Abstract: The control of bone resorption is crucial in osteolytic diseases. Once attached to bone, osteoclasts (OCs) initiate the resorption process through the activation of a complex cascade of morphological and biochemical changes. Hyaluronan (HA), an extracellular glycosaminoglycan long non-branching polysaccharide, is expressed in bone matrices. Here we demonstrate that HA counter-balances the erosion activity of human mature OCs by significantly reducing their degradative potential. HA treatment of fully differentiated OCs derived from human peripheral blood monocytes inhibited migration on collagen as well as bone resorption. HA-mediated effects were primarily due to TRAcP, MMP-9, and cathepsin K down-regulation and to the increased levels of TIMP-1, a natural MMP-9 inhibitor. Binding of HA to mature OCs was entirely mediated by CD44: function-blocking anti-CD44 antibodies fully abrogated HA effects, and the engagement of HA receptor caused a rapid de-phosphorylation of Ser325 in the CD44 cytoplasmic tail. The inhibitory action by HA was associated with a transient up-phosphorylation of Pyk2, a novel persistent phosphorylation of p38 and the down-regulation of NFATc1 transcription factor. Our results provide a direct evidence for the involvement of CD44 in the HA-dependent regulation of OC activity and suggest a signaling pathway that could be unique in OC function inhibition.
Abstract: The determination of functional Antithrombin is a central part of thrombophilia screening. In this multicenter study, a new FXa-based method (INNOVANCE® Antithrombin) was evaluated on four different analyzers.
Abstract: We evaluated efficacy and safety of early and short-term prophylaxis with acenocumarine or dalteparin in the prevention of non-occlusive or occlusive central vein catheter-related thrombosis (CVCrT).
Abstract: In 2007, an Italian cancer research group proposed a specific concerted action aimed at the "analytical and clinica validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis." The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators' meeting, "Analytical and clinical validation of new biomarkers for early diagnosis," was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.
Abstract: BACKGROUND: Bronchioloalveolar carcinoma (BAC) is a histological subtype of non-small cell lung cancer (NSCLC), particularly of adenocarcinoma. Given its multifocality and the poor activity of chemotherapy, there is no established treatment for BAC, although promising results have been achieved with inhibitors of the epidermal growth factor receptor (EGFR). No tumor marker has been validated in the diagnosis and follow-up of lung cancer, in particular to predict the outcome of treatment with EGFR inhibitors. PURPOSE: As CA 15-3 antigen serum levels are reported to be pathologically abnormal in adenocarcinoma of the lung, we chose this tumor marker to monitor treatment with EGFR inhibitors of patients affected by adenocarcinoma with BAC features or pure BAC. PATIENTS AND METHODS: We collected data from 26 consecutive Caucasian patients with BAC, mostly women and never smokers, who received EGFR inhibitors. RESULTS: We noticed that all patients with normal CA 15-3 serum levels at baseline (15/26, 57.7%) showed a response to EGFR inhibitors, whereas all patients with abnormal CA 15-3 serum levels (11/26, 42.3%) did not. CONCLUSION: Our data suggest that CA 15-3 levels might be a predictive factor for the response to EGFR inhibitors in patients with BAC.
Abstract: Passenger leucocytes transfused with allogenic blood are responsible for potential adverse effects. The impact of pre-storage leucodepletion (in-line filtration) of all whole blood units on transfusion reaction rate among patients suffering from cancer was retrospectively studied, comparing all reactions following red blood cell (RBC) transfusions during 2 years of pre-storage vs. 2 years of selective (bedside) leucodepletion. During selective leucodepletion, 5165 RBC units - of which 2745 were bedside filtered units- were transfused to 866 patients. Twenty-eight reactions were recorded: 22 (15 in the bedside group) febrile non-haemolytic transfusion reactions (FNHTR) and six allergic reactions (five in the bedside group). The overall percentage of reactions was 0.54 (0.76 for bedside) and 0.42 for FNHTR (0.54 for bedside). During pre-storage leucodepletion, 4116 RBC units were transfused to 841 patients. Eleven reactions were recorded: four FNHTR and seven allergic reactions (urticaria). The percentage of reactions for transfused RBC units was 0.26 (0.09 for FNHTR). Comparison between pre-storage filtration and bedside filtration with regard to FNHTR showed an odds ratio of 2.80 (95% confidence interval = 0.83-14.87) for bedside filtration. The study suggests that, for transfused patients affected by cancer, pre-storage leucodepletion is more effective than selective (bedside) filtration in reducing the incidence of transfusion reactions (FNHTR).
