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.

Hematological complications in anorexia nervosa.

BACKGROUND/OBJECTIVES: Anemia, leukopenia and, although less frequently, thrombocytopenia are possible hematological complications of anorexia nervosa considered strictly secondary to chronic malnutrition. This is a retrospective study on the prevalence of these disorders in a large cohort of 318 female patients with AN (20.4±5.6 years, body mass index (BMI) 15.9±1.6 kg/m2), recruited in the Outpatient Unit for Malnutrition secondary to Eating Disorders at the Department of Clinical Medicine and Surgery, Federico II University Hospital, since February 1991 to December 2012. SUBJECTS/METHODS: Patients were studied on an outpatient basis after obtaining medical history, clinical examination, routine hematobiochemical and endocrine tests, electrocardiography, psychiatric interview and bioelectrical impedance analysis and, in particular, phase angle determination. All patients with other comorbidities, in particular with mean corpuscular volume <80 fl, were excluded for suspected genetic alteration in the synthesis of hemoglobin. RESULTS: Hematologic data showed that 16.7% of patients had anemia, 7.9% neutropenia and 8.9% thrombocytopenia. These abnormalities were strictly related to the duration of illness (P=0.028), and to protein energy malnutrition, in particular, BMI and phase angle (P<0.001). CONCLUSIONS: Our study offers description of the incidence of hematologic defects in a selected and large sample of AN female patients, suggesting that its incidence is related to the degree and duration of protein energy malnutrition.

Blood cell flow cytometry in paroxysmal nocturnal hemoglobinuria: a tool for measuring the extent of the PNH clone.

The membrane expression of nine glycosyl phosphatidyl inositol (GPI)-linked molecules was analyzed by flow cytometry on circulating cells from 18 patients affected by paroxysmal nocturnal hemoglobinuria (PNH). The results allowed us to select CD66b, CD14, CD59, CD24 and CD59 monoclonal antibodies as the most suitable reagents for discriminating between normal and PNH cells in PMN, monocytes, RBC and B or T lymphocytes, respectively. In order to assess whether the analysis of distinct cell populations could provide differential information on the extent of the disease, we compared the proportion of residual normal cells in RBC, monocyte and PMN populations. The mean percentage of unaffected cells was higher in RBC as compared to PMN (50.5 +/- 18.7 vs 17.7 +/- 19.7, P < 0.0001). The proportion of normal PMN was, in turn, significantly greater than that of normal monocytes (17.7 +/- 19.7 vs 8.7 +/- 11.0; P < 0.05). The percentage of CD14+ monocytes was directly related to Hb concentration and platelet (Plt) count, and inversely to percent lysis at the Ham's test. The percentage of CD66b+ PMN was directly related to Plt count and Hb level, while the percentage of CD59+ RBC was associated, in an inverse fashion, only to the Ham's test. No significant correlation was found between cell marker expression and PMN count, reticulocytosis, bilirubin and serum LDH. By dividing the patients into two groups, according to high (> 10 percent) or low (< 10 percent) percentage of CD14+ monocytes, a statistical analysis showed that the main hematological parameters were significantly different.

T cell growth-promoting activity of interferon-gamma. Mitogenic effect of the recombinant cytokine on cells from a human T-chronic lymphocytic leukemia.

Interferon-gamma (IFN-gamma) has previously been described as exerting a growth factor activity for murine and human stimulated normal T lymphocytes, in addition to its established role in regulating the cytotoxic activity of T and NK cells. We analyzed the effect of human recombinant IFN-gamma on the proliferation of leukemic lymphocytes isolated from the peripheral blood of a patient affected by a T-cell chronic lymphocytic leukemia (T-CLL). Incubation with IFN-gamma induced the proliferation of unstimulated leukemic cells. Cell proliferation was maximal after 6 days of culture with the cytokine; the half-maximal effect of IFN-gamma was observed at a concentration of approximately 800 U/ml. We also measured the production of IFN-gamma by leukemic cells. Cells incubated in control medium released small quantities of IFN-gamma activity, while the addition of low doses of the exogenous cytokine to the cell cultures induced high levels of IFN-gamma mRNA and protein production. Furthermore, anti-HLA class I monoclonal antibodies, that exert a mitogenic effect on these neoplastic lymphocytes, also induced the IFN-gamma gene expression in the same cells. These results indicate that IFN-gamma may stimulate the proliferation of human neoplastic T cells and suggest that this cytokine might have a role in the expansion of T-leukemic cell clones in vivo.

Membrane proteins in paroxysmal nocturnal haemoglobinuria.

