24 results on '"Bianca Serio"'
Search Results
2. SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency
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Albino Carrizzo, Concetta Iside, Angela Nebbioso, Vincenzo Carafa, Antonio Damato, Sebastiano Sciarretta, Giacomo Frati, Flavio Di Nonno, Valentina Valenti, Michele Ciccarelli, Eleonora Venturini, Mariarosaria Scioli, Paola Di Pietro, Tommaso Bucci, Valentina Giudice, Marianna Storto, Bianca Serio, Annibale Alessandro Puca, Giuseppe Giugliano, Valentina Trimarco, Raffaele Izzo, Bruno Trimarco, Carmine Selleri, Lucia Altucci, Carmine Vecchione, Carrizzo, Albino, Iside, Concetta, Nebbioso, Angela, Carafa, Vincenzo, Damato, Antonio, Sciarretta, Sebastiano, Frati, Giacomo, Di Nonno, Flavio, Valenti, Valentina, Ciccarelli, Michele, Venturini, Eleonora, Scioli, Mariarosaria, Di Pietro, Paola, Bucci, Tommaso, Giudice, Valentina, Storto, Marianna, Serio, Bianca, Puca, Annibale Alessandro, Giugliano, Giuseppe, Trimarco, Valentina, Izzo, Raffaele, Trimarco, Bruno, Selleri, Carmine, Altucci, Lucia, and Vecchione, Carmine
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Pharmacology ,Endothelium ,MTHFR ,Nitric oxide ,SIRT1 ,Vascular function ,Animals ,Genotype ,Homocystinuria ,Humans ,Methylenetetrahydrofolate Reductase (NADPH2) ,Mice ,Muscle Spasticity ,Psychotic Disorders ,Resveratrol ,Sirtuin 1 ,Thrombosis ,ndothelium ,Cell Biology ,Article ,Cellular and Molecular Neuroscience ,Molecular Medicine ,Molecular Biology - Abstract
Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/–) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/– mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.
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- 2022
3. Mesenchymal Stem Cells from the Wharton’s Jelly of the Human Umbilical Cord: Biological Properties and Therapeutic Potential
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Carmine Selleri, Maria Antonietta Castaldi, Rosa Vitolo, Bianca Serio, R Rosamilio, Enrico Ragni, Maurizio Guida, Caterina Fulgione, Salvatore Giovanni Castaldi, Luigi Marino, and Rosario Bianco
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Human leukocyte antigen ,Review Article ,Umbilical cord ,Regenerative medicine ,Immunomodulation ,03 medical and health sciences ,0302 clinical medicine ,Wharton's jelly ,medicine ,Human umbilical cord blood ,030304 developmental biology ,GvHD ,0303 health sciences ,business.industry ,Mesenchymal stem cell ,Cell Biology ,Embryonic stem cell ,Haematopoiesis ,medicine.anatomical_structure ,WJ-MSCs ,Immunology ,Mesenchymal stem cells ,Stem cell ,business ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Wharton's jelly mesenchymal stem cells (WJ-MSCs) are a class of stem cells with high differentiative potential, an immuno-privileged status and easy access for collection, which raise no legal or ethical issues. WJ-MSCs exhibit several features of embryonic stem cells, both in the phenotypic and genetic aspects, with only a few differences, such as a shorter doubling time and a more extensive ex vivo expansion capacity. WJ-MSCs have immunomodulatory properties, involving both innate and adaptive immune responses. This review focuses on the role of WJ-MSCs in the management of graft-versus-host disease (GvHD), a life-threatening complication of the allogenic transplantation of hematopoietic stem cells. Different studies documented the beneficial effect of the infusion of WJ-MSCs, even when not fully HLA identical, in patients with severe GvHD, refractory to standard treatment. Finally, we summarized current ongoing clinical trials with WJ-MSCs and their potential in regenerative medicine.
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- 2019
4. Low-Density Granulocytes Are Decreased in Acute Myeloid Leukemia and in Myelodysplastic Syndromes with Negative Prognostic Factors
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Valentina Giudice, Rossella Marcucci, Maddalena Langella, Rita Pepe, Carmine Selleri, Maria Teresa Buonanno, Rosa Vitolo, Matteo D'Addona, Maria Carmen Martorelli, Bianca Serio, Marisa Gorrese, Idalucia Ferrara, Angela Bertolini, and Paola Manzo
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business.industry ,Myelodysplastic syndromes ,Immunology ,medicine ,Cancer research ,Low density ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,business ,Biochemistry - Abstract
Introduction. Myelodysplastic syndromes (MDS), a group of clonal hematological diseases, are characterized by ineffective hematopoiesis, progressive peripheral blood (PB) cytopenia(s), and increased risk of developing acute myeloid leukemia (AML). Classification and risk stratification are constantly under revision for a better estimation of prognosis in those patients. Investigation of immune biomarkers is needed, because immune dysregulation also plays an important role in dysplastic hemopoiesis and immunological escape of neoplastic clones. Here, we studied frequency of low-density granulocytes (LDGs), a neutrophil subset with immunoregulatory functions, in MDS and AML at diagnosis and during treatments. Methods. A total of 17 patients (M/F, 14/12; median age, 69 years old; range, 21-84 years) and seven healthy subjects were enrolled at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy, between October 2020 and July 2021. Patients were diagnosed with AML (N = 7), or MDS (N = 10) according to the 2016 World Health Organization criteria. For immunophenotyping, fresh EDTA whole PB was stained with the ollowing antibodies: CD45; HLA-DR; CD15; CD3; CD56; CD19; CD11b; CD33; CD34; CD14; and CD16 (all from Beckman Coulter, Brea, CA). Acquisition was carried out using a Navios EX flow cytometer, and Navios software v1.3 (Beckman Coulter). Post-acquisition compensation and analysis were performed using FlowJo software (v.10.7.1, Becton Dickinson). LDGs were identified as CD3-CD56-CD19-CD11b+CD33+CD14-CD15+ cells, following previously published gating strategies (Rahman S, et al. Ann Rheum Dis. 2019). Data were analyzed using Prism (GraphPad software, La Jolla, CA). A P < 0.05 was considered statistically significant. Results. Frequencies of circulating LDGs were significantly reduced in AML patients at diagnosis compared to controls (P = 0.0018) and MDS (P = 0.0077) and were slightly decreased compared to AML in complete remission (P = 0.1605). MDS patients were then divided based on Revised International Prognostic Scoring System (IPSS-R), and very-low and low-risk MDS patients displayed significantly higher circulating LDG frequencies compared to AML at diagnosis (P = 0.0083), while no differences were described between AML at baseline and intermediate-risk MDS (P = 0.1103). Subsequently, LDGs were correlated with clinical and phenotypic features by correlation analysis showing significant negative correlations between LDGs and blasts identified by flow cytometry (r = -0.5463; P = 0.0057) but not by cytology (P = 0.1346), between LDGs and lymphocytes (r = -0.4407; P = 0.0311) or flow cytometric normalized blast count (NBC; r = -0.5283; P = 0.0096) as previously defined (Giudice V, et al. Biomedicines. 2021). A slight negative correlation was described between LDGs and WT1 expression levels (r = -0.5369; P = 0.0719), particularly evident in MDS patients (r = -0.9980; P = 0.0402), supporting our previous findings of negative prognostic impact of WT1 expression in MDS and AML. Finally, we investigated CD16 expression on LDGs, because CD16 is essential for neutrophil degranulation. Despite no differences were described between percentage of LDG subsets among patients' groups, various correlations were identified by Pearson analysis. In particular, CD16+ LDGs negatively correlated with blasts (P = 0.0229), while positively correlated with lymphocytes (P = 0.0404) detected by flow cytometry. Conversely, CD16int and CD16- LDGs negatively correlated with lymphocytes (P = 0.0109 and P = 0.0021, respectively) and positively correlated with granulocytes identified by flow cytometry (P = 0.0024 and P = 0.0008, respectively). In addition, CD16int LDGs negatively correlated with blasts detected by flow cytometry (r = -0.65; P = 0.0414). Conclusions. Our preliminary results suggested a possible role of LDGs in prognostic definition of AML and MDS patients especially when combined with other biomarkers, such as WT1 expression levels or NBC. Moreover, our data supported the hypothesis of biological heterogeneity of granulocytes, as LDG subsets variously correlated with lymphocytes and leukemic cells suggesting different roles in suppression or activation of immune responses. However, our findings need further validation in larger cohorts and in in vitro studies. Disclosures No relevant conflicts of interest to declare.