Abstract: Studies of gene expression profiling (GEP) have been successfully used for the identification of molecules to be employed as potential prognosticators. With the aim of identifying the immunophenotypic profile of B-CLL subsets with different prognoses, we investigated by flow cytometry the expression of 36 surface antigens in 117 cases, 113 with survival data. In analogy with GEP, results were analyzed by applying unsupervised hierarchical algorithms (surface-antigen expression profiling, SEP). Distinct immunophenotypic groups (A, B1, B2 and C) were identified, group C (57/117) with longer survivals, as compared to groups A (23/117), B1 (16/117) and B2 (21/117). The immunophenotypic signatures of these groups were characterized by the coordinated and differential over-expression of: i) CD62L, CD54 and CD49c (group C); ii) CD38 and CD49d (group A); iii) none of the above markers (group B1 and B2). Other molecules were either not expressed, widely expressed by all samples, or were variably expressed within the observed B-CLL subgroups, although without a clearly distinguishable pattern. By employing an identical approach for investigating the reactivity of B-cell panel monoclonal antibodies (B-mAbs) in B-CLLs (29 cases) and in 19 B and non-B leukemia/lymphoma cell lines, we found mAbs (B012, B001, B006, B018, B019, B020, B017) mainly unreactive in all the samples, mAbs (B002, B010, B013, B014, B015) strongly reactive in B-CLLs and B-cell lines but not in non-B-cell lines, and mAbs recognizing antigens variably expressed in cell lines and B-CLLs. A hierarchical clustering focused on B-CLLs alone, combining reactivity values for B-mAbs with the expression of CD62L and CD38, these latter antigens identified as leader markers of B-CLL subsets with different prognosis, demonstrated a correlation between CD62L expression and the reactivity of B007, B003, B011 and B005 mAbs. These mAbs may represent potentially novel markers with prognostic relevance in B-CLLs.
Abstract: Mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes, along with CD38 expression, is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL). Configuration of IgVH genes displaying > 2% mismatch has been shown to correlate with longer survivals. In a series of 64 B-CLLs, we failed to confirm the prognostic value of the IgVH gene mutational status by using the suggested cutoff. However, the IgVH mutational status maintained its prognostic value only when evidence of antigen-driven selection could be documented. This was accomplished by applying statistical methods aimed at evaluating a significant skewing of replacement mutations from framework to complementary determining regions, as it occurs during germinal center differentiation of B cells. These data caution against wide application of the 2% somatic mutation cutoff as a prognostic determinant without demonstration of antigen-driven selection.
Abstract: Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.
Abstract: The CAG repeat (CAGn) present in the N-terminal region of the androgen receptor (AR) inversely correlates with AR transactivation activity. The aim of this study was to investigate whether polymorphic variation in the CAGn length is associated with the risk of developing ovarian cancer. Using a case-control study design 121 women with histologically confirmed ovarian cancer and 100 controls (healthy women) were genotyped for AR-CAG length. No marked difference in the mean length of CAGn was observed between ovarian cancer patients and controls. However, when considering patients with positive personal or family history of tumor (PPFHT), the mean lengths of the long allele, the short allele and the average of the 2 alleles were longer than in the controls. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were computed after allowance for age. We observed an increase in the risk of ovarian cancer, in terms of OR, in women with CAGn >or=22 (OR=2.17, 95% CI:1.10-4.27). The increase of relative risk was particularly high in women with CAGn >or=22 belonging to the PPFHT group: OR=3.52 (95% CI 1.18-10.47). We also found a statistically significant trend (chi2 trend=4.91; p=0.03) towards an increased risk of ovarian cancer with increasing CAGn length (from <or=21 to 22-23, 24-25 and >or=26). Again, a strong association between increase in CAGn and risk of ovarian cancer was observed in PPFHT patients (chi2 trend=6.38; p=0.01). The results suggest that AR-CAG repeat length could play a role as modifier of the ovarian cancer risk conferred by highly penetrant genes rather than itself conferring a low risk.
