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1.
J Clin Immunol ; 2022 Jul 09.
Article in English | MEDLINE | ID: mdl-35809212

ABSTRACT

BACKGROUND AND PURPOSE: Waning immunity and the surge of SARS-CoV-2 variants are responsible for breakthrough infections, i.e., infections in fully vaccinated individuals. Although the majority of vaccinated infected subjects report mild or no symptoms, some others require hospitalization. The clinical and immunological features of vaccinated hospitalized COVID-19 patients are currently unknown. METHODS: Twenty-nine unvaccinated and 36 vaccinated hospitalized COVID-19 patients were prospectively enrolled and clinical and laboratory data were gathered. Immunophenotyping of leukocytes' subsets, T and B cell SARS-CoV-2-specific responses were evaluated via flow cytometry. Anti-IFN-α autoantibodies were measured via ELISA. RESULTS: Despite vaccinated patients were older and with more comorbidities, unvaccinated subjects showed higher levels of pro-inflammatory markers, more severe disease, and increased mortality rate. Accordingly, they presented significant alterations in the circulating leukocyte composition, typical of severe COVID-19. Vaccinated patients displayed higher levels of anti-Spike IgGs and Spike-specific B cells. Of all participants, survivors showed higher levels of anti-Spike IgGs and Spike-specific CD4+ T cells than non-survivors. At hospital admission, 6 out of 65 patients (9.2%) displayed high serum concentrations of autoantibodies targeting IFN-α. Remarkably, 3 were unvaccinated and eventually died, while the other 3 were vaccinated and survived. CONCLUSION: Despite more severe pre-existing clinical conditions, vaccinated patients have good outcome. A rapid activation of anti-SARS-CoV-2-specific immunity is fundamental for the resolution of the infection. Therefore, prior immunization through vaccination provides a significant contribution to prevention of disease worsening and can even overcome the presence of high-risk factors (i.e., older age, comorbidities, anti-IFN-α autoantibodies).

2.
Front Neurol ; 13: 872396, 2022.
Article in English | MEDLINE | ID: mdl-35693002

ABSTRACT

Relapsing-remitting multiple sclerosis (RRMS) is a demyelinating disease in which pathogenesis T cells have a major role. Despite the unknown etiology, several risk factors have been described, including a strong association with human leukocyte antigen (HLA) genes. Recent findings showed that HLA class I-G (HLA-G) may be tolerogenic in MS, but further insights are required. To deepen the HLA-G role in MS inflammation, we measured soluble HLA-G (sHLA-G) and cytokines serum level in 27 patients with RRMS at baseline and after 12 and 24 months of natalizumab (NTZ) treatment. Patients were divided into high (sHLA-G>20 ng/ml), medium (sHLA-G between 10 and 20 ng/ml), and low (sHLA-G <10 ng/ml) producers. Results showed a heterogeneous distribution of genotypes among producers, with no significant differences between groups. A significant decrease of sHLA-G was found after 24 months of NTZ in low producers carrying the +3142 C/G genotype. Finally, 83.3% of high and 100% of medium producers were MRI-activity free after 24 months of treatment, compared to 63.5% of low producers. Of note, we did not find any correlation of sHLA-G with peripheral cell counts or cytokines level. These findings suggest that serum sHLA-G level may partly depend on genotype rather than peripheral inflammation, and that may have impacted on MRI activity of patients over treatment.