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- 2021
5. Hemopoiesis and Immune Cell Perturbations during Venetoclax Plus Azacytidine Treatment in Acute Myeloid Leukemia
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Serena Luponio, Angela Bertolini, Francesca D'Alto, Bianca Cuffa, Bianca Serio, Roberto Guariglia, Carmine Selleri, Laura Mettivier, Idalucia Ferrara, L Pezzullo, Marisa Gorrese, Valentina Giudice, Matteo D'Addona, and Danilo De Novellis
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Venetoclax ,business.industry ,Immunology ,Cell ,Myeloid leukemia ,Cell Biology ,Hematology ,Biochemistry ,chemistry.chemical_compound ,Haematopoiesis ,medicine.anatomical_structure ,Immune system ,chemistry ,medicine ,Cancer research ,business - Abstract
Treatment of acute myeloid leukemia (AML) in elderly is still challenging. Indeed, high-dose chemotherapy followed by hematopoietic stem cell transplantation with myeloablative regimens is not always feasible because patients are often unfit and have several comorbidities; however, they frequently show multiple negative prognostic factors and have a worse overall survival compared to younger adults. Venetoclax, the first-in-class Bcl-2 antagonist and first approved for treatment of chronic lymphocytic leukemia, inhibits the anti-apoptotic functions of Bcl-2 inducing apoptosis and tumor growth arrest. Venetoclax is also used in combination with azacytidine, or decitabine, or low-dose cytarabine for treatment of elderly newly diagnosed AML. However, several mechanisms of resistance have already been described, such as increased expression of other anti-apoptotic proteins by the leukemic clone. In this case series, we investigated hematopoiesis and immune cell perturbations during venetoclax plus azacytidine treatment in elderly AML patients. A total of six AML patients (M/F, 2/4; median age, 71 years old; range, 63-79 years) were retrospectively evaluated at the Hematology and Transplant Center, University Hospital "San Giovanni di Dio e Ruggi d'Aragona", Salerno, Italy. Patients received a diagnosis of AML based on the 2016 World Health Organization (WHO) criteria and chemotherapy with azacytidine 75 mg/m 2 daily for 7 days per cycle and venetoclax 70 mg/daily. Two patients were NPM1 mutated (one of them also had mutated IDH1, VAF 27.2%), while all subjects had FLT3 wild type. Based on the 2017 European LeukemiaNet risk classification, two patients had favorable risk and four intermediate. Median follow-up was 10.1 months (range, 4.9-16.6 months), and all patients were in partial or complete remission at the time of writing. Flow cytometry immunophenotype, complete blood counts (CBCs), and WT1 expression levels were performed at diagnosis and after every cycle of therapy as per our institutional guidelines. In our case series, leukemic cells were already decreased after the first cycle of therapy (blasts by flow cytometry + SD, 54.7+39.9% vs 4.2+5.4%, diagnosis vs post I cycle; P = 0.0671; paired t-test performed), while normal granulocytes detected by flow cytometry recovered only after the third cycle of therapy (20.7+23.7% vs 53+6.6%, diagnosis vs post III cycle; P = 0.1396; uncorrected Fisher's mixed model performed). Treated patients also displayed a contextual decreased in WT1 expression levels (normalized WT1 copy number + SD, 1810+2723 copies vs 201+132.9 copies, diagnosis vs post I cycle; P = 0.2660; paired t-test performed). Platelet count tended to increase after the first cycle (P = 0.0680); however, at the end of the second cycle, half of patients were again thrombocytopenic (platelets < 100 x 10 3/µL). Interestingly, percentage of lymphocytes detected by flow cytometry were significantly increased after the second cycle of azacytidine plus venetoclax compared to baseline and after the first cycle of therapy (mean+SD, 13.5+13.3% vs 48+8.7%, diagnosis vs post II cycle; P = 0.0167; and vs 28+11.3%, vs post III cycle; P = 0.0480), likely because an increase in Natural Killer (NK) cell frequency peaking after the second cycle (mean+SD, 19.4+4.4% vs 32.5+15.1%, diagnosis vs post II cycle; P = 0.1383). Moreover, five out of six patients displayed expansion of plasma cells detected by flow cytometry in the bone marrow after the first cycle: in particular, one case patient had expansion of aberrant CD45-/dimCD38++CD138++CD56+CD19- plasma cells, while one subject showed only a transient appearance of clonal plasma cells after the second cycle. No differences in bone marrow monocyte frequencies were described during treatment. Our preliminary results added evidence to efficacy and safety of the combination of venetoclax and azacytidine in treatment of elderly AML in a real-world setting. These drugs might synergistically function on hematopoiesis by inducing apoptosis of neoplastic cells while favoring differentiation of other lineages, as suggested by the expansion of plasma cells, or triggering NK-mediated immunosurveillance. However, prognostic and clinical significance of plasma cell and NK cell expansion in the setting of AML treatment needs to be further explored in larger prospective cohorts. Disclosures No relevant conflicts of interest to declare.
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- 2021
6. Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization
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Pia Ragno, Patrizia Ricci, Nunzia Montuori, Guido Rossi, Carmine Selleri, Francesco Blasi, Bianca Serio, Maria Vincenza Carriero, Bruno Rotoli, Valeria Visconte, Nicolai Sidenius, C., Selleri, Montuori, Nunzia, P., Ricci, V., Visconte, M. V., Carriero, N., Sideniu, Serio, Bianca, F., Blasi, Rotoli, Bruno, Rossi, Guido, and P., Ragno
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medicine.medical_specialty ,Sialic Acid Binding Ig-like Lectin 3 ,Immunology ,Lipopolysaccharide Receptors ,Antigens, Differentiation, Myelomonocytic ,Receptors, Cell Surface ,Biochemistry ,CXCR4 ,Receptors, Urokinase Plasminogen Activator ,Antigens, CD ,Cell Movement ,Internal medicine ,Granulocyte Colony-Stimulating Factor ,medicine ,Humans ,Progenitor cell ,Hematopoietic Stem Cell Mobilization ,Chemistry ,uPAR ,hematopoietic stem cell ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Receptors, Formyl Peptide ,Tissue Donors ,Up-Regulation ,Urokinase receptor ,Haematopoiesis ,Endocrinology ,medicine.anatomical_structure ,SuPAR ,Cancer research ,Stem cell - Abstract
We investigated the involvement of the urokinase-type plasminogen-activator receptor (uPAR) in granulocyte–colony-stimulating factor (G-CSF)–induced mobilization of CD34+ hematopoietic stem cells (HSCs) from 16 healthy donors. Analysis of peripheral blood mononuclear cells (PBMNCs) showed an increased uPAR expression after G-CSF treatment in CD33+ myeloid and CD14+ monocytic cells, whereas mobilized CD34+ HSCs remained uPAR negative. G-CSF treatment also induced an increase in serum levels of soluble uPAR (suPAR). Cleaved forms of suPAR (c-suPAR) were released in vitro by PBMNCs and were also detected in the serum of G-CSF–treated donors. c-suPAR was able to chemoattract CD34+ KG1 leukemia cells and CD34+ HSCs, as documented by their in vitro migratory response to a chemotactic suPAR-derived peptide (uPAR84-95). uPAR84-95 induced CD34+ KG1 and CD34+ HSC migration by activating the high-affinity fMet-Leu-Phe (fMLP) receptor (FPR). In addition, uPAR84-95 inhibited CD34+ KG1 and CD34+ HSC in vitro migration toward the stromal-derived factor 1 (SDF1), thus suggesting the heterologous desensitization of its receptor, CXCR4. Finally, uPAR84-95 treatment significantly increased the output of clonogenic progenitors from long-term cultures of CD34+ HSCs. Our findings demonstrate that G-CSF–induced upregulation of uPAR on circulating CD33+ and CD14+ cells is associated with increased uPAR shedding, which leads to the appearance of serum c-suPAR. c-suPAR could contribute to the mobilization of HSCs by promoting their FPR-mediated migration and by inducing CXCR4 desensitization.
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- 2005
7. The Role of B Regulatory Cells in the Immunological Escape of Tumor Cells in Hodgkin Lymphoma
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M Rocco, G Villani, Valentina Giudice, Carmine Selleri, R Rosamilio, L Pezzullo, Bianca Serio, and Idalucia Ferrara
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biology ,business.industry ,T cell ,Dacarbazine ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,CD19 ,Vinblastine ,Interleukin 10 ,medicine.anatomical_structure ,biology.protein ,Medicine ,Cytotoxic T cell ,IL-2 receptor ,business ,CD8 ,medicine.drug - Abstract
Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin Lymphoma (HL) are surrounded by a rich inflammatory infiltrate which aids in their survival and escape from cytotoxic CD8+ T cells (CTLs) and Natural Killer cells (NKs). Within HL environment, T regulatory cells (Tregs) directly suppress the activity of CTLs and NKs, enhancing the tolerance against HRS cells. B regulatory cells (Bregs) have been shown to support the differentiation of Tregs through IL-10 production; thus, we hypothesized that they could have a role in the pathophysiology of HL. We evaluated 30 classic HL patients (M/F: 18/12; median age, 31 years, range 15-62) and 5 healthy controls (HC) for circulating peripheral blood (PB) Bregs, Tregs, CTLs, NKs, and NKTs. Twenty-four of them were new-diagnosed patients (NwHL) and 6 received a previous diagnosis of HL but were in complete remission (CR) for more than 12 months (PvHL). NwHL patients were divided according to the International Prognostic Score (IPS) and the Ann-Arbor Staging System. All subjects were treated following the ABVD protocol (doxorubicin 25 mg/m2, bleomycin 10 U/m2, vinblastine 6 mg/m2, and dacarbazine 375 mg/m2). Flow cytometry was performed on heparinized PB samples with a 5-color Beckman Coulter Cytomics FC500 flow cytometer. Breg (CD19+CD24+), Treg (CD3+CD4+CD25+), CTL (CD3+CD8+), NK (CD3-CD56+), and NKT (CD3+CD56+) levels were measured simultaneously with the PET/CT evaluations, ie at diagnosis, at the end of the second ABVD administration, at the end of treatment, and at 6 and/or 12 months off-therapy. Moreover, Breg levels were compared to IPS and Ann-Arbor staging groups, and also were correlated to the erythrocyte sedimentation rate (ESR) and to the absolute lymphocyte count (ALC). We found decreased circulating Bregs in NwHL and PvHL patients compared to controls (0.39% vs 0.875% vs 1.813%, respectively, p Our preliminary data suggest involvement of Bregs in the escape and survival of HRS cells during active disease. Peripheral blood may mirror disease activity in lymphoid tissues. Thus, the decrease of circulating Bregs may be related to the recruitment of these cells to the tumor site; amplification of the Bregs/Tregs ratio may result in a greater Breg-dependent Treg activation with subsequent inhibition of CTL and NK function. Additionally, the normalization of Bregs and the Bregs/Tregs ratio after chemotherapy could be used to predict disease remission. While larger prospective studies are required to validate these results, we present intriguing evidence of the involvement of Bregs in the pathophysiology of HL. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