Abstract: Chronic renal failure often is associated with abnormal bleeding that may represent an important complication of this disorder. The hemorrhagic tendency currently is attributed to altered primary hemostasis, mainly platelet dysfunction. However, von Willebrand factor (vWF) also seems to be involved, even though the nature of its abnormalities is still controversial. To gain insight into the role of vWF in determining uremic bleeding, we studied 11 patients with stable, chronic renal failure. We found a significant increase in plasma factor VIII (FVIII), vWF:antigen (Ag), and vWF:ristocetin cofactor (Rco) levels, associated with a mean decrease in platelet vWF:Ag. Plasma vWF multimer pattern was characterized by increased representation of all oligomers in all patients, but five patients also showed a slight decrease in large vWF multimers. In addition, platelet vWF multimer pattern displayed a decrease in all components, especially those with high molecular weight. Despite normal bleeding time, collagen-induced platelet aggregation was defective in almost all patients, whereas vWF collagen binding capacity was normal. The levels of glycocalicin, the circulating fragment of glycoprotein Ib-IX, the major platelet vWF receptor, were also normal. In six patients who also were studied after initiation of dialysis, collagen-induced platelet aggregation was impaired further. Moreover, plasma vWF, and especially FVIII levels, were increased additionally, in association with a normalized platelet vWF content and an improved vWF multimer pattern. The results suggest that vWF abnormalities are present in uremia. Moreover, thrombopathy caused by impaired collagen-induced platelet aggregation is constantly present and apparently not improved by dialytic treatment.
Abstract: Primary effusion lymphoma (PEL) is a peculiar B-cell lymphoma characterized by infection by human herpesvirus type-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV) and by preferential growth in the serous body cavities. Histogenetic studies have suggested that PEL originates from B cells at a late stage of differentiation. In this study, we have investigated PEL for the expression status of MUM1/IRF4 (multiple myeloma 1/interferon regulatory factor 4) protein, which is involved in physiological B-cell maturation and represents a histogenetic marker of late B-cell differentiation. Using multiple detection assays, all cases of PEL (n = 22) were found to express MUM1/IRF4 molecules. MUM1/IRF4 expression was a selective feature of PEL among lymphomas involving the serous body cavities as secondary lymphomatous effusions generally failed to express the protein. In reactive lymphoid tissues, MUM1/ IRF4 expression clustered with advanced stages of B-cell differentiation. Comparison of MUM1/IRF4 expression with that of other histogenetic markers defined two phenotypic variants of PEL, i.e. MUM1/IRF4+, CD138/syndecan-1+, B-cell antigen- (20 out of 22 cases) and MUM1/IRF4+, CD138/syndecan-1-, B-cell antigen+ (2 out of 22 cases), suggesting a certain degree of heterogeneity in the disease histogenesis. The implications of these data are threefold. First, MUM1/IRF4 expression corroborates the notion that PEL originates from post-germinal centre, preterminally differentiated B-cells. Second, MUM1/IRF4 may help in the differential diagnosis of PEL among other lymphomas involving the serous body cavities. Finally, MUM1/IRF4 may interact with HHV-8/KSHV-encoded interferon regulatory factors (IRFs) and thus contribute to PEL escape from interferon-mediated control of viral infection.