A review of recent information on the abnormalities of the blood cell membrane in paroxysmal nocturnal haemoglobinuria (PNH) is presented, with a detailed analysis of biochemical and flow cytometry findings. The complex patterns observed in the various cell lineages of which the PNH clone consists are described, and a simplified monoclonal antibody panel is defined for diagnostic purposes. Available data on in vitro culture of progenitor cells and on the recent establishment of PNH cell lines are summarized. Finally, we discuss speculative hypotheses on the growth advantage of the PNH clone.

Trisomy 13 in a patient with leukemic progression of myelodysplasia.

Four months after the diagnosis of refractory anemia, a 60-year-old patient developed acute leukemia with blast cells that were poorly differentiated by morphology and clearly myeloid by immunophenotyping. Cytogenetic analysis performed at leukemization showed trisomy 13. An extra copy of chromosome 13 has already been reported in a few cases of acute leukemia and myelodysplastic syndrome.

Cytoplasmic GpIIb-IIIa and cytokine secretion by blasts in a case of megakaryoblastic transformation of essential thrombocythemia.

A 49-year-old woman with a four year history of therapy resistant essential thrombocythemia, progressed to acute leukemia that also proved refractory to chemotherapy. Blast cell features including immunophenotype, cytogenetics and in vitro cell cultures, suggested megakaryoblastic leukemia. In serum-free culture, blasts released GM-CSF and IL-6 which sustained autocrine growth and promoted normal myeloid and megakaryocytic colony formation.

Triphasic helical CT in Budd-Chiari syndrome: patterns of enhancement in acute, subacute and chronic disease.

AIM: To retrospectively evaluate helical computed tomography (CT) findings in a series of consecutive patients with Budd-Chiari syndrome. METHODS: Patterns of enhancement observed at contrast-enhanced helical CT in 10 consecutive patients (six women, four men; aged 27-51 years) with either acute, subacute or chronic Budd-Chiari syndrome were retrospectively evaluated along with the status of the hepatic veins. All patients underwent triphasic helical CT (10 mm beam collimation, 7 mm rec. intervals, 120 kV, 200-250 mA, pitch = 1.0) performed at 20-25, 70-75 and 300 s after i.v. bolus (3 ml/s) injection of 150 ml iodinated non-ionic contrast media. RESULTS: Abnormal patterns of enhancement were identified in eight patients. In all patients with acute Budd-Chiari disease (3/3) abnormal arterial enhancement of the caudate lobe, the so-called "fan-shaped pattern" was observed, whereas visible venous thrombosis was only depicted in two. Conversely, a "patchy pattern" of enhancement was observed in five out of seven patients with either sub-acute (2) or chronic Budd-Chiari disease (5) along with a strip-like appearance or lack of visualization of hepatic veins. CONCLUSIONS: The "fan-shaped" pattern of enhancement represent a characteristic finding of acute Budd-Chiari disease, and it may help to suggest the correct diagnosis even in absence of visible venous thrombosis.

T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules.

Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T-CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T-cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti-CD3 MoAbs and enhance that of both types of cells stimulated with anti-CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9-1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.

Immunoregulatory cytokine polymorphisms in Italian patients affected by paroxysmal nocturnal haemoglobinuria and aplastic anaemia.

We investigated regulatory variants of five cytokine genes [tumour necrosis factor (TNF)-alpha, interferon (IFN)-gamma, transforming growth factor (TGF)-beta, interleukin (IL)-6 and IL-10] in 40 Italian patients affected by paroxysmal nocturnal haemoglobinuria (PNH) and aplastic anaemia (AA). Genotypes associated with high production of TGF-beta and IFN-gamma were more frequent in patients than in controls. Genetic regulation of the immunological pathways involved in the pathogenesis of bone marrow failure is suggested.

Heterogeneity in the mitogenic response of peripheral blood mononuclear cells to a pan T monoclonal antibody.

Peripheral blood mononuclear cells (PBMC) from 80 normal donors were studied for their capacity to proliferate in response to Pan T2, an IgG1 monoclonal antibody (MoAb), that recognizes the CD3 complex. Forty percent of this population, regardless of sex or age, were found to be non-responders. However, the binding of MoAb Pan T2 to T cells as studied by indirect immunofluorescence was positive in all the donors. The addition of IL 1 or IL 2 to Pan T2-stimulated non-responder lymphocytes did not activate T cell proliferation, while the addition of responder monocytes restored the proliferation capacity in non-responder PBMC. The data indicate the existence of a heterogeneous responsiveness among normal individuals to a mitogenic IgG1 MoAb, and are in agreement with reports obtained using other anti-T3 MoAbs of IgG1 isotype, i.e. UCHT1, Leu4 and WT31. This defect is reported to be a function of monocytes, related to a polymorphism of Fc receptors for mouse IgG1 on human monocytic cells.