8. How to Improve the Definition of Chronic Lymphocytic Leukemia Outcome Using a Simple Flow Cytometric Score Based on CD49d and Homing Marker Expression
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Bianca Serio, G Villani, Luigi Marino, Valentina Giudice, S Annunziata, Carmine Selleri, Idalucia Ferrara, R Rosamilio, L Pezzullo, R Fontana, and M Rocco
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Oncology ,medicine.medical_specialty ,business.industry ,Chronic lymphocytic leukemia ,Immunology ,Cell Biology ,Hematology ,CD49d ,medicine.disease ,Biochemistry ,Chemotherapy regimen ,Immunophenotyping ,immune system diseases ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Platelet Count measurement ,Hemoglobin measurement ,business ,Homing (hematopoietic) - Abstract
The identification of new molecular markers in Chronic Lymphocytic Leukemia (CLL) allowed to better define prognosis and clinical outcome. The actual staging systems could estimate the prognosis, but not the rapidity of disease evolution. Neither the identification of new molecular markers did allow to foresee the evolution and clinical response, because discordant findings were mostly reported. The aim of the present study was (1) to confirm the independent prognostic role of CD49d as a single marker in CLL patients, (2) to investigate the relationship between CD49d and other well-established CLL-membrane predictor markers (CD5, CD11c, CD20 and CD38) or clinical staging systems and (3) to evaluate the role of an immunophenotypic score based on the flow-cytometric detection of CD5, CD11c, CD20, CD38 and CD49d in the work up of CLL staging. Heparinized whole blood was collected from 68 CLL patients for immunophenotyping using the following antibodies: anti kappa, anti-lambda, CD5, CD11c, CD19, CD20, CD23, CD38, CD45, CD49d. A scoring system was elaborated combining 5 membrane markers: CD5, CD11c, CD20, CD38 and CD49d. Antigens were divided in two groups, favorable (CD5 and CD20) and unfavorable (CD11c, CD38 and CD49d) prognostic markers, and the cut-off of positivity was chosen according to the literature (30% for CD5, CD11c, CD20 and CD38, and 45% for CD49d). A value of "0" or "1" ("2" only for CD49d positivity) was assigned according to antigen expression. Finally, we defined a favorable phenotype when the sum of all the cytometric features was equal or less than 2, conversely the unfavorable phenotype was defined for a sum equal or greater than 3 (between 3 and 6). Flow cytometric analysis showed high CD49d expression in CD19+ cells in 47% of patients (n=32), and high CD38 expression in 44% of subjects (n=30), simultaneously expressed in 28% of patients (n=19). The 19% (n=13) of all CLL patients were CD5-, and interestingly the 85% of them showed higher expression of CD49d. Linear correlation was found between CD49d and CD38 (r2=0.08772, p=0.0142), and between CD49d and CD20 expression (r2=0.2490, p45% of CD49d positive cells. Four patients with Unfavorable Phenotype received chemotherapy with an ORR of 25%. Furthermore, a small population (n=16) of our CLL cohort was also studied for genetic abnormalities using FISH technique. According to FISH analysis, 25% of studied patients were classifies as very low-risk and, interestingly, no one of them showed an Unfavorable Phenotype (only one patient carried CD49d as unique negative marker). In our cohort, 50% of patients were low-risk with no genetic abnormalities or +12, but 63% of them showed an Unfavorable Phenotype with high CD49d and CD38 expression in 100% and 60% of cases, respectively. Our data confirm the independent negative prognostic role of CD49d and suggest a stronger prognostic power compared to CD38 in the definition of CLL outcome, because of its complex activity as homing marker, signaling receptor and anti-apoptotic molecule. Thus, the prognostic significance of CD49d may be enhanced when considered in comparison with other established markers, as CD11c and CD38. In conclusion, our results propose the use of the CD49d marker in combination with other B-cell membrane antigens as an additional tool for routine diagnosis and risk-stratification of CLL patients. Identification of high-risk phenotype with a simple scoring method could improve the treatment of these patients, who could take advantage of the most recent molecular targeting therapies. Disclosures No relevant conflicts of interest to declare.
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- 2016
9. Cytomegalovirus reactivation prophylaxis with low dose valgancyclovir after hematopoietic stem cell transplantation
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Ciro De Luca, R Fontana, Carmine Selleri, M Rocco, Valentina Giudice, S Annunziata, Mariarosaria Sessa, R Rosamilio, L Pezzullo, Bianca Serio, and Idalucia Ferrara
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medicine.medical_specialty ,Acute leukemia ,business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Asymptomatic ,Gastroenterology ,Surgery ,Cyclosporin a ,Internal medicine ,medicine ,Methotrexate ,medicine.symptom ,Complication ,business ,Viral load ,Multiple myeloma ,medicine.drug - Abstract
Cytomegalovirus (CMV) reactivation is one of the most frequent complication after hematopoietic stem cell transplantation (HSCT). Pre-transplant CMV-positive recipient serostatus is the most significant independent variable for viral reactivation. Oral valgancyclovir (VGCV) is a prodrug of intravenous gancyclovir (GCV) and is an effective and safety alternative for the management of CMV reactivation prophylaxis and preemptive therapy. However, VGCV at standard dose (900 mg twice a day) increases risk of myelosuppression in HSCT recipients. The efficacy of low dose (LD, 450 mg daily) oral VGCV was retrospectively evaluated in 30 allogeneic HLA-matched related patients and 2 unrelated, with a median age of 40 years (range, 18-59) and a median follow-up of 30 months (range, 3-56). Primary diseases were acute myeloid leukemia (AML, n=19), acute lymphoblastic leukemia (n=4), non-Hodgkin’s lymphoma (n=3), multiple myeloma (n=3) and myelodysplastic syndrome (n=3). Seventeen of twenty-three acute leukemia (AL) patients were transplanted in first complete remission (CR), while the remaining (n=6) were transplanted in 2nd CR. Five patients suffered from AML secondary to long-lasting MDS (n=3) or Hodgkin disease and breast cancer (n=2). Based upon CMV serostatus (D/R, donor/recipient), thirty (94%) of HSCT recipients were classified as high risk (D-/R+ = 3 and D+/R+ = 27) for CMV reactivation and only 6% as low risk (D-/R- = 2); none of the patients was in the intermediate risk group (D+/R-). Fifteen and 17 patients received a myeloablative and RIC regimens, respectively. Twenty-one patients received GvHD prophylaxis with cyclosporin A (CsA, 1 mg/kg intravenously from day -1 to +21, then 8 mg/kg orally for at least 6 months) and short-course methotrexate (MTX, 10 mg/kg on days +1, +3, +6 and +11). The others (n=11) received CsA with MTX and antithymocyte globulin (ATG, as a part of the conditioning regimen at 10 mg/kg at days -3, -2 and -1). According to the Glucksberg scoring system, thirteen patients experienced grade I-II and two grade III-IV acute GvHD, while 7 patients developed limited (n=6, 18%) and extensive (n=3, 10%) chronic GvHD. Starting from time of engraftment, LD oral VGCV was given prophylactically for at least 6 months. CMV infection was monitored weekly using polymerase chain reaction (PCR) in high risk seropositive recipients and we started preemptive therapy when the peak viral load exceeding 1000 copies/mL in two consecutive plasma samples. Six patients (4 early and 2 late) developed a positive PCR after a mean of 59 days post-HSCT successfully treated with 900 mg of VGCV twice a day for at least when PCR negative (in a median of 12 days). Only one patient developed late fatal gastrointestinal CMV disease. Indeed, asymptomatic early and late CMV-DNA PCR reactivation occurred only in 17% (n=5) of high risk seropositive HSCT recipients, in contrast to 37% and 18% of early and late CMV reactivation observed in matched gender, disease phase, graft source and CMV serostatus cohort of 32 HSCT recipients treated prophylactically with oral acyclovir (ACV, 15 mg/kg daily) and high dose intravenous immunoglobulins (IVIG, 0.4 gr/kg weekly for at least 6 months) . Seven patients presented hematological toxicity do not requiring drug discontinuation. The rate of non CMV-related infections was 25% and was similar in both groups with and without CMV reactivation. At the end of the follow-up, 18 of 32 (56%) patients were alive with a median follow-up of 31 months (range, 2-56). Relapsed-related mortality was 20%, transplant-related mortality was 9% and did not differ between group with and without CMV reactivation. Our data provide evidence that LD-VGCV is safe and effective as CMV reactivation prophylaxis in allogeneic HSCT recipients. These results require further validation in randomized studies. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
10. Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab
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Lucio Luzzatto, Patrizia Ricci, Giorgio Fratellanza, Anna Paola Iori, Angela Amendola, Antonio M. Risitano, Giacomo Gianfaldoni, Francesco Rodeghiero, Andrea Camera, Rosario Notaro, Filippo Barbano, Eros Di Bona, Fiorella Alfinito, Ludovica Marando, Bruno Rotoli, Carla Boschetti, Elisa Seneca, Bianca Serio, Carmine Selleri, Alberto Zanella, Danilo Ranaldi, Risitano, ANTONIO MARIA, Notaro, R, Marando, L, Serio, B, Ranaldi, D, Seneca, E, Ricci, P, Alfinito, Fiorella, Camera, A, Gianfaldoni, G, Amendola, A, Boschetti, C, Di Bona, E, Fratellanza, Giorgio, Barbano, F, Rodeghiero, F, Zanella, A, Iori, Ap, Selleri, Carmine, Luzzatto, L, and Rotoli, B.