Abstract: Chronic isolated hereditary macrothrombocytopenia (CHMT) is a congenital form of macrothrombocytopenia that seems to be due to defective production secondary to a disturbance in megakaryocyte fragmentation. To better understand the pathogenesis of thrombopoiesis in this hereditary thrombocytopenic disorder, we determined the percentage of reticulated platelets (RP), plasma glycocalicin (GC) and thrombopoietin (TPO) levels in 29 patients with CHMT, 23 patients with immune thrombocytopenic purpura (ITP), and 17 patients with thrombocytopenia secondary to decreased bone marrow megakaryocytes (hypoplasia). The % RP was similar in CHMT (2.27 +/- 1.33) and hypoplasia (1.98 +/- 1.35) patients and markedly lower than that in ITP patients (8.80 +/- 7.97; p <0.001), suggesting that the production of new platelets is reduced in CHMT. Plasma GC was within the normal range (0.84 +/- 0.16 microg/mL) both in patients with CHMT (0.63 +/- 0.20 microg/mL) and ITP (0.82 +/- 0.90 microg/mL), while it was significantly decreased in patients with hypoplasia (0.16 +/- 0.04 microg/mL; p < 0.001). When the GC value was normalized for platelet count, the GC index was normal in CHMT patients (2.05 +/- 1.1) and in patients with hypoplasia (0.85 +/- 0.10) while it was significantly increased in ITP patients (10.88 +/- 18.00; p<0.001); thus, patients with CHMT seem to have a normal platelet turnover. TPO was significantly increased in CHMT (195 +/- 72 pg/ml) as compared with normal (80 +/- 53 pg/ml; p < 0.002); however, the mean level was not as high as in ITP patients (345 +/- 167 pg/mL; p < 0.001). This finding suggests that CHMT syndrome is not secondary to a defective production of TPO and that megakaryocyte mass is nearly normal.
Abstract: BACKGROUND: Excessive bleeding may complicate congenital cardiac defects. To explain the pathogenesis of this abnormality, we evaluated selected parameters of primary hemostasis in patients with aortic valve stenosis before and after corrective surgery. METHODS AND RESULTS: We examined shear-induced platelet aggregation with the filter aggregometer test and von Willebrand factor (vWF) structure by evaluating the multimeric distribution and extent of subunit proteolysis. The platelet count was reduced before corrective surgery, and shear-induced platelet aggregation was impaired. Moreover, vWF multimers of higher molecular mass were decreased, and proteolytic subunit fragments were increased. After correction of the cardiac defect, all of these parameters returned to normal. CONCLUSIONS: Alterations of vWF and platelet function may contribute to the bleeding diathesis in patients with aortic valve stenosis. Improvement after corrective surgery suggests that the passage of blood through a stenosed aortic valve may result in shear forces that induce vWF interaction with platelets in the circulation and, in turn, trigger platelet clearance, vWF degradation, and the impairment of primary hemostasis.
Abstract: Thrombocytopenia is frequently reported in type 2B von Willebrand disease (vWD), and thought to be related to the abnormally high affinity of 2B von Willebrand factor (vWF) for platelet GPIb-IX. To gain an insight into the nature of this thrombocytopenia, we measured plasma glycocalicin (GC) levels (as a marker of platelet turnover), and platelet surface expression of the alpha granule protein P-selectin (as a marker of platelet activation) in 9 patients with type 2B vWD before, and in 4 patients also following the infusion of 1-desamino-8-d-arginine vasopressin (DDAVP). Three patients presented a persistent decrease of platelet counts in the resting condition. GC levels were within the normal range, regardless of the platelet counts, in all but one patient who presented, on the other hand, a normal platelet count. Moreover, platelets expressed normal amounts of P-selectin on their surface, regardless of platelet counts. These findings suggest that the thrombocytopenia observed in type 2B vWD is not due to platelet activation and subsequent consumption in circulation. Despite a significant, albeit transient, decrease in platelet count, DDAVP did not induce an increase in plasma GC levels, nor enhance P-selectin expression. These observations indicate that the acute post-DDAVP thrombocytopenia in type 2B vWD is not related to platelet activation and consumption. We advance that the post-DDAVP 2B vWF is hemostatically more active, and able to induce agglutination but not aggregation of circulating platelets. This would explain both the prompt recovery of basal platelet counts after the post-DDAVP decrease, and the lack of reported thrombotic complications in this disorder. Therefore, even though 2B vWF is characterized by an enhanced affinity for the platelet surface, its binding to platelet GPIb-IX in the soluble phase is not able to induce true platelet aggregation: vWF thus appears to be mainly an adhesive protein, rather than an aggregating agent.