3.
Am J Hematol ; 97(7): 846-855, 2022 07.
Article in English | MEDLINE | ID: mdl-35338671

ABSTRACT

Prognostic modeling in myelofibrosis (MF) has classically pursued the integration of informative clinical and hematological parameters to separate patients' categories with different outcomes. Modern stratification includes also genetic data from karyotype and mutations. However, some poorly standardized variables, as peripheral blood (PB) blast count by morphology, are still included. In this study, we used multiparameter flow cytometry (MFC) with the aim of improving performance of existing scores. We studied 363 MF patients with available MFC files for PB CD34+ cells count determination at diagnosis. We adapted Ogata score to MF context including 2 parameters: absolute CD34+ cells count (/µL) and granulocytes to lymphocytes SSC ratio. A score of 1 was attributed to above-threshold values of each parameter. Accordingly, patients were categorized as MFClow (score = 0, 62.0%), MFCint (score = 1, 29.5%), and MFChigh (score = 2, 8.5%). MFClow had significantly longer median OS (not reached) compared to MFCint (55 months) and MFChigh (19 months). We integrated MFC into established models as a substitute of morphological PB blasts count. Patients were reclassified according to MFC-enhanced scores, and concordance (C-) indexes were compared. As regards IPSS, C-indexes were 0.67 and 0.74 for standard and MFC-enhanced model, respectively (Z score - 3.82; p = 0.0001). MFC-enhanced MIPSS70+ model in PMF patients yielded a C-index of 0.78, outperforming its standard counterpart (C-index 0.73; Z score - 2.88, p = 0.004). Our data suggest that the incorporation of MFC-derived parameters, easily attainable from standard assay used for CD34+ cells determination, might help to refine the current prognostic stratification models in myelofibrosis.


Subject(s)
Primary Myelofibrosis , Antigens, CD34 , Flow Cytometry , Humans , Mutation , Primary Myelofibrosis/diagnosis , Primary Myelofibrosis/genetics , Prognosis
4.
J Clin Med ; 10(8)2021 Apr 14.
Article in English | MEDLINE | ID: mdl-33919958

ABSTRACT

Relapsed/refractory (R/R) acute myeloid leukemia (AML) is a largely unmet medical need, owing to the lack of standardized, effective treatment approaches, resulting in an overall dismal outcome. The only curative option for R/R AML patients is allogeneic hematopoietic stem cell transplantation (HSCT) which is only applicable in a fraction of patients due to the scarce efficacy and high toxicity of salvage regimens. Recently, a number of targeted agents with relatively favorable toxicity profiles have been explored in clinical trials for R/R AML patients. The Bcl-2 inhibitor venetoclax, in combination with hypomethylating agents or low dose cytarabine, has produced impressive results for newly diagnosed AML, while its role in R/R disease is not well defined yet. We retrospectively analyzed the clinical outcomes of 47 R/R AML patients treated with venetoclax-based regimens between March 2018 and December 2020 at our institution. Overall, we report a composite complete response rate of 55% with an overall acceptable toxicity profile. Outcomes were particularly favorable for NPM1 mutated patients, unlike for FLT3-ITD positive patients irrespective of NPM1 status. For patients treated with intention to transplant, the procedure could be finally performed in 54%. These findings suggest a role for venetoclax-based regimens in R/R AML patients and support the design of prospective studies.

5.
Methods Mol Biol ; 2285: 99-109, 2021.
Article in English | MEDLINE | ID: mdl-33928546

ABSTRACT

T-cell receptor (TCR)-Vß repertoire analysis is a sensitive method for detection of T-cell clonality. This type of analysis has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens and in neoplastic T cells. Here, we describe the flow cytometric methods to perform this analysis.


Subject(s)
Flow Cytometry , Receptors, Antigen, T-Cell, alpha-beta/metabolism , T-Lymphocyte Subsets/metabolism , Animals , Humans , Immunophenotyping , Phenotype , Research Design , T-Lymphocyte Subsets/immunology , Workflow
6.
Cancers (Basel) ; 12(11)2020 Oct 30.
Article in English | MEDLINE | ID: mdl-33143086

ABSTRACT

Acute myeloid leukemia (AML) "with myelodysplasia-related changes (MRC)" is considered a separate entity by the World Health Organization (WHO) classification of myeloid neoplasms. While anamnestic and cytogenetic criteria provide objective attribution to this subset, with clear unfavorable prognostic significance, the actual role of multi-lineage dysplasia (MLD) as assessed by morphology is debated. The aim of our work was to study MLD by a technique alternative to morphology, which is multiparameter flow cytometry (MFC), in a large series of 302 AML patients intensively treated at our Center. The correlation with morphology we observed in the unselected analysis reiterated the capability of the MFC-based approach at highlighting dysplasia. MLD data, estimated through an immune-phenotypic score (IPS), provided no insight into prognosis when considered overall nor within well-defined genetic categories. Of interest, IPS-related dysplasia conveyed significant prognostic information when we focused on genetically undefined patients, triple-negative for NPM1, FLT3 and CEBPA (TN-AML). In this context, the lack of dysplastic features (IPS_0) correlated with a significantly higher CR rate and longer survival compared to patients showing dysplasia in one or both (neutrophil and erythroid) cell lineages. The impact of IPS category maintained its validity after censoring at allogeneic HSCT and in a multivariate analysis including baseline and treatment-related covariates. In a subgroup featured by the lack of genetic determinants, our data could help address the relative unmet needs in terms of risk assessment and treatment strategy, and provide insight into prediction of response in the rapidly evolving therapeutic scenario of AML.