- Subjects
Hemolytic anemia ,Male ,medicine.medical_specialty ,Erythrocytes ,Cell Survival ,Immunology ,Hemoglobinuria, Paroxysmal ,CD59 ,Antibodies, Monoclonal, Humanized ,Biochemistry ,Blood cell ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Humans ,Hematology ,biology ,business.industry ,Antibodies, Monoclonal ,hemic and immune systems ,Cell Biology ,Complement C3 ,Eculizumab ,medicine.disease ,Flow Cytometry ,Red blood cell ,medicine.anatomical_structure ,Paroxysmal nocturnal hemoglobinuria ,biology.protein ,Female ,Immunotherapy ,Antibody ,business ,circulatory and respiratory physiology ,medicine.drug - Abstract
In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59−). The proportion of C3+ RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom 51Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess 51Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.
- Published
- 2009
11. Role of the Urokinase Receptor (uPAR) in the Cross-Talk of Hematopoietic Stem Cells with the Bone Marrow Microenvironment
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Claudio La Penna, Pia Ragno, Nunzia Montuori, Antonio M. Risitano, Valeria Visconte, Patrizia Ricci, Guido Rossi, Carmine Selleri, Bruno Rotoli, Ada Pesapane, Bianca Serio, Montuori, Nunzia, Ricci, P, Serio, B, Visconte, V, La Penna, C, Pesapane, A, Risitano, ANTONIO MARIA, Rotoli, B, Rossi, G, Ragno, P, and Selleri, C.
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Stromal cell ,Immunology ,Hematopoietic stem cell ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Cell biology ,Urokinase receptor ,medicine.anatomical_structure ,SuPAR ,medicine ,Stem cell ,Progenitor cell ,Cell adhesion ,Homing (hematopoietic) - Abstract
The urokinase-type plasminogen activator receptor (uPAR) is a cell-surface receptor involved in cell adhesion and migration. uPAR binds urokinase (uPA) and vitronectin (VN) and interacts with integrins and chemotaxis receptors. Soluble forms of uPAR (suPAR) have been detected in human plasma and urine. A cleaved form of suPAR (c-suPAR), lacking the N-terminal domain and exposing the sequence SRSRY (aa 88–92), stimulates cell migration by activating fMLP receptors. We recently demonstrated uPAR involvement in G-CSF-induced CD34+ hematopoietic stem cell (HSC) mobilization. We also demonstrated that c-suPAR could induce mobilization of hematopoietic stem/progenitor cells in mice. Since HSC mobilization and homing to bone marrow (BM) are mirror image processes which utilize the same mediators and similar signaling pathways, we investigated whether uPAR and its ligands could play a role in regulating CD34+ HSC interactions with the BM stroma, thus also contributing to HSC homing and engraftment to the BM. We found expression of uPA and VN in cultures of human BM stroma cells. Interestingly, stroma cells also produced suPAR and high amounts of c-suPAR, exposing the chemotactic SRSRY sequence. The role of the different soluble forms of uPAR produced by stroma cells in regulating HSC interactions with the BM microenvironment was analyzed by long term cultures (LTC) of BM and G-CSF mobilized CD34+ HSCs, in the presence of suPAR or the uPAR-derived uPAR84–95 peptide, corresponding to the active site of c-suPAR. Both suPAR and the uPAR84–95 peptide increased the number of adherent and released clonogenic progenitors from LTC of BM and G-CSF mobilized HSCs. To elucidate the mechanism of suPAR and c-suPAR effects on CD34+ HSC interactions with the stromal microenvironment, in vitro adhesion and proliferation assays were performed on CD34+ KG1 cells. suPAR treatment determined a significant increase in CD34+ KG1 cell adhesion whereas c-suPAR increased cell proliferation. Taken together, our results indicate that BM stroma produces soluble forms of uPAR that regulate CD34+ HSC interactions with BM microenvironment, their local proliferation and trafficking from and to BM.
- Published
- 2008
12. Involvement of nitric oxide in farnesyltransferase inhibitor-mediated apoptosis in chronic myeloid leukemia cells
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Bianca Serio, Valeria Visconte, Bruno Rotoli, Jaroslaw P. Maciejewski, Nunzia Montuori, Luigiana Luciano, Patrizia Ricci, Carmine Selleri, Selleri, C., Maciejewski, J. P., Montuori, Nunzia, Ricci, P., Visconte, V., Serio, B., Luciano, L., Rotoli, Bruno, Selleri, Carmine, J. P., Maciejewski, P., Ricci, V., Visconte, B., Serio, L., Luciano, and B. R. o. t. o. l., I.
- Subjects
Farnesyltransferase ,Immunology ,Nitric Oxide Synthase Type II ,Antigens, CD34 ,Apoptosis ,Bone Marrow Cells ,Biology ,Protein Serine-Threonine Kinases ,Nitric Oxide ,Biochemistry ,Fas ligand ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,hemic and lymphatic diseases ,RhoB GTP-Binding Protein ,Cytotoxic T cell ,Farnesyltranstransferase ,Humans ,RNA, Messenger ,fas Receptor ,Enzyme Inhibitors ,rhoB GTP-Binding Protein ,Alkyl and Aryl Transferases ,Caspase 3 ,Farnesyltransferase inhibitor ,Myeloid leukemia ,Cell Biology ,Hematology ,Caspase Inhibitors ,Enzyme Activation ,Proto-Oncogene Proteins c-bcl-2 ,Caspases ,Cancer research ,biology.protein ,Neoplastic Stem Cells ,Nitric Oxide Synthase ,K562 Cells ,rhoA GTP-Binding Protein ,K562 cells - Abstract
The mechanism of action of farnesyltransferase inhibitors (FTIs) has not been fully clarified. We investigated the cytotoxic effects of various FTIs in chronic myeloid leukemia (CML), using LAMA cells and marrow cells from 40 CML patients in chronic phase. FTI-mediated cytotoxic effect was observed in LAMA cells and in 65% of primary CML cells, whereas marrow cells from controls were only weakly affected. Cytotoxic effects were partially related to enhanced apoptosis; however, Fas-receptor (FasR) and Fas-ligand (FasL) expression were not modified by FTIs. Susceptibility to FTI-mediated inhibition did not correlate with FasR/FasL expression in CD34+ CML cells. Moreover, intra-cellular activation of caspase-1 and -8 were not altered by FTIs, and their blockade did not reverse FTI toxicity. However, we observed FTI-induced activation of caspase-3, and its inhibition partially reverted FTI-induced apoptosis. FTIs did not modulate bcl2, bclxL, and bclxS expression, whereas they increased inducible nitric oxide (iNOS) mRNA and protein levels, resulting in higher NO production. Furthermore, C3 exoenzyme, a Rho inhibitor, significantly increased iNOS expression in CML cells, suggesting that FTIs may up-regulate NO formation at least partially through FTI-mediated inhibition of Rho. We conclude that FTIs induce selective apoptosis in CML cells via activation of iNOS and caspase-3.
- Published
- 2003
13. Induction Therapy With Continuous Alternate-Day Low Dose Lenalidomide Combined With Low-Dose Prednisone In Octogenarian Multiple Myeloma Patients
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Bianca Serio, M Rocco, Mariarosaria Sessa, G Villani, Idalucia Ferrara, Valentina Giudice, Carmine Selleri, R Fontana, and L Pezzullo
- Subjects
medicine.medical_specialty ,Aspirin ,Performance status ,business.industry ,Immunology ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Gastroenterology ,Discontinuation ,Surgery ,Zoledronic acid ,Prednisone ,Internal medicine ,Medicine ,business ,Adverse effect ,Multiple myeloma ,Lenalidomide ,medicine.drug - Abstract
About 30% of patients with newly diagnosed multiple myeloma (NDMM) are older than 75 years. Immunomodulatory drugs (IMIDs) have improved response rates and outcomes of NDMM, except for patients older than 75 years more vulnerable to side effects of IMIDs because of their frailty and comorbidities. We evaluated efficacy, toxicity and health-related quality of life (HRQOL) associated with continuous alternate-day low dose lenalidomide (LD-R, 10 mg on alternate days) and low dose prednisone (15 mg/day) (LD-RP) in 7 octogenarian NDMM patients (5 males and 2 females) with a median age of 82 years (range 80-87). All octogenarian patients had IgG MM, except 1 oligosecretory lambda chain MM; all were in Durie-Salmon stage III, except 1 in stage II, and had poor WHO performance status (median: 2, range 1-3). Patients were evaluated at baseline and every 6 months for HRQOL according to MM-specific questionnaire QLQ-MY20 of European Organisation for Research and Treatment of Cancer (EORTC). All patients received aspirin thromboprophylaxis, 57% of them requiring from diagnosis erythropoietin and zoledronic acid treatment. In these 7 octogenarian NDMM patients completing at least three months of therapy, the overall response rate (ORR) was 86%, including 1 complete remission (CR), 2 very good partial remission (VgPR) and 3 PR. After a median follow-up of 12 months (range 3-24), the quality of response improved with continuous LD-RP treatment with a cumulative median reduction in monoclonal protein levels of 85% (range 20-100%); none of the patients required discontinuation of treatment secondary to specific hematologic and/or extra-hematologic toxicity. In addition, QLQ MY-20 questionnaires revealed that 70% of patients treated with continuous LD-RP reported improvements of QOL scores. Two out of 7 octogenarian patients died (1 for progression after 12 months and 1 for sepsis no treatment-related), and 2-year overall survival and progression-free survival estimates were 41% and 75%, respectively. Noteworthy, all patients treated with continuous alternate-day LD-RP showed a progressive increase in the percentage of CD3+ CD56+ NK cells during the first 6 months of LD-RP therapy reaching a plateau maintained until +12 months after initiation of therapy: the median percentage of NK cells was 4% before LD-RP treatment versus 10%, 13%, 30%, 31%, and 27% at +1, +3, +6, +9 and +12 months, respectively. Mean fold increase of NK cells during LD-RP therapy was 1.5, 2.5, and 6.5 at +1, +3 and +6 months, respectively. Progressive increase of NK cells was concomitantly associated with reduction in tumor-linked monoclonal immunoglobulin in all patients and increased circulating NK cells further support that this drug may mediate its anti-MM effect, at least in part by modulating NK-cell number and function. Our data provide evidence that continuous alternate-day low dose lenalidomide is a manageable and effective frontline treatment for octogenarian NDMM patients and increases circulating NK cells. These preliminary results require further validation in prospective larger studies. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