Abstract: EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.
Abstract: We studied glycocalicin (GC), expressed as plasma GC concentration and as GC index (ratio to platelet count), in 129 thrombocytopenic patients (platelet count < 100 x 10(9)/l) and 60 sex- and age-matched controls. Seventy-two patients had idiopathic immune thrombocytopenia, 32 secondary immune thrombocytopenia, 8 microangiopathic thrombocytopenia and 17 thrombocytopenia secondary to bone marrow aplasia. Patients with immune thrombocytopenia (ITP) were also subclassified, according to their clinical behaviour, as having active disease or being in spontaneous or therapy-induced partial remission. A significant correlation was found between glycocalicin levels and platelet count both in normals and in patients with bone marrow aplasia (r = 0.75). ITP patients showed a GC index significantly higher than controls (6.02+/-7.87 vs. 0.9+/-0.2, p<0.001). When ITP patients with similar platelet count (30-50 x 10(9)/l) were studied, the mean level of GC and the GC index were significantly higher in those patients with active disease than in those in remission (0.97+/-0.38 vs. 0.58+/-0.17 microg/ml, p <0.05; 6.41+/-2.64 vs. 3.44+/-0.94, p<0.05, respectively). A longitudinal study performed in 10 patients with different subtypes of ITP suggested a positive correlation between GC index and the activity of the disease. The GC value and GC index were significantly higher in patients with microangiopathic thrombocytopenia than in controls (1.44+/-0.73 vs. 0.8+/-0.16 microg/ml, p < 0.01; and 18.77+/-22.23 vs. 0.9+/-0.2, p<0.001, respectively). The GC value was significantly lower in bone marrow failure (0.15+/-0.04 microg/ml, p<0.01) compared to controls, while no difference was observed in the GC index. Our data confirm that the GC index is helpful in differentiating thrombocytopenia due to increased platelet destruction from the one due to impaired production. In addition, the assay has been proven useful in the differential diagnosis of different ITP subtypes and their follow-up.
Abstract: When studying autoantibody specificity by the indirect antiglobulin test with column agglutination techniques ether and xylene elution techniques result in haemoglobin stained eluates which give a red colouration to the gel or glass beads and do not allow the identification of positive reactions. Xylene eluates were incubated with commercially available group O-test red cell panels at 37 degrees C for 45 min in the wells of a microtitre plate in a 3:1 eluate:red cell ratio. After washing with normal saline, sensitized red cells, resuspended in low ionic strength solution (LISS), were applied onto the microtubes containing the antiglobulin serum and positive reactions were recorded after centrifugation. We studied the specificity of 35 autoantibody containing eluates from 12 patients with lymphoproliferative disorders (six having autoimmune haemolysis) and 23 HIV patients without autoimmune haemolysis. All patients had a gel or column positive (IgG) direct antiglobulin test while the tube direct antiglobulin test failed to show red cell bound IgG. We found a reactive indirect antiglobulin test in 20/23 eluates from HIV infected patients (with a panreactive specificity), in all patients with autoimmune haemolysis (one with anti-C, two with anti-E, one with anti-K and two with a panreactive specificity) and in all patients with positive direct antiglobulin test but without immune mediate haemolysis (in all cases with panreactive specificity). The method proposed is a promising tool for the study of the specificity of antibody containing haemoglobin stained eluates; in this study it allowed us to confirm that some HIV patients have specific binding of IgG on their RBC and to identify the specificity of tube test non-reactive eluates.