7.
Immunology ; 161(4): 345-353, 2020 12.
Article in English | MEDLINE | ID: mdl-32870529

ABSTRACT

SARS-CoV-2 is responsible for a new infectious disease (COVID-19) in which individuals can either remain asymptomatic or progress from mild to severe clinical conditions including acute respiratory distress syndrome and multiple organ failure. The immune mechanisms that potentially orchestrate the pathology in SARS-CoV-2 infection are complex and only partially understood. There is still paucity of data on the features of myeloid cells involved in this viral infection. For this reason, we investigated the different activation status profiles and the subset distribution of myeloid cells and their correlation with disease progression in 40 COVID-19 patients at different stages of disease. COVID-19 patients showed a decrease in the absolute number of plasmacytoid and myeloid dendritic cells, different subset distribution of monocytes and different activation patterns of both monocytes and neutrophils, coupled to a significant reduction of HLA-DR monocyte levels. We found that some of these alterations are typical of all COVID-19 patients, while some others vary at different stages of the disease and correlate with biochemical parameters of inflammation. Collectively, these data suggest that not only the lymphoid, but also the myeloid compartment, is severely affected by SARS-CoV-2 infection.


Subject(s)
COVID-19/immunology , Dendritic Cells/immunology , Myeloid Cells/immunology , Adult , Aged , COVID-19/pathology , Dendritic Cells/pathology , Female , Flow Cytometry , Humans , Intensive Care Units , Male , Myeloid Cells/pathology
8.
J Clin Invest ; 130(9): 4694-4703, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32463803

ABSTRACT

BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy).


Subject(s)
Betacoronavirus , Coronavirus Infections/immunology , Cytotoxicity, Immunologic , Interleukin-6/immunology , Pneumonia, Viral/immunology , Adult , Aged , Aged, 80 and over , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19 , Coronavirus Infections/blood , Coronavirus Infections/epidemiology , Critical Care , Cytokines/blood , Cytokines/immunology , Female , Granzymes/blood , Granzymes/immunology , Humans , Interleukin-6/blood , Killer Cells, Natural/immunology , Male , Middle Aged , Models, Immunological , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/epidemiology , SARS-CoV-2
9.
Front Immunol ; 11: 559, 2020.
Article in English | MEDLINE | ID: mdl-32328061

ABSTRACT

Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the post-treatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naïve and memory CD4+ and CD8+) across 15 RRMS patients before and after two years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multi-dimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis and treatment regimes. Natalizumab (NTZ) and autologous hematopoietic stem cell transplantation (AHSCT) are two successful treatments for relapsing-remitting multiple sclerosis (RRMS), an autoimmune T-cell-driven disorder affecting the central nervous system that is characterized by relapses interspersed with periods of complete or partial recovery. Both RRMS treatments have been documented to impact T-cell subpopulations and the T-cell receptor (TCR) repertoire in terms of clone frequency, but, so far, the link between T-cell naive and memory populations, autoimmunity, and treatment outcome has not yet been established hindering insight into the posttreatment TCR landscape of MS patients. To address this important knowledge gap, we tracked peripheral T-cell subpopulations (naive and memory CD4+ and CD8+) across 15 RRMS patients before and after 2 years of continuous treatment (NTZ) and a single treatment course (AHSCT) by high-throughput TCRß sequencing. We found that the two MS treatments left treatment-specific multidimensional traces in patient TCRß repertoire dynamics with respect to clonal expansion, clonal diversity, and repertoire architecture. Comparing MS TCR sequences with published datasets suggested that the majority of public TCRs belonged to virus-associated sequences. In summary, applying multidimensional computational immunology to a TCRß dataset of treated MS patients, we show that qualitative changes of TCRß repertoires encode treatment-specific information that may be relevant for future clinical trials monitoring and personalized MS follow-up, diagnosis, and treatment regimens.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Multiple Sclerosis, Relapsing-Remitting/therapy , Receptors, Antigen, T-Cell, alpha-beta/immunology , Adult , CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Female , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunologic Factors/therapeutic use , Male , Middle Aged , Natalizumab/therapeutic use , Receptors, Antigen, T-Cell, alpha-beta/drug effects , Transplantation, Autologous
10.
Haematologica ; 102(3): 529-540, 2017 03.
Article in English | MEDLINE | ID: mdl-28250006