14. Permissive Conditions for Evolution of PNH Clones Are Characterized by Overproduction of IFN-γ by Clonal CD4 and CD8 T Cells, Fas-L by CTLs, and Promoted by Immunogenetic Background
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Anna M. Jankowska, Jaroslaw P. Maciejewski, Heather Cazzolli, Ronald Paquette, Thomas P. Loughran, Susan B. Nyland, Alan F. List, Bianca Serio, Pearlie K. Epling-Burnette, Christine L. O'Keefe, Marcin W. Wlodarski, and Michael J. Clemente
- Subjects
education.field_of_study ,T cell ,Immunology ,Population ,Clone (cell biology) ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Immune system ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Paroxysmal nocturnal hemoglobinuria ,medicine ,HLA-DR ,Cytotoxic T cell ,education ,CD8 - Abstract
The association between immune-mediated aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) has been well documented, yet the related immune pathophysiology remains ill-defined. Response of AA patients with PNH clones to immunosuppressive agents such as anti-thymocyte globulin (ATG) and cyclosporine A provides clinical evidence for the involvement of the immune system in the evolution of PNH. The similar immunobiology of PNH and AA is also exemplified by the overrepresentation of HLA-DR*15 in AA, AA/PNH and PNH. To discern the relationship between T cell responses and effector mechanisms we applied a battery of immune tests to patients with these rare diseases. First, using T cell receptor Vβ flow cytometry in a cohort of patients with AA (N=42), AA/PNH (N=15), or PNH (N=18), we identified cytotoxic CD8 T cell (CTL) clonal expansions in 28/42 (66.7%), 9/15 (60%), and 7/18 (38.8%) patients, respectively. CD4 T cell expansions were present in 6/42 of AA (14.3%), 2/15 (13.3%) of AA/PNH, and 2/18 (11.1%) of PNH patients. We then studied whether expanded clones were associated with production of inflammatory cytokines; across the entire cohort, patients with clonal CD8 Vβ expansions demonstrated a significantly increased proportion of IFN-γ producing T cells as well as elevated levels of circulating Fas-L when compared to patients without clonal skewing (p=.032 CD4+IFN-γ+, p=.008 CD8+IFN-γ+, p=.097 sFAS-L). Even more pronounced was the increase in the proportion of IFN-γ producing CD4 T cells in patients with clonal CD4 Vβ expansions (p=.010). Furthermore, while a strong trend toward increased sFAS-L as detected by ELISA was found in patients with CD8 Vβ skewing vs. those without, patients with pronounced CD4 expansions did not produce elevated Fas-L levels, consistent with different effector mechanisms employed by CD4 vs. CD8 T cells. Based on these results we hypothesized that the presence of PNH clones will be associated with activation of immune effector mechanisms. Linear regression analysis of the size of the PNH clone vs. proportion of IFN-γ CTLs displayed a positive correlation that nearly reached statistical significance at α=0.05 (p=.067). A high proportion of CD4 IFN-γ cells (defined by a value above 95% mean confidence intervals of controls) was also associated with the presence of PNH (p=.048). Genetic analysis revealed further clues as to the increased propensity of patients with AA and PNH clones to produce elevated levels of IFN-γ; the hypersecretor genotype T/T for IFN-γ was over-represented in AA (28% vs. 10% in controls, p=.02) and correlated with presence of a PNH clone (35% vs. 14%, p=.01). An essential role of T cells in generating permissive conditions for the evolution of PNH clones is also supported by the immunogenetic relationship of PNH to HLA-DR*15, a relationship which was confirmed in our population of patients: phenotype frequency of HLA DR*15 was 42.8% AA, 40% AA/PNH, 27.8% PNH vs. 17.2% in control group. When HLA DR*15 positive and DR15 negative patients were compared, those with DR*15 displayed a strong trend toward increased proportion of CD8+IFN-γ producing cells (p=.094), previously shown to be elevated in patients with PNH clones. Our results reveal insights into the nature of permissive conditions involving oligoclonal T cell responses, oversecretion of proinflammatory cytokines, and immunogenetic background which together may promote the expansion of PNH clones. Conversely, it remains possible that the cytotoxic milieu may be a result of an immune response directed against intrinsically abnormal PNH clones.
- Published
- 2008
15. Subcutaneous Alemtuzumab Is a Safe and Effective Treatment for Global or Single-Lineage Immune-Mediated Marrow Failures: a Survey from the EBMT-WPSAA
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Giulia Scalia, Gérard Socié, Joerg Halter, Judith C. W. Marsh, Bruno Rotoli, Andrea Bacigalupo, Sébastien Maury, A. D. Kulagin, Antonio M. Risitano, Vikas Gupta, Elisa Seneca, Annapaola Iori, André Tichelli, Bianca Serio, Luigi Del Vecchio, Ludovica Marando, Hubert Schrezenmeier, Jakob Passweg, and Carmine Selleri
- Subjects
medicine.medical_specialty ,business.industry ,Standard treatment ,medicine.medical_treatment ,Immunology ,Bone marrow failure ,Valganciclovir ,Immunosuppression ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Gastroenterology ,Regimen ,Internal medicine ,medicine ,Alemtuzumab ,Aplastic anemia ,business ,medicine.drug - Abstract
Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in aplastic anemia (AA), or may selectively involve single lineages, as in pure red cell (PRCA) or in pure white cell aplasias (PWCA). Because of their common cellular immune-mediated pathophysiology, standard treatment for these conditions includes immunosuppression (IS), which may differ according to the specific disease. We investigated an experimental IS regimen based on the anti-CD52 antibody alemtuzumab (MabCampath®, ALE); the study included a phase II/III prospective trial, as well as a collection of retrospective cases. A total of 32 patients have been treated by ALE (18 SAA, 10 PRCA and 4 PWCA), fourteen of them (mostly PRCA) having not received previous IS. The most utilized schedule (as defined in the prospective trial) was 3,10,30,30,30 mg (total dose 103), administered subcutaneously in consecutive days, with adequate premedication; the last dose was amended in PRCA and PWCA patients (total dose 73 mg). In the prospective trial, all patients also received oral low dose cyclosporine A (1 mg/kg) from day 7, and an intensive anti-infectious prophylaxis, which included oral valgancyclovir and cotrimoxazol. All patients completed the treatment with unrelevant injection-related side effect (fever and/or rash in some cases) and absence of laboratory abnormalities. Complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; transient worsening of neutropenia and/or thrombocytopenia were observed in some cases. The median follow up was 12 months; there were 5 deaths, only one was possibly related to the treatment. In the prospective trial (n=23), infectious events were rare: a single FUO, associated with fatal complication of an underlying atrial fibrillation, other four viral infections (1 VZV with shingles, 2 HSV and 1 flu), all resolving quickly. No CMV or EBV disease was observed, even if 3 border-line CMV reactivations were documented (after discontinuation of the antiviral prophylaxis), promptly resolved by preemptive valganciclovir. One HBV reactivation without hepatitis required lamivudine. The response rate was globally 61% (42% CR and 19% PR), which raised to 73% (50% CR and 23% PR) when only patients with a follow up of at least 4 months were considered. In the more homogeneous cohort of the prospective trial, response rate was analyzed according to the underlying disease. Among 10 AA treated (5 as first line), 7 had an adequate follow up and showed 4 CR (57%) and 2 PR (29%). Response rate was even higher in 10 PRCA (8 as first line): 7 were evaluable for response, with 5 CR (71%) and 1 PR (14%); the 1 non responding patient subsequently showed evolution to MDS. Finally, 2 of 3 PWCA achieved a CR (66%), with the remaining showing early progression to MDS. Among the responding patients, relapses were quite frequent, even while on cyclosporine: 3/6 SAAs, 5/6 PRCAs and 1/2 PWCA. Relapses were successfully treated by additional ALE (as single shoots or complete courses). Immune reconstitution was delayed up to several months, especially affecting the CD4+ compartment; this was also due to additional ALE needed to treat or to prevent relapses. In conclusion, subcutaneous ALE is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Preliminary results suggest excellent efficacy, even if responses may be quite late (3–4 months); relapses often occur, but can be easily managed by ALE retreatment. ALE is an excellent alternative to standard IS regimen, and deserves systematic investigation in bone marrow failure patients.