Autocrine growth function of human interleukin 1 molecules on ROHA-9, an EBV-transformed human B cell line.

In this study we report that the ROHA-9 cell line, an IL 1-secreting EBV-transformed human B cell line, exhibits an autocrine pathway of growth. In fact, ROHA-9 cells spontaneously secreted an autoregulatory growth factor that co-purified with the constitutively secreted IL 1-like molecules. Accordingly, monocyte-derived human IL 1, free of other known biological activities, also stimulated the growth of ROHA-9 cells in a dose-dependent way. Human recombinant interleukin 2, recombinant IFN-alpha or IFN-gamma and purified IFN-beta were ineffective when used at concentrations up to 1 X 10(3) U/ml. Furthermore, mouse recombinant IL 1, HPLC-purified multi-colony stimulating factor and partially purified preparations of BCGF were ineffective when assayed for growth-promoting activity on ROHA-9 cells. Moreover, a rabbit polyclonal antibody and a mouse monoclonal antibody to human IL 1 molecules blocked the growth of ROHA-9 cells induced by the autologous growth factor and by human IL 1. Lastly, purified human IL 1 increased the clonal efficiency of ROHA-9 cells seeded at a low cell concentration, allowing the isolation of the ROHA-9MC3 subclone, which showed similar growth response specificity and was particularly sensitive to the mitogenic activity of human IL 1.

Molecular characterization of PK-LR gene in pyruvate kinase-deficient Italian patients.

We studied the PK-LR gene in 15 unrelated Italian patients with congenital hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency. Fourteen different mutations were detected among 26 mutated alleles identified: a five-nucleotide (nt) deletion (227 to 231), two splice-site (1269C and IVS3(-2)c), 10 missense (514C, 787T, 823A, 993A, 994A, 1168A, 1456T, 1529A, 1552A, and 1594T) and one nonsense mutation(s) (721T). Eight of these (deletion 227-231, 1269C, IVS3(-2)c, 514C, 787T, 823A, 1168A, and 1552A) were novel. Moreover, a new polymorphic site was detected in the 3' untranslated region of the mRNA (C/T, nucleotide 1738). The deletion 227-231 causes a stop codon after amino acid 77, probably resulting in an unstable gene product. Mutations 1269C and IVS3(-2)c lead to an alteration of the 5' and 3' splice-site consensus sequence, respectively; cDNA analysis failed to reveal any abnormal transcript, suggesting that these mutations generate an unstable mRNA that is rapidly degraded. Of the five new missense mutations, 823A (Gly275-Arg) and 1168A (Asp390-Asn) involve highly conserved amino acids, 514C (Glu172-Gln) and 1552A (Arg518-Ser), although found in less conserved regions, affect the balance of the electric charges of the protein. Mutation 787T (Gly263-Trp) is likely to determine strong modifications in the local structure of the molecule. The most frequent mutation in Italy appears to be 1456T (seven of 30 alleles), followed by 1529A (three of 30) and 994A (three of 30). A correlation was found between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Study of the molecular defects in glucose phosphate isomerase-deficient patients affected by chronic hemolytic anemia.

We have studied four unrelated Italian patients with chronic hemolytic anemia associated with glucose phosphate isomerase (GPI) deficiency. Using intronic primers, we were able to detect the gene alterations on the genomic DNA of the patients. Five different mutations were identified among the eight mutated alleles found: three missense mutations (301A,584T,1028G), one nonsense mutation (286T), and a four nucleotides deletion [Del 1473-IVS16(+2)]. All of these were new except for mutation 1028G, which was previously identified in a Japanese variant (GPI Narita). Two patients were homozygotes (301A/301A and 1028G/1028G), whereas the other two were compound heterozygotes sharing a common mutation [286T/584T and Del 1473-IVS16(+2)/584T]. The missense mutations were found to involve highly conserved amino acids, suggesting that these residues are crucial for the maintenance of the enzyme function. The mutation 286T results in a truncated protein of 95 amino acids in comparison with the 558 of the normal one. The four nucleotides deletion located at the junction of exon/intron 16(5'-TTGGTCGgtgagt-3') is the first GPI mutation affecting a splice site. Moreover one difference from the published sequence (473T-->G) was found in exon five in all of the eight alleles studied and in 30 normal subjects. Correlation was made between mutations, biochemical characteristics of the enzyme, and clinical course of the disease.

Low plasma cholesterol: a correlate of nondiagnosed celiac disease in adults with hypochromic anemia.