ABSTRACT

Mutations in CCAAT/enhancer binding protein α (CEBPA) occur in 5-10% of cases of acute myeloid leukemia. CEBPA-double-mutated cases usually bear biallelic N- and C-terminal mutations and are associated with a favorable clinical outcome. Identification of CEBPA mutants is challenging because of the variety of mutations, intrinsic characteristics of the gene and technical issues. Several screening methods (fragment-length analysis, gene expression array) have been proposed especially for large-scale clinical use; although efficient, they are limited by specific concerns. We investigated the phenotypic profile of blast and maturing bone marrow cell compartments at diagnosis in 251 cases of acute myeloid leukemia. In this cohort, 16 (6.4%) patients had two CEBPA mutations, whereas ten (4.0%) had a single mutation. First, we highlighted that the CEBPA-double-mutated subset displays recurrent phenotypic abnormalities in all cell compartments. By mutational analysis after cell sorting, we demonstrated that this common phenotypic signature depends on CEBPA-double-mutated multi-lineage involvement. From a multidimensional study of phenotypic data, we developed a classifier including ten core and widely available parameters. The selected markers on blasts (CD34, CD117, CD7, CD15, CD65), neutrophil (SSC, CD64), monocytic (CD14, CD64) and erythroid (CD117) compartments were able to cluster CEBPA-double-mutated cases. In a validation set of 259 AML cases from three independent centers, our classifier showed excellent performance with 100% specificity and 100% sensitivity. We have, therefore, established a reliable screening method, based upon multidimensional analysis of widely available phenotypic parameters. This method provides early results and is suitable for large-scale detection of CEBPA-double-mutated status, allowing gene sequencing to be focused in selected cases.


Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/genetics , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Mutation , Phenotype , Adolescent , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor , Bone Marrow/pathology , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cluster Analysis , Cytogenetic Analysis , DNA Mutational Analysis , Female , Genetic Association Studies , Genotype , Humans , Immunophenotyping , Kaplan-Meier Estimate , Leukemia, Myeloid, Acute/mortality , Male , Middle Aged , Prognosis , Reproducibility of Results , Sensitivity and Specificity , Young Adult
11.
Brain Behav Immun ; 50: 78-86, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26130058

ABSTRACT

Fingolimod affords protection from MS by sequestering lymphocytes in secondary lymphoid organs via down regulation of their sphingosine 1 phosphate receptor (S1P1). Unexpectedly, accumulating evidence indicates that patients who discontinue fingolimod treatment may be at risk of rehearsal of magnetic resonance (MR) and clinical disease activity, sometimes featuring dramatic rebound. We therefore developed in vivo and in vitro models of post-fingolimod MS rebound to unravel its cellular and molecular mechanisms. The impact of fingolimod withdrawal on T regulatory lymphocytes was also investigated by means of cytofluorimetric analysis and antigen-specific lymphocyte proliferation assays. We show that mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE) undergo extremely severe, chronic disease rebound upon discontinuation of fingolimod. Remarkably, rebound is preceded by a burst of S1P1 overexpression in lymph node-entrapped lymphocytes that correlates with subsequent massive lymphocyte egress and widespread CNS immune infiltration. Also, consistent with the ability of S1P1 to counteract polarization and function of T regulatory lymphocytes their number and suppression of effector T cells is reduced by fingolimod suspension. Data disclose the first pathogenic mechanisms of post-fingolimod rebound that may be targeted for therapeutic intervention.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Fingolimod Hydrochloride/administration & dosage , Fingolimod Hydrochloride/adverse effects , Receptors, Lysosphingolipid/metabolism , Signal Transduction/drug effects , T-Lymphocytes, Regulatory/immunology , Animals , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Female , Mice , Mice, Inbred C57BL , Receptors, Lysosphingolipid/agonists , Signal Transduction/immunology , Spinal Cord/drug effects , Spinal Cord/immunology , Spinal Cord/metabolism , T-Lymphocytes, Regulatory/metabolism
12.
Exp Hematol ; 43(10): 869-879.e22, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26101160