- Published
- 2008
16. C3-Mediated Extravascular Hemolysis as Additional Mechanism of Disease in Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients Treated by the Complent Inhibitor Eculizumab
- Author
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Franco Iuliano, Antonio M. Risitano, Bianca Serio, Antonio Marino, Eros Di Bona, Francesco Pietrogrande, Carla Boschetti, Elisa Seneca, Silvana Bonfigli, Annapaola Iori, Stefano Pulini, Angelo Michele Carella, Elisabetta Antonioli, Angela Amendola, Filippo Barbano, Bruno Rotoli, Silvana Capalbo, Danilo Ranaldi, Giacomo Gianfaldoni, Filippo Milano, Wilma Barcellini, Rosario Notaro, Ludovica Marando, Lucio Luzzatto, Francesco Fabbiano, Alberto Zanella, and Francesco Rodeghiero
- Subjects
education.field_of_study ,medicine.medical_specialty ,medicine.diagnostic_test ,Anemia ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,Eculizumab ,medicine.disease ,Biochemistry ,Gastroenterology ,Hemolysis ,Coombs test ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Paroxysmal nocturnal hemoglobinuria ,Hemoglobin ,education ,business ,Complement membrane attack complex ,medicine.drug - Abstract
PNH is a hematological disorder characterized by complement-mediated intravascular hemolysis due to lack on RBCs of the complement regulators CD55 and CD59 and subsequent activation of the membrane attack complex (MAC). Eculizumab (EC) is an anti-complement fraction 5 antibody which abolishes intravascular hemolysis in PNH patients, leading to reduction of transfusion requirement and of anemia. Although the effector complement pathway is completely blocked by eculizumab in all patients, response to this agent in terms of transfusion requirement and hemoglobin level varies considerably. We have investigated the notion that patients with suboptimal hematological response may suffer from residual hemolysis mediated by mechanisms other than intravascular hemolysis via MAC. After the initial observation of a positive C3d Coombs test in some PNH patients on EC, we extensively studies C3 coating by flow cytometry in 56 PNH patients (41 of them while receiving EC). We found that in all cases on EC treatment a significant proportion of RBCs were coated with complement fraction 3 (C3); by converse, in 28 untreated PNH patients we found no evidence of C3 on red cells. C3 coating was strictly limited to CD59-neg RBCs, as CD59+/C3+ RBCs (as those seen in cold agglutinine disease patients, positive control) were never found. C3 coating was quite different among EC-treated patients, and correlated with the PNH RBC population. The percentage of C3+ cells within the PNH RBC population (the only subjected to C3 coating) was quite different in individual patients (0.5–61.3%, median 22.6%) and substantially preserved over time. We compared the level of C3 coating with the hematological response: all the 41 EC-treated patients showed marked LDH reduction with a substantial improvement of anemia, leading to transfusion independence in 34/41 patients (83%) and stable resolution of anemia in 14 (34%, defined optimal responders). The optimal responders showed a lower percentage of C3+ cells in comparison to suboptimal responders (20.9±19.0 vs 32.2±17.8; p=0.04). Indeed, patients with lower C3 coating (below the median value of 22.6%) showed a significantly higher rate of optimal response (51% in comparison to 15% of those with coating above the median, p=0.01). C3 coating also correlated with the absolute reticulocyte count (p=0.03), clearly suggesting that C3 coating was associated with ongoing residual extravascular hemolysis in vivo, and with pre-treatment LDH level (p=0.001). To confirm the presence of extravascular hemolysis, in 3 index patients with suboptimal response reduced RBC half-life was demonstrated in vivo by 51Cr RBC survival study, which showed reduced RBC half-life and excess uptake in liver and spleen. One of these patients underwent video-laparoscopic splenectomy, which led to transfusion independence and significant increase in Hb level. These data demonstrate that C3 coating of PNH RBCs is a common phenomenon in PNH patients on EC; in addition, we provide evidence that this leads to C3-mediated extravascular hemolysis through the reticulo-endothelial system. This novel mechanism of disease may account for residual hemolysis and suboptimal clinical benefit in some EC treated PNH, paving the way for additional therapeutic strategies to optimize the hematological response to this agent.
- Published
- 2008
17. Decreased Numbers of Tregs in Aplastic Anemia Is Detected by Immunohistochemistry and Flow Cytometry
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Jennifer Powers, Jaroslaw M.D. Maciejewski, Ziad Peerwani, Ramon V. Tiu, Erik Hsi, and Bianca Serio
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Pathology ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,Immunology ,hemic and immune systems ,chemical and pharmacologic phenomena ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Pancytopenia ,Flow cytometry ,medicine ,Immunohistochemistry ,Aplastic anemia ,business - Abstract
Idiopathic aplastic anemia (AA) is characterized by immune-mediated destruction of hematopoietic stem cells, leading to peripheral pancytopenia. Immune pathogenesis in AA is supported by experimental data, as well as clinical observations and may be related to the breach of peripheral or central tolerance. Regulatory T cells (Treg) constitute one of the most important mechanisms of central tolerance engaged in the down-modulation of autoreactive T cells. Tregs have been found to be reduced in several autoimmune diseases and decreased frequencies of Tregs were also reported in AA and MDS. Overexpression of the high affinity IL-2 receptor alpha chain (CD25) and the forkhead family transcription factor P3 (FoxP3), required for the development and function of Tregs, serve as phenotypic markers for Tregs. We investigated Treg levels in a cohort of AA patients (N=21) and healthy individuals (N=15); flow cytometric quantification of Treg was carried out after surface/intracellular staining of whole blood for Treg markers (CD3, CD4, CD25, FoxP3). After proper gating (light scatter properties, CD3, CD4, CD25), CD4+ T cells were subdivided into CD25−, CD25int and CD25hi populations, and the co-expression of CD25hi and Foxp3 was analyzed. In comparison to controls, AA patients (N=12) show not only lower frequencies of CD4+CD25hi+ T cells within the total lymphocyte population (median 0.07% vs. 0.21%; p=.03), but also absolute lower absolute numbers (1.31/uL vs. 5.78/uL, p=.0002). Similarly, CD4+CD25hi+FoxP3+ T cells were found to be depressed in untreated AA patients in comparison to controls (median 0.07% vs. 0.21% and 1.06/uL vs. 4.76/uL; p=.03 and p=.003). While Tregs were lower in patients with active disease unresponsive to immunosuppressive treatment (responder 0.1% vs non responder 0.07%, CD4+CD25hi Tcells, p=.02), serial testing performed in 6 patients treated with ATG/CsA did not reveal correlation between hematologic improvement and recovery of Treg numbers. When double immunohistochemical staining for CD3 and Foxp3 was performed in pre-treatment bone marrow core biopsies of AA patients (N=3) and controls (N=2) a mean of 3 CD3+Foxp3+ cells/10 high power fields (hpf) were counted (vs. mean 28/10 hpf, p
- Published
- 2007
18. Analysis of Immunogenetic Factors in Myelodysplastic Syndromes (MDS) Reveals Potential Pathogenic Role Cytokine Genotypes Such as TGF-β
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Ramon V. Tiu, Zach Nearman, Mikkael A. Sekeres, Bianca Serio, Ania Jankowska, Jaroslaw M.D. Maciejewski, and Christine O’Keefe
- Subjects
education.field_of_study ,Immunology ,Population ,Haplotype ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Human leukocyte antigen ,Immunogenetics ,Biology ,Biochemistry ,Molecular biology ,Genotype frequency ,hemic and lymphatic diseases ,Genotype ,Genetic predisposition ,education - Abstract
Pathophysiologic and clinical features of MDS are related to the phenotype of the dysplastic hematopoietic clone. The immunogenetic background resulting from complex genetic predisposition traits may influence the quality of the immune response and shape the clinical features of MDS, including the severity of cytopenias and speed of malignant evolution. To test this hypothesis we selected the following immunogenetic factors: KIR and KIR-ligand (KIR-L) genotype, as well as cytokine/receptor single nucleotide polymorphisms (SNP). We genotyped a cohort of 90 patients with MDS/sAML (30 RA/RCMD, 30 RARS/ RCMD-RS, 30 RAEB/sAML); 60 healthy donors matched for ethnicity were analyzed as a control. A group of 66 patients with aplastic anemia (AA) was used as a reference. HLA type, KIR, KIR-L haplotypes, and the following SNPs were analyzed: IL-1α (−889 T/C),IL2 (−330 T/G +166 G/T), IL4 (−1098 T/G -590 T/C -33 T/C, IL-1R (−1970 C/T), IL-1RA (mspa 111100 T/C), IL-4RA (+ 190 G/A), IL-1 β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, -819 C/T), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF- γ (+874 A/T), TNF- α (−308 G/A, -238 G/A), CTLA-4 (exon 1, +49 A/G), FcgIIIR (+559 G/T) as well as SNPs in the CD45 gene (exon 4 +77 C/G, +138 A/G). In the MDS cohort, no difference in the frequency of KIR genotype constellations was identified. However, a higher frequency of 2DS5 (66% vs. 26%, p=.01) and a decreased frequency of 2DL3 (62% vs. 87% p=.02) was found when patients with hypocellular MDS (N=10) were analyzed separately. No significant difference in KIR-L C1/C2 genotype frequency between the group was found. However, an increased incidence of C2/C2 was found in high grade MDS/sAML (RAEB/sAML 44% vs. 13%, p=.02). In MDS, there was a decreased frequency of stimulatory 2DS1/C2 mismatch consistent with potentially enhanced cytotoxicity (17% vs. 44%, p=.01). No significant difference in the MDS cohort compared to control and when MDS subgroup were compared to each other, was found for the SNPs in IL-4RA, IL-12, IL-1α, IFN-γ, IL-2, IL-1 α, IL-1R, and IL4. However, when we examined the frequency of TGF- β genotypes, the MDS population showed a higher rate of TT codon 10 variant (59% vs. 32% in controls, p=.002) and of GG codon 25 variant (71% vs. 35% in controls, p=.0001), consistent with a “high secretor phenotype”. Of note is that, when AA was examined and compared to controls, a higher frequency of TGF-β high secretor genotype was found (GG codon 25 variant; 61% vs 35% in controls, p=.03). We also found a higher incidence of A/A genotype for CTLA-4 in MDS (47 vs 27, p=.001). This relationship was even more pronounced in hypocellular MDS. Moreover, hypocellular MDS was characterized by a higher prevalence of IL10 -819 T/T and -592 A/A phenotypes (40% vs 12% p=.03), which are functionally associated with a lower secretion. In sum, our findings demonstrate that various immunogenetic factors may be demonstrated in MDS patients, which may likely influence the quality of immune response and shape clinical features of MDS. Certain genotypic constellations (e.g., TGF gene variants) resemble, in particular in hypocellular MDS, a constellation seen in AA.