Abstract: The presence and specificity of antiplatelet autoantibodies in 32 patients with primary and 18 patients with secondary autoimmune thrombocytopenic purpura (AITP), as well as 11 non-thrombocytopenic patients with systemic autoimmune diseases, were studied. By means of the direct and indirect monoclonal antibody immobilization of platelet antigen (MAIPA) assay, antiplatelet autoantibodies were detected using monoclonal antibodies specific for platelet glycoproteins (GPs) Ib, IIb/IIIa, Ia/IIa, and IV. Serum antiplatelet autoantibodies were found in 18 of 32 primary AITP patients (56%), 6 of 18 secondary AITP patients (33%), and 5 of 11 nonthrombocytopenic patients (45%). Platelet-associated autoantibodies were detected in five of eight patients with primary (62%) and in four of eight patients with secondary AITP (50%) and in two of four patients without thrombocytopenia (50%). Multiple antibody reactivity, mainly against GPs IIb/IIIa and Ib and, in a few patients, against Ia/IIa, was found. Using MAIPA, platelet xylene eluates from 20 patients were also studied. Antiplatelet elutable autoantibodies were related to thrombocytopenia; autoantibodies against membrane GPs Ib and IIb/IIIa were demonstrable in 84 and 63% of eluates from patients with primary and secondary AITP, respectively, but not in eluates from nonthrombocytopenic patients. The presence of antiplatelet antibodies thus appears to be a common feature of many autoimmune diseases apart from the thrombocytopenia, but the (primary or secondary) etiology of the immune thrombocytopenia cannot be differentiated on the grounds of their specificity.
Abstract: BACKGROUND: Threonine145/methionine145 dimorphism in platelet glycoprotein (GP) Ib alpha defines the human platelet antigen (HPA)-2 system that has been implicated in refractoriness to HLA-matched platelet transfusion and in neonatal immune thrombocytopenic purpura. STUDY DESIGN AND METHODS: The occurrence of this amino acid dimorphism was investigated in 379 Italian blood donors by studying their genomic DNA. Two oligonucleotide primers, Ib alpha-3 (5'-GGACGTCTCCTTCAACCGGC-3') and Ib alpha-4 (5'-GCTTTGGTGGGGAACTTGAC-3'), were used in a polymerase chain reaction to generate a 591-base pair fragment that was digested with the restriction enzyme Acy I. To investigate whether this dimorphism is involved in the binding of von Willebrand factor (vWF) to GPlb, the binding of vWF to the GPlb/IX complex was measured in two Met145/Met145 and two Thr145/Thr145 subjects. RESULTS: The genotypic frequencies are 78.9% for Thr/Thr, 19.8% for Thr/Met), and 1.3% for Met/Met; the allelic frequencies are 88.8% for Thr145 and 11.2% for Met145. Estimates for binding of subunit molecules per platelet at saturation and inhibition constant in mol per L, respectively, follow. In the presence of ristocetin (0.5 mg/mL), they are 11,460 +/- 2,040 and 1.26 +/- 0.44 x 10(-8) for normals and 11,230 +/- 2,330 and 1.29 +/- 0.48 x 10(-8) for patients. In the presence of botrocetin (2.5 micrograms/mL), they are 64,260 +/- 7,760 and 2.99 +/- 0.96 x 10(-8) for normals and 65,770 +/- 11,570 and 2.47 +/- 0.22 x 10(-8) for patients. Platelet aggregation responses obtained using platelet-rich plasma from donors with Met145/Met145 or Thr145/Thr145 genotype were within normal limits. CONCLUSION: Genotypic and phenotypic frequencies in the HPA-2 system in this population are consistent with those reported among the white population. Furthermore, the HPA-2 system is not involved in the binding of vWF to GPlb.
Abstract: Acquired abnormalities of red cell membrane protein composition in 37 patients with a positive direct antiglobulin test have been studied: 17 patients had true autoimmune haemolytic anaemia and 20 were HIV-infected subjects with a positive direct antiglobulin test but without signs of haemolysis. The study was carried out by performing sodium dodecyl sulphate polyacrylamide gel electrophoresis of ghost proteins followed by densitometric evaluation of the areas under the peaks, normalized by the total (alpha + beta) spectrin content. Results show a significant decrease of bands 3, 4.1 and 4.2 over spectrin in patients with autoimmune haemolysis as compared to controls; at least in a small subset of patients, different specificities recognized by autoantibodies do not seem to account for these abnormalities which are reproducible independently from the molecular size of bands immunoprecipitated by autoantibodies. A similar decrease of protein 4.2 but not of band 3 staining intensity is also noticeable in HIV patients with a positive direct antiglobulin test. These results are consistent with the hypothesis that, following interactions between autoantibodies and autoantigens, modifications occur on membrane proteins resembling a variety of quantitative defects described in inherited haemolytic anaemias, and mainly the "vertical interaction defects' of hereditary spherocytosis. Moreover, the decrease of band 3 staining intensity seems to represent a feature of patients with immune mediated haemolysis and not only with autoantibody binding.