ABSTRACT

The presence of multilineage dysplasia (MLD) by morphology at diagnosis in acute myeloid leukemia (AML) defines a separate subset in the World Health Organization classification with still-debated prognostic value. A major controversy concerns MLD's role in NPM1-mutated (NPM1⁺) AML, which correlates with good prognosis. We used flow cytometry (FC), an emerging technique for assessing dysplasia, to investigate MLD in NPM1⁺ AML by an immunophenotypic score (IPS), a technique previously adopted in myelodysplastic syndrome. Eighty-five intensively treated NPM1⁺ AML cases were studied. Patients were grouped according to the combination of data in maturing cell compartments. FC-assessed dysplasia showed a significant correlation with morphology-assessed dysplasia, showing the efficacy of this method in highlighting dysplasia in AML. Except for MLD, IPS did not influence any patient- or disease-related characteristics at diagnosis. Furthermore, IPS did not influence complete remission rate, disease-free survival, or overall survival. By investigating NPM1 status on separated cell compartments, we established a correlation between FC-assessed MLD and belonging to AML clone. This study shows that dysplasia evaluated by immunophenotype has no impact on clinical-biological characteristics or on outcome of NPM1⁺ AML. Dysplasia is part of the spectrum of NPM1⁺ AML, and the prognostic stratification of this category of patients should not be based upon it.


Subject(s)
Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/mortality , Mutation , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/mortality , Nuclear Proteins/metabolism , Adult , Aged , Disease-Free Survival , Female , Humans , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Nuclear Proteins/genetics , Survival Rate
13.
Int Med Case Rep J ; 6: 77-80, 2013.
Article in English | MEDLINE | ID: mdl-24204177

ABSTRACT

INTRODUCTION: Pleural effusion as the first clinical manifestation of acute lymphoblastic leukemia (ALL) is a relatively rare event. An early and accurate diagnosis of this clinical picture is very important for adequate patient management. CASE PRESENTATION: We report the atypical onset of T-lineage ALL in a 31-year-old man. The patient was admitted to the emergency room due to lung failure; at that moment, the patient's initial blood count was normal; the chest X-ray radiography showed a massive pleural effusion and a thoracentesis was carried out. Routine investigations performed on the pleural fluid using a new technology system and digitalized cell analysis demonstrated infiltration by immature cells. Therefore, bone marrow aspirate and flow cytometry analyses were performed, leading to the diagnosis of T-lineage ALL. A cord blood transplantation procedure was performed at the first hematological remission following chemotherapy regimens. The patient died of septic shock. CONCLUSION: The case we reported underlines the usefulness of using automated instruments to identify abnormal lymphoid cells in body fluids.

14.
Haematologica ; 99(2): 262-6, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24038027

ABSTRACT

Complement blockade by eculizumab is clinically effective in hemolytic paroxysmal nocturnal hemoglobinuria. However, the response is variable and some patients remain dependent on red blood cell transfusions. In 72 patients with hemolytic paroxysmal nocturnal hemoglobinuria on eculizumab we tested the hypothesis that response may depend on genetic polymorphisms of complement-related genes. We found no correlation between the complement component C3 genotypes and the need for blood transfusions. On the other hand, we found a significant correlation with the HindIII polymorphism of a complement regulatory gene, the complement receptor 1 (CR1) gene. At this locus two co-dominant alleles are known, of which H (common) is associated with high expression, whereas L (rare) is associated with low expression of CR1 on red blood cells. Patients who still needed blood transfusion on eculizumab accounted for 18% of the H/H homozygotes, 33% of the H/L heterozygotes and 68% of the L/L homozygotes (P=0.016). Thus, patients with paroxysmal nocturnal hemoglobinuria who have the L/L genotype are seven times more likely to be sub-optimal responders to eculizumab. Both in vitro and in vivo we found that the CR1 HindIII genotype correlates with the abundance of paroxysmal nocturnal hemoglobinuria red cells that have bound C3, and with the kinetics of C3 binding. These results are consistent with the notion that by affecting C3 binding the CR1 genotype influences the response to eculizumab treatment, and this emerges as a novel example of pharmacogenetics.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Blood Transfusion , Genotype , Hemoglobinuria, Paroxysmal , Polymorphism, Genetic , Receptors, Complement 3b , Complement C3/genetics , Complement C3/metabolism , Complement C4/genetics , Complement C4/metabolism , Female , Follow-Up Studies , Hemoglobinuria, Paroxysmal/blood , Hemoglobinuria, Paroxysmal/genetics , Hemoglobinuria, Paroxysmal/therapy , Humans , Male , Receptors, Complement 3b/genetics , Receptors, Complement 3b/metabolism
15.
PLoS One ; 8(1): e54046, 2013.
Article in English | MEDLINE | ID: mdl-23342069