- Published
- 2007
19. Subcutaneous Alemtuzumab Is Safe and Effective for Treatment of Global or Single-Lineage Immune-Mediated Marrow Failure: A Pilot Study
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Lucio Catalano, Antonio M. Risitano, Bruno Rotoli, Bianca Serio, Andrea Camera, Ludovica Marando, Luigi Del Vecchio, Giulia Scalia, and Carmine Selleri
- Subjects
medicine.medical_specialty ,business.industry ,medicine.medical_treatment ,Immunology ,Pure red cell aplasia ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Neutropenia ,medicine.disease ,Biochemistry ,Gastroenterology ,Surgery ,Transplantation ,Regimen ,Internal medicine ,Injection site reaction ,medicine ,Alemtuzumab ,Chills ,medicine.symptom ,business ,medicine.drug - Abstract
Acquired marrow failure syndromes may globally affect all hematopoietic lineages, as in severe aplastic anemia (SAA), or may selectively involve single lineages, as in pure red cell aplasia (PRCA) or in agranulocytosis (AGR). All these conditions share a cellular immune-mediated pathophysiology, which is supported by many experimental data; thus, various immunosuppressive (IS) strategies have been exploited. Alemtuzumab (MabCampath®) (ALE) is a lympholytic MoAb with strong and prolonged IS activity, more reliable than ATG or ALG as for activity, dosing and commercial availability. Here we report a phase II/III pilot study with ALE followed by low-dose cyclosporine A (CsA) on 11 patients suffering from SAA (n=4), PRCA (n=5) or AGR (n=2). All patients received ALE as s.c. injection premedicated by betamethasone, clorpheniramine and paracetamol, with a dose escalating schedule of 3-10-30-30-(30) mg in consecutive days; the total dose was 103 mg for SAA, and 73 for PRCA and AGR. Six patients received one or more additional courses as a result of relapse, so a total of 18 courses were administered. All patients on day 7 started oral low dose CsA (1 mg/kg). Valgancyclovir 450 mg bi-daily and trimethoprim-sulphamethoxazole bi-daily thrice a week were administered as anti-CMV and anti-Pneumocystis Carinii prophylaxis, respectively. All patients completed the treatment, with severe or moderate infusion-related side effect (fever, chills and/or injection site reaction) occurring in 1 (not premedicated) and 3 cases, respectively. No significant abnormality of routine biochemical testing, nor other medically significant adverse events were reported. A complete lympho-ablation was observed in all patients within 2–3 days, which persisted for several weeks; in addition, transient worsening of neutropenia (managed by occasional G-CSF support) and of thrombocytopenia were observed in some patients. At a median follow-up of 6 months, infectious events were irrelevant: in cumulative 75 patient-months, 1 HSV and 1 flu have been recorded (globally 1 day of fever), all resolving quickly. No CMV reactivation was demonstrated. Immune reconstitution was delayed up to several months, with absolute lymphocyte count ranging between 30–200/uL, 100–400/uL, 250–800/uL and 500–2000/uL at months +1, +3, +6 and +12 from the treatment. The CD4+ compartment was significantly more affected than the CD8+, with a persistent inversion of the CD4/CD8 ratio. As for efficacy, the 4 SAA patients showed 1 CR, 1 PR (both relapsing at 6 months and re-treated with additional ALE courses), 1 NR at 3 months (addressed to early stem cell transplantation due to life-threatening hemorrhages); 1 is not evaluable yet. In the 5 PRCAs, there were 4 CR and 1 NR (at 3 months); 3 responding patients relapsed and were successfully managed by further courses of ALE. The 2 AGRs showed both CR, followed by late relapse (at 18 months) in one case (now receiving a second course). In conclusion, ALE administered as subcutaneous injection is a feasible and safe IS regimen for patients suffering from immune-mediated marrow failure syndromes. Infectious complications were unremarkable, and preliminary results suggest good efficacy, especially in lineage-restricted forms; as with other IS regimens, the hematological response is late (3–4 months) and relapses may occur, which are sensitive to further ALE courses. Such favorable risk-to-benefit ratio predicts for this regimen a leading position in the future IS strategies.
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- 2007
20. Immunogenetic Analysis Reveals the Association of INF-γ (+874 A/T) Hypersecretor Genotype in AA and a Low Frequency of KIR-2DL3/C1 Mismatch in Responders to Immunosuppression
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Ramon V. Tiu, Giridharan Ramsingh, Antonio M. Risitano, Mikkael A. Sekeres, Bianca Serio, and Jaroslaw M.D. Maciejewski
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Genetics ,Immunology ,Haplotype ,Cell Biology ,Hematology ,Human leukocyte antigen ,Biology ,Biochemistry ,Molecular biology ,Interleukin 10 ,Immune system ,Aldesleukin ,Genotype ,Interleukin 12 ,Cytotoxic T cell - Abstract
Clinical and laboratory evidence support an immune pathogenesis in most cases of idiopathic aplastic anemia (AA) and closely related disorders such as paroxysmal nocturnal hemoglobinuria (PNH). While external triggers are likely necessary, a complex constellation of immunogenetic factors may determine disease susceptibility. Many immunogenetic factors can influence the quality of immune response and affect the propensity to immune-mediated attack on hematopoietic stem cells in AA. Here we investigated whether KIR and KIR-L (HLA-A) genotype and cytokine/receptor gene variants are over-represented in AA and PNH. We studied a cohort of 77 patients with AA (23 AA, 20 AA/PNH and 34 PNH), 10 with hypocellular MDS and 175 healthy controls. The following SNPs in immunoregulatory genes were analyzed: IL-1α (−889 T/C), IL-2 (−330 T/G +166 G/T), IL-4 (−1098 T/G −590 T/C −33 T/C), IL-1R (−1970 C/T), IL-1Rα (mspa111100 T/C), IL-4RA (+ 190 G/A), IL-1β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, −819 C/T, −592 C/A), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), INF-γ (+874 A/T), TNF-α (−308 G/A, −238 G/A) and immunomodulatory receptor genes including CTLA-4 exon 6 (+49 G/A), FcRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of NK and cytotoxic T cells, we examined the combined impact of KIR/KIR-L genotypes on the risk of AA and PNH syndrome. In AA we found a decreased frequency of inhibitory KIR-2DL3 genes (68% vs. 89%, p=.0002); analysis of the KIR genotype in correlation with the corresponding KIR-L profile, revealed a decreased frequency of stimulatory 2DS1/C2 mismatch resulting in a potentially enhanced cytotoxic activity (14% vs.44%, p=.003). No association was found for most of the SNPs tested. However, when we examined the frequency TGF-β genotypes, increased frequency of GG variant in codon 25 (61% vs. 35% in controls, p=.03), associated with the “high secretor” phenotype, was found in AA. This relationship was also present in hypocellular MDS (82% vs. 32%, p=.007). Additionally, we found a lower incidence of TT genotypes for the IL-1Rα gene (33% vs. 62% p=.02). We confirm that the hypersecretor genotype T/T of INF-γ was over-represented in AA (28% vs. 10% in controls, p=.02). Subgroup analysis revealed that the T/T genotype of IFN-γ (35% vs. 14% p=.01) correlated with presence of a PNH clone. Previously, we have shown the association of HLA-DR15 with responsiveness to immunosuppression. When AA patients were subgrouped according to response to ATG/CsA, therapy refractoriness correlated with the presence of the C2/C2 haplotype (30% vs. 0% p=.02) and inhibitory KIR-2DL3/C1 mismatch (70% vs. 0%, p=.01) which may result in a greater propensity to breach of self-tolerance. In comparison, in the total AA group, C2/C2 haplotype and KIR-2DL3/C1 mismatch were present in 17% vs. 24% and 8% vs. 16% of controls, respectively. An increase in the frequency of 2DL3 and a decrease in 2DS1 mismatch may result in imbalance between cytotoxicity and KIR inhibition. In sum, our findings demonstrate that complex inherited traits involving immunogenetic factors may genetically determine propensity to bone marrow failure syndromes.