Abstract: It has been suggested that acquired abnormalities of the red cell membrane due to various injuries [azidothymidine (AZT) therapy, immunoglobulin coating of red cells, differentiation abnormalities of erythroid precursors] contribute to the onset of anaemia in HIV-infected patients. In vitro proteolysis of erythrocyte membrane proteins is regarded as a molecular marker of membrane damage induced in vivo by different agents. We therefore investigated in vitro proteolysis of ghosts derived from red blood cells of 30 HIV-infected patients. Considered collectively, there was no significant increase in in vitro proteolysis in ghosts from anaemic HIV patients. However, a significantly higher degree of in vitro self-digestion of RBC membrane proteins was evident in HIV-infected patients with spleen enlargement, but not in splenomegalic patients suffering from liver cirrhosis. Neither AZT therapy nor the presence of a positive direct antiglobulin test seemed to be directly associated with increased in vitro protein breakdown. The results seem to suggest damage of the red cell membrane in HIV infection, induced by injuries on red cells during their prolonged retention inside an enlarged spleen, while it seems unlikely that AZT therapy or immunoglobulin coating of red cells play major roles in red cell damage.
Abstract: Several 'capture' assays are currently employed to identify specific platelet antibodies, but all require the use of murine monoclonal antibodies (MoAbs) against the antigen of interest. We have developed a new antigen capture assay for the detection of platelet reactive antibodies, based on platelet surface sialoglycoprotein labelling with biotin hydrazide, and a following immobilization of the biotinylated platelet proteins to microtiter wells that had been coated with streptavidin. The resulting solid phase can then be used in a simple ELISA to detect serum and platelet associated antibodies. We describe here two versions of this biotin-avidin immobilization of platelet glycoproteins (BAIPG) assay. In BAIPG assay type I, the test sera are directly incubated in microtiter wells previously coated with streptavidin plus biotinylated platelet proteins. The BAIPG type II procedure involves the incubation of sera with biotinylated platelets before platelet solubilization, and, after platelet lysis, the immobilization of the immune complexes to streptavidin-coated wells. In both cases, the bound antibodies are determined by alkaline phosphatase conjugated anti-human IgG. Using BAIPG type I, positive results were obtained in 7/33 patients with idiopathic thrombocytopenic purpura (ITP), 1/10 patients with secondary immune thrombocytopenia (SIT) and 4/17 with non-immune thrombocytopenia (NIT). The BAIPG type II test was positive in 13 out of 33 patients with ITP, in six out of ten patients with SIT, and in three out of the 17 patients with NIT. A comparison between BAIPG and monoclonal antibody immobilization of platelet antigens (MAIPA) assays showed a high degree of correlation between the two methods. These results suggest that the BAIPG assay is a valuable new tool for the detection of anti-platelet antibodies.
Abstract: Erythrocyte surface was labelled by means of biotin; immunoprecipitation technique was then used to localise antigens recognised on red cell membrane proteins by: a) autoantibodies from 13 patients with antierythrocyte autoimmunity; b) commercially available anti-D and anti-k (Cellano) antierythrocyte alloantibodies. Results with alloantibodies are comparable to those obtained using radiochemical probes. Immunoprecipitations with autoantibody containing eluates showed reactivity at different molecular weights (the most common at 34-50 kD, others at 100 and 45 kD and a newly described one at 80 kD), thus confirming that many membrane proteins may act as target antigens for erythrocyte autoimmunity. We found a higher percentage of reactive immunoprecipitates than previously reported using the same labelling method. However, critical conditions to allow valuable results seem to be a threshold amount of autoantibody to precipitate any recognisable band and the sensitivity of the detection method. Hence methodological variables must be taken into consideration before concluding that "non protein" antigens trigger the autoimmune process.