ABSTRACT

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10⁻6 to 37.5×10⁻6, with a median of 4.9×10⁻6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.


Subject(s)
Granulocytes/metabolism , Adult , Female , Humans , Male , Membrane Proteins/genetics , Middle Aged , Mutation , Point Mutation/genetics , Young Adult
16.
Cytometry B Clin Cytom ; 84(2): 71-81, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23281097

ABSTRACT

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is a unique disorder caused by a PIG-A gene mutation in a stem cell clone. Its clinical picture can sometimes make challenging the distinction from other disorders, and especially from myelodysplastic syndromes (MDS), since both diseases correlate with cytopenias and morphological abnormalities of bone marrow (BM) cells. Recently, flow cytometry (FC) has been proposed to integrate the morphologic assessment of BM dysplasia, and thus to improve the diagnostics of MDS. METHODS: In the present study, we have analyzed systematically FC data resulting from the study of BM cells from patients with PNH and MDS. RESULTS: Our data demonstrated abnormalities in PNH beyond the deficiency of glycosylphosphatidylinositol-linked proteins and the application of a systematic approach allowed us to separate effectively MDS and PNH in a cluster analysis and to highlight disease-specific abnormalities. Indeed, the parallel evaluation of some key parameters, i.e. patterns of expression of CD45 and CD10, provided information with practical diagnostic usefulness in the distinction between PNH and MDS. Moreover, the hypo-expression of CD36 that we observed on monocytes might be related to the thrombotic tendency in PNH. CONCLUSIONS: We investigated systematically the phenotypic profile of BM cells from patients with PNH; our data provide useful antigenic patterns to solve between PNH and MDS, sometimes morphologically overlapping. Moreover, some PNH-related phenotypic changes might be involved in the physiopathology of the disease and further studies addressing this issue are warranted.


Subject(s)
Bone Marrow Cells/cytology , Flow Cytometry , Hemoglobinuria, Paroxysmal/diagnosis , Myelodysplastic Syndromes/diagnosis , Adult , Aged , Aged, 80 and over , CD36 Antigens/biosynthesis , CD55 Antigens/genetics , CD55 Antigens/immunology , CD59 Antigens/genetics , CD59 Antigens/immunology , Cluster Analysis , Diagnosis, Differential , Female , GPI-Linked Proteins , Glycosylphosphatidylinositols/deficiency , Hemoglobinuria, Paroxysmal/genetics , Humans , Karyotype , Leukocyte Common Antigens/biosynthesis , Male , Membrane Proteins/genetics , Middle Aged , Neprilysin/biosynthesis , Phenotype , Seizures , Young Adult
17.
Blood ; 121(2): 360-8, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23129323

ABSTRACT

Increased microvessel density contributes to abnormal BM and spleen microenvironment in myelofibrosis (MF). Taking advantage of the JAK2V617F mutation as a marker of malignancy, in the present study, we investigated whether splenic endothelial cells (ECs) obtained from capillaries by laser microdissection or from fresh spleen tissue by cell culture or cell sorting harbored such mutation in patients bearing the mutation in their granulocytes and undergoing splenectomy for therapeutical reasons. To extend the analysis to the ECs of large vessels, endothelial tissue from the splenic vein was also studied. We found JAK2V617F(+) ECs in 12 of 18 patients also bearing the mutation in their granulocytes. In 3 patients, the mutation was found in at least 2 different EC samples obtained by laser microdissection, cell culture, or cell sorting. The mutation was detected in the splenic vein ECs of 1 of 6 patients investigated. In conclusion, we provide evidence that some ECs from the spleen and splenic veins of patients with MF bear the JAK2V617F mutation. We suggest that splenic ECs are involved in the process of malignant transformation in MF.