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- 2007
21. Non Synonymous SNP-Array-Based Disease Association Analysis in Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria
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Bianca Serio, Aaron D. Viny, Alan E. Lichtin, Ramon V. Tiu, Bartlomej Przychodzen, Christine L. O'Keefe, and Jaroslaw M.D. Maciejewski
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Genetics ,Immunology ,Clone (cell biology) ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Human leukocyte antigen ,Biology ,Biochemistry ,Genotype ,SNP ,TAP1 ,Genotyping ,SNP array - Abstract
The pathogenesis of aplastic anemia (AA) includes external triggers that induce an immune-mediated attack in susceptible individuals. A complex genetic background is likely the basis for AA predisposition; various genetic factors, including HLA, KIR and SNPs in immune response genes, were studied in AA. However, such an empiric approach, despite the rational target selection, is inefficient. SNP array (SNP-A) genotyping technology allows for investigation of complex genetic traits and is a suitable hypothesis-generating technology. We stipulated that application of SNP-A in AA will allow for identification of SNPs in pathogenic loci. We applied the Illumina 12K non-synonymous SNP-A to study 77 AA and ethnically matched controls (ctr; N=60; +170 historical ctr). The power of this technique is demonstrated by our ability to obtain >2.6 million genotypes with a fidelity (against PCR) of 98%. The training set included 64 AA patients and 56 controls. Initially, Exemplar automated analysis was used; due the Bonferroni correction this study underpowered. However, our strategy included ranking SNPs based on their P value, low frequencies of pathogenic genotypes in ctr, and high case/ctr ratio narrowing the selection of potentially informative SNPs to be tested in a validation set. We also applied Random Forests, a nonparametric tree method, whereby all SNP were used multivariately to predict disease association; this method does not relay on Bonferroni correction and more closely reflects complex polygenic traits. Results pointed towards many SNPs recognized by both approaches and a number of SNP was chosen for further analysis. E.g., rs8022805 (GA) in telomerase-associated protein-1 (TEP1) was found in 19% of AA patients but only in 2.7% of controls (N=220, p
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- 2007
22. Phospho-IkappaB Is Abnormally Expressed in Bone Marrow of CMML Patients
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Jaroslaw P. Maciejewski, Ziad Peerwani, Eric D. Hsi, Bianca Serio, Ramon V. Tiu, and Andrew E. Schade
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Pathology ,medicine.medical_specialty ,Myeloid ,biology ,Immunology ,Cell ,Chronic myelomonocytic leukemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Pathogenesis ,medicine.anatomical_structure ,Fibrosis ,hemic and lymphatic diseases ,medicine ,biology.protein ,Immunohistochemistry ,Bone marrow ,Antibody - Abstract
Introduction: Chronic myelomonocytic leukemia (CMML) is a heterogeneous group of bone marrow disorders currently grouped into the MDS/MPD overlap by WHO classification. Its clinical and laboratory features includes the presence of up to 20% blast in the bone marrow and the peripheral blood, a PB monocyte count >1000/mL, splenomegaly, variable reticulin fibrosis in the bone marrow and cytopenias. The exact pathogenesis remains in question and there are no effective therapies. Recent studies in certain myeloid disorders suggest that the nuclear transcription factor NFkB regulates cell survival, proliferation, and differentiation. It has been found to be highly expressed in AML and may serve as an important therapeutic target. Little is known about NFkB in other hematologic disorders, including CMML. Examination of NFkB activation in situ has been technically difficult due to lack of quality antibody reagents suitable for fixed tissues. Recently, phosphospecific antibodies have become available, which reflect the functional status of proteins. IkB regulates NFkB subunits by sequestering them in the cytoplasm. Phosphorylation of IkB by IKK results in release of NFkB and translocation to the nucleus. Thus, phospho-IkB (pIkB) is an indicator of NFkB activation. We studied the pattern of pIkB expression in CMML as a surrogate of NFkB activation. Methods: We identified a cohort of 24 CMML (CMML1=17; CMML2=7) patients and 9 healthy controls. Cases were characterized clinically and pathologically. Immunohistochemistry (IHC) for pIkB was performed in trephine biopsies using a phosphor-specific antibody (Cell Signaling). The staining pattern was compared to normal bone marrow. We utilized JMP 5.1.2 statistical software to compare a variety of clinical and laboratory parameters. Results: The mean age of patients at diagnosis was 61 (range:38–72). Median WBC, Hgb, PLT, absolute monocytes were 18.6K/ul, 10.2g/dL, 93K/ul, 4K/ul respectively. As expected, the overall survival (OS) was short (mean OS = 11.1 months). Compared to normal bone marrow, pIkB was found to be abnormally activated in maturing granulocytic cells and was found to be present in cytoplasmic as well as nuclear locations. The mean % neutrophils that expressed pIkB was 36.6 compared to 14.9 for normal bone marrow (P Conclusions: An abnormal in situ pIkB expression pattern is present in CMML compared to normal bone marrow, suggesting abnormal activation of NFkB. Interestingly, maturing myeloid cell expression of pIKB was associated with higher WBC and absolute monocyte count. Further studies are warranted in examining the role of NFkB activation in CMML and potential therapeutic intervention in this pathway, such as with proteasome inhibitors.
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- 2007
23. Hemochromatois-Associated Gene Mutations in Patients with Myelodysplastic Syndromes with Refractory Anemia and Ringed Sideroblasts
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Hadrian Szpurka, Bianca Serio, Mikkael A. Sekeres, Alan E. Lichtin, Jaroslaw P. Maciejewski, Ilka Warshawsky, and Zachary P. Nearman
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Thrombocytosis ,Myelodysplastic syndromes ,Immunology ,Cell Biology ,Hematology ,Gene mutation ,Biology ,Refractory anemia with ringed sideroblasts ,medicine.disease ,Biochemistry ,hemic and lymphatic diseases ,Genotype ,medicine ,Restriction fragment length polymorphism ,Allele ,Hemochromatosis - Abstract
Complex interaction between a multitude of genetic variants may be responsible for differential susceptibility to specific diseases, and be responsible for phenotypic variability and heterogeneity of clinical presentations. Such a variability in clinical features confounded for many years investigations into the pathogenesis of myelodysplastic syndromes (MDS). We made a curious observation of increased ferritin levels in some newly diagnosed patients with MDS RARS (refractory anemia with ringed sideroblasts) in whom transfusional iron-overload was unlikely due to very low transfusion burden. Hence, we hypothesized that RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this particular subset of MDS. We studied a cohort of 109 MDS patients; 42 with RARS, and 67 with other forms of MDS (18 RA, 12 RAEB, 7 RAEB-T, 1 CMML, and 29 MDS/MPD overlap). All patients were genotyped using restriction fragment length polymorphism (RFLP) method, designed to detect presence of C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygozity for the C282Y mutation in 21% of RARS patients (vs 9% in control population, n=2016, p= 0.017) while H63D genotype was not increased. The possible pathogenic role of this finding in RARS was supported by the normal distribution of mutant HFE alleles in patients with other forms of MDS (5% vs. 9%, p =0.35). Interestingly, 3/7 patients with RA not fulfilling the RARS criteria, but having increased numbers of ringed sideroblasts (
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- 2006
24. Impact of Genetic Polymorphisms on Immune Response and Clinical Features in MDS
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Anna M. Jankowska, Ramon V. Tiu, Giridharan Ramsingh, Bianca Serio, Jaroslaw P. Maciejewski, Mikkael A. Sekeres, Christine O’Keefe, Hadrian Szpurka, and Zach Nearman
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Genetics ,medicine.medical_specialty ,medicine.medical_treatment ,Immunology ,Haplotype ,Single-nucleotide polymorphism ,Cell Biology ,Hematology ,Human leukocyte antigen ,Biology ,Biochemistry ,Gastroenterology ,Pathogenesis ,Interleukin 10 ,Immune system ,Cytokine ,hemic and lymphatic diseases ,Internal medicine ,Genotype ,medicine - Abstract
The pathogenesis of cytopenias in myelodysplastic syndrome (MDS) is not fully explained by evolution of the clonal displacement of normal hematopoiesis. The quality of immune surveillance may contribute to generalized hematopoietic inhibition. E.g., certain cases may present with hypocellular marrow reminiscent of idiopathic aplastic anemia (AA). Immunogenetic background resulting from predisposing complex traits can influence the quality of immune response and shape clinical features of MDS. Immunogenetic factors include: KIR and KIR-ligand (KIR-L) genotype, as well as cytokine/receptor single nucleotide polymorphisms (SNP). We genotyped 130 patients with MDS (32 RA/RAMCD, 24 RARS/ RAMCD-RS, 51 RAEB/sAML, 23 MDS/MPD and CMML). Controls included current (n=87) and historical controls. We studied HLA type, KIR, KIR-L genotypes, and immunomodulatory SNPs, including: IL-1α (−889 T/C), IL-1R (−1970 C/T), IL-1RA (mspa111100 T/C), IL-4RA (+ 190 G/A), IL-1β (−511 C/T, +3962 T/C), IL-6 (−174 C/G, nt565 G/A), IL-10 (−1082 G/A, −819 C/T), IL-12 (−1188 C/A), TGF-β (+10 C/T, +25 G/C), TGF-βR2 (+358 A), INF-γ (+874 A/T), TNF-α (−308 G/A, −238 G/A), CTLA-4 (exon 1, +49 A/G), FcγIIIR (+559 G/T) as well as SNPs in CD45 gene (exon 4 +77 C/G, +138 A/G). In all MDS, no difference in the frequency of KIR genotype constellations was identified. However, higher frequency of stimulatory KIR2DS2 in low grade MDS (72% vs 48% in controls, p=.01) and 2DS5 in hypocellular MDS (62% vs 26%, p=.02) was found. When KIR-L C1/C2 genotype was studied, no significant difference in haplotype frequency was observed compared to controls. However, an increased incidence of C2/C2 was found in high grade MDS (70% vs 24%, p=.003). Analysis of the resulting KIR/KIR-L combination demonstrated increased frequency of inhibitory KIR2DL3/C1 mismatch (34% vs 16%, p=.04) in the MDS cohort consistent with higher cytotoxicity. A similar observation was made when we compared advanced and low-grade MDS (70% vs 23%, p=.02). A higher proportion of 2DS2/C1 mismatches (50% vs 10%, p=.006) in advanced MDS could reflect lesser degree of immune surveillance facilitating clonal expansion. No significant difference in MDS cohort, nor in any of subgroups compared to control and each other, was found for the SNPs in IL-4Rα, IL-12, IL-1β, IL-6, TGF-βR2, IL-10, IL-12, IL-1α, IL-1R, FcγIIIR, and CD45. However, when we examined the frequency of INF-γ and TGF-β genotypes, increased frequency of INF-γ AT variant (62% vs 33% in controls, p=.015) and a decrease frequency of TGF-β genotype T/T-G/G or T/C-G/G (53% vs 74%, p=.04) were found, consistent with high secretor phenotype. In addition, higher incidence of G/G phenotype for the CTLA-4 gene (p=.0001) was found in the MDS cohort and in low grade MDS (p=.003). Heightened immune response could result in similar hematopoietic suppression as observed in AA, and lead to hypoplasia. Consequently, we subdivided all MDS patients according to marrow cellularity and found that hypoplastic variant of MDS (n=10) was characterized by higher prevalence of A/T phenotype of INF-γ gene (70% vs 33%, p=.02), similar to a cohort of AA patients (n=26, 50% vs 33%, p=.05). An analogous observation was also made for G/A genotype of TNF-α. In sum, our findings demonstrate that a large number of immunogenetic predisposition factors associated with more risk immune response may exist in MDS, which may influence certain clinical features and phenotype.
- Published
- 2006
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