining region 3 (CDR3) in order to investigate T cell repertoire diversity among immune thrombocytopenia patients who had either responded or not responded to splenectomy, and compared them to normal controls. ITP patients who had had a durable platelet response to splenectomy showed a mean 2.8 +/- 2.1 abnormal CDR3 size patterns per patient, similar to healthy volunteers (2.9 +/- 2.0 abnormal CDR3 size patterns). In contrast, patients unresponsive to splenectomy demonstrated evidence of significantly more clonal T cell expansions than patients who had responded to splenectomy or controls (11.3 +/- 3.3 abnormal CDR3 size patterns per patient; P < 0.001). Of the VB subfamilies analysed, VB3 and VB15 correlated with response or non-response to splenectomy, each demonstrating oligoclonality in non-responding patients (P < 0.05). These findings suggest that removal of the spleen may lead directly or indirectly to reductions in T cell clonal expansions in responders, or that the extent of T cell clonality impacts responsiveness to splenectomy in patients with ITP.