Abstract: By means of immunoblotting and monoclonal antibody immobilization of platelet antigens (MAIPA) we have studied the specificity of antiplatelet antibodies in patients with antiphospholipid antibodies and thrombocytopenia defined as presence of anticardiolipin IgG and a platelet count below 100 x 10(9)/l. The study group consisted of 10 patients with systemic lupus erythematosus (SLE), 8 patients with primary anti-phospholipid syndrome (PAPS) and 16 patients with idiopathic thrombocytopenic purpura (ITP). The comparison group was formed by 17 patients with classical chronic ITP without anticardiolipin IgG. We identified the 80-100, 130-150 and 150-170 KD surface proteins that comigrate with GPIIIa, GPIIb and GPIb and a 50-70 KD cytoplasm band by immunoblot. In patients with classical chronic ITP, the prevalence of the antiplatelet antibodies against GPIIIa was 53% on immunoblot assay and 47% on MAIPA. In ITP patients who had also anti-phospholipid antibodies in serum, the percentage of reactivity to GPIIIa declined to 37% on immunoblot and 21% on MAIPA but it was not statistically different from the percentage observed in patients with classical ITP. Autoantibodies to platelet surface glycoproteins were almost absent in SLE and PAPS patients, who showed a significant prevalence (78%) of IgG reactivity to the 50-70 KD internal platelet protein which was frequently encountered also in patients with ITP and aPL (56%). Our study provides additional evidence that platelet antigens in patients with phospholipid-associated secondary immune thrombocytopenia are different from those of primary ITP, and that surface glycoproteins were not involved.
Abstract: BACKGROUND. In sera and platelet eluates of ITP patients, antigen specificity was widely studied by means of sensitive methods including immunoprecipitation, monoclonal antibody immobilization, and immunoblot. These studies indicated that GPIIb-IIIa were the main epitopes of ITP autoantibodies. METHODS. We studied the specificity of antiplatelet autoantibodies in 45 patients with acute and chronic ITP. Patient sera were tested by Western blot on separated platelet proteins in non-reducing conditions; antibody binding was identified using biotinconjugated anti-human IgG and avidin-peroxidase. RESULTS. Two main nonspecific bands of 200 and 125 kD were visible using normal serum; the first referred to platelet IgG, and the second was due to a naturally occurring antibody towards an internal protein. Twenty-five sera (55%) stained one (n = 11), two (n = 7), three (n = 3) or four (n = 4) specific bands. In patients with chronic ITP there was a prevalence of multiple bands. The relative molecular weights of the recognized antigens were in the range of 140-160, 80-100, 50-70 and 40 kd. The 80-100 epitope was recognized as a membrane protein in only 40% of sera, and it was partially characterized as GPIIIa in 4 patients. The other stained epitopes were absorbed by platelet lysate and then identified as internal proteins. CONCLUSIONS. This finding might be related to sensitization to antigens exposed by platelets during immune damage, and may pose an important problem in the identification with the immunoblot technique of target antigens responsible for immune sequestration.
Abstract: Twenty-seven patients with peripheral atherosclerotic disease were randomized into two therapy regimens consisting of indobufen (Indo) (400 mg/day) and dipyridamole (Dip) (225 mg/day) plus acetylsalicylic acid (ASA) (1 g/day), respectively. Maximal walking distance (MWD) and ankle-arm systolic pressure ratios were measured before and after three and six months of therapy; bleeding time, beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and serum thromboxane B2 (TXB2) were also assessed. The two treatment groups showed a significant and progressive increase in pain-free walking distance at both three and six months of therapy, but patients taking indobufen showed a greater improvement. On the contrary, the pressure doppler ratio at rest was statistically improved only in the ASA plus Dip group. Basal beta-TG and PF4 levels were normal and no changes occurred during the study in either group, while in all patients bleeding times showed a significant increase above basal values and serum TBX2 decreased.