OBJECTIVE: Hypochromic anemia is at times attributable to nondiagnosed celiac disease. The aim of this study was to define the correlates of celiac disease in anemic adults without overt malabsorption. METHODS: One hundred patients with hypochromic anemia and without diarrhea underwent a complete diagnostic work-up, including screening for celiac disease, i.e., upper endoscopy with duodenal biopsy and search of antiendomysium antibodies. RESULTS: Patients with hypochromic anemia were from two different Divisions and were analyzed as a single group because they were not significantly different for any variable. Hypochromic anemia was attributable to celiac disease in 10 patients. Compared to anemic patients without celiac disease, anemic patients with celiac disease had significant or borderline significant differences for plasma cholesterol (-17.9%), albumin (-9.4%), and body mass index (-11.8%), but not for gender distribution, age, weight, height, blood hemoglobin, mean corpuscolar volume, plasma iron, and ferritin. All anemic patients with celiac disease had plasma cholesterol < 156 mg/100 ml. Within the entire cohort of anemic patients, plasma cholesterol inversely related to prevalence of celiac disease (p < 0.001); also plasma albumin and body mass index inversely related to celiac disease, but coefficients were borderline significant (p = 0.056 and 0.052, respectively). CONCLUSIONS: The data suggest that among patients with hypochromic anemia, plasma cholesterol in the high-to-normal range could be used to exclude the presence of celiac disease. Other nutritional markers are less sensitive as indices of risk of celiac disease. Hematological indices are not of help to define the risk of celiac disease in anemic patients without signs of malabsorption.

Lymphocyte proliferative response to mitogenic monoclonal antibodies in systemic sclerosis. Evidence for unresponsiveness to murine monoclonal antibodies of IgG1 isotype.

Proliferative response of peripheral blood mononuclear cells to phitohemoagglutinin and anti-CD3 mitogenic monoclonal antibodies (MoAbs) of the IgG2a (OKT3) and IgG1 (PanT2, CLB T3/4.1) isotypes was studied in 39 patients with systemic sclerosis (SSc) and in 82 control subjects. The effect of IL-2 on this response was also investigated. No difference in the response to PHA and to IgG2a anti-CD3 MoAb OKT3 was seen between scleroderma patients and controls. Both the patient and control groups contained responders and non-responders to IgG1 anti-CD3 MoAbs. The percentage of non-responders was significantly higher in scleroderma patients than in controls. When purified lymphocytes from non-responder scleroderma patients were cultured with monocytes from control responders, proliferative response to IgG1 MoAbs was restored. Our results show that monocytes from patients with systemic sclerosis bear a defect leading to IgG1 unresponsiveness by T lymphocytes.

Glucose phosphate isomerase (GPI) "Morcone": a new variant from Italy.

Here we report the 4th Italian case of glucose phosphate isomerase (GPI) deficiency. The propositus is a young man suffering from chronic haemolytic anaemia since birth with occasional transfusion requirement. Biochemical characterization of the defective enzyme revealed increased affinity for F-6-P, decreased affinity for G-6-P and marked thermoinstability. Electrophoretic mobility appeared normal. GPI from both parents showed similar but less pronounced biochemical alterations. The variant described here seems to be different from those previously reported. Thus, we propose the provisional name of GPI "Morcone".

Molecular basis of chronic non-spherocytic haemolytic anaemia: a new G6PD variant (393 Arg----His) with abnormal KmG6P and marked in vivo instability.

More than 80 genetic variants of glucose-6-phosphate dehydrogenase (G6PD) are associated with chronic non-spherocytic haemolytic anaemia (CNSHA). In order to help clarify the molecular basis of this association, we have carried out a detailed biochemical and genetic characterization of two G6PD deficient brothers affected by CNSHA. The G6PD from the two patients has altered electrophoretic mobility, abnormally elevated Michaelis constant (Km) for G6P, and extreme instability in vivo and in vitro. By comparison with published information we found that this is a new G6PD variant which we have designated G6PD Portici. The entire coding region of the gene has been sequenced, and a single point mutation, a G----A transition, was found at position 1178 in exon X, causing a substitution of histidine for arginine at residue 393 in the polypeptide chain. By polymerase chain reaction (PCR) amplification followed by diagnostic restriction enzyme analysis and allele-specific oligonucleotide hybridization we have demonstrated the inheritance of this mutation in the patient's family. Our results support the notion of a causative link between this mutation in the G6PD gene and CNSHA. Our data, in combination with previous data in the literature, suggest that the three-dimensional structure of G6PD is such as to cause interaction in the binding of its two substrates, G6P and NADP.

Molecular characterization of G6PD deficiency in Southern Italy: heterogeneity, correlation genotype-phenotype and description of a new variant (G6PD Neapolis).

We report on the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty-one unrelated G6PD-deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A-, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A- and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467(Pro-->Arg) substitution responsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.