Subject(s)
Endothelial Cells/pathology , Janus Kinase 2/genetics , Primary Myelofibrosis/genetics , Spleen/pathology , Aged , Cell Separation , Comparative Genomic Hybridization , Female , Flow Cytometry , Humans , In Situ Hybridization, Fluorescence , Laser Capture Microdissection , Male , Middle Aged , Mutation , Reverse Transcriptase Polymerase Chain Reaction
18.
Cancer Cell ; 22(2): 250-62, 2012 Aug 14.
Article in English | MEDLINE | ID: mdl-22897854

ABSTRACT

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) regulate normal development and homeostasis and drive disease progression in many forms of cancer. Both proteins signal by binding to receptor tyrosine kinases and heparan sulfate (HS) proteoglycans on target cell surfaces. Basic residues comprising the primary HS binding sites on HGF and VEGF provide similar surface charge distributions without underlying structural similarity. Combining three acidic amino acid substitutions in these sites in the HGF isoform NK1 or the VEGF isoform VEGF165 transformed each into potent, selective competitive antagonists of their respective normal and oncogenic signaling pathways. Our findings illustrate the importance of HS in growth factor driven cancer progression and reveal an efficient strategy for therapeutic antagonist development.


Subject(s)
Gene Targeting , Heparitin Sulfate/metabolism , Hepatocyte Growth Factor/metabolism , Neoplasms/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism , Animals , Antigens, CD34/metabolism , Cell Proliferation/drug effects , Cluster Analysis , Dogs , Enzyme Activation/drug effects , Hepatocyte Growth Factor/antagonists & inhibitors , Hepatocyte Growth Factor/chemistry , Humans , Magnetic Resonance Spectroscopy , Mice , Neoplasm Metastasis , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Protein Binding/drug effects , Protein Folding/drug effects , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Multimerization/drug effects , Proto-Oncogene Proteins c-met/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/pharmacology
19.
Int J Biol Markers ; 27(3): e195-202, 2012 Oct 08.
Article in English | MEDLINE | ID: mdl-22865297

ABSTRACT

Several immunological fecal occult blood tests (FOBT) are currently available for colorectal cancer (CRC) screening. We compared the HM Jack (Jack) (Kiowa, Japan), with the OC-Hemodia (OC) (Eiken, Japan) in use in the Florence screening program. Aims of the study were: (i) to investigate the diagnostic performance and the best cutoff value for Jack; (ii) to evaluate the handiness of sampling tubes; (iii) to compare costs. A total of 5,044 subjects were screened with both tests. Sampling tube investigation was performed running each sample on both instruments. A number of 352 subjects positive for at least one test (175 OC, 310 Jack) were selected for further investigations, while 46 subjects refused further assessments. Analysis of costs related to the assessment phase was performed on the basis of Tuscany region's fares. Amongst the 306 subjects investigated, 9 CRC and 67 advanced adenomas (AdA) were detected. Detection rates (DR) were 1.4‰ for CRC and 9.6‰ for AdA. After Jack cutoff optimization, DR for CRC+AdA resulted in 11.1‰ for OC and 13.3‰ for Jack (p=0.041). Sensitivity of the methods was 73.7 for OC and 88.2 for Jack; specificity was 97.6 for OC and 96.0 for Jack, resulting in an increase of the required assessments from 3.5% to 5.1%. No differences were observed between sampling methods. Despite the lower specificity of Jack, its greater sensitivity makes the method attractive for screening programs. An increase of the costs of 30% for every subject investigated for pathological lesion (CRC+AdA) may be thus foreseen.


Subject(s)
Biomarkers, Tumor/blood , Colorectal Neoplasms/blood , Latex/chemistry , Occult Blood , Colonoscopy , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/prevention & control , Early Detection of Cancer/methods , Feces/chemistry , Female , Humans , Italy , Male , Middle Aged
20.
Mol Cell Probes ; 24(6): 401-2, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20655380

ABSTRACT

Complement C3 'slow' and 'fast' allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 'slow' and 'fast' alleles by a single tube reaction.


Subject(s)
Alleles , Complement C3/genetics , DNA Primers/metabolism , Mutation/genetics , Polymerase Chain Reaction/methods , Cell Line , Genotype , Humans , Polymorphism, Single Nucleotide/genetics
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