ABSTRACT
BACKGROUND: Bone marrow stem cells (BMSC) can participate to liver regeneration. However, conflicting results have been reported on this topic in patients undergoing liver resection. AIMS: To assess the impact of liver resection extent and presence of underlying liver disease in modulating BMSC mobilization. METHODS: We enrolled 29 patients undergoing liver resection of different extents, 5 surgical controls and 10 blood donors. Circulating CD133+ BMSC were measured by flow cytometry at different time-points after surgery. The hepatic commitment of mobilized BMSC was investigated by polymerase chain reaction. Liver specimens were collected during surgery for histopathological analysis. Hepatocyte growth factor and granulocyte-colony stimulating factor serum levels were measured by enzyme-linked immunosorbent assay. RESULTS: BMSC mobilization was found in patients undergoing major liver resection, especially in the presence of underlying disease. Ductular reactions were noted in patients with chronic hepatopathy and the hepatic progenitor-like cells expressed CD133, NCAM, cytokeratin-19, and alpha-fetoprotein. Hepatocyte growth factor and granulocyte-colony stimulating factor levels increased following liver resection and the contemporaneous presence of liver disease was associated with their highest raise. CONCLUSIONS: Liver repair is mainly an endogenous process. BMSC become important in case of extensive resection, especially in the presence of underlying hepatopathy and hepatic progenitor-like cells activation. Hepatocyte growth factor and granulocyte-colony stimulating factor seem to be involved in the dynamics underlying hepatic regeneration and BMSC recruitment.
Subject(s)
Antigens, CD/blood , Glycoproteins/blood , Hematopoietic Stem Cell Mobilization , Hepatectomy/methods , Liver Regeneration , Peptides/blood , AC133 Antigen , Biomarkers/blood , Case-Control Studies , Female , Granulocyte Colony-Stimulating Factor/blood , Hepatocyte Growth Factor/blood , Humans , Keratin-19/blood , Liver Diseases/surgery , Male , Middle Aged , Neural Cell Adhesion Molecules/blood , alpha-Fetoproteins/metabolismABSTRACT
UNLABELLED: Background The MPL(Ser505Asn) mutation has been reported to be a cause of hereditary thrombocythemia. Recently, we detected this mutation in a large proportion of children with familial thrombocythemia, suggesting that in Italy the incidence of MPL(Ser505Asn) mutation could be underestimated. DESIGN AND METHODS: We extended the search for this mutation to all patients with essential thrombocythemia who had a positive family history for thrombocytosis or essential thrombocythemia. We identified eight Italian families positive for the MPL(Ser505Asn) mutation. Clinical and hematologic data were available for members of seven families, including 21 patients with a proven mutation and 20 relatives with thrombocytosis. RESULTS: Fifteen major thrombotic episodes, nine of which were fatal, were recorded among 41 patients. The thrombotic manifestation was stroke in four cases, myocardial infarction in seven cases, fetal loss in two cases, deep vein thrombosis of the leg in one case and Budd Chiari syndrome in one case. Almost all patients over 20 years old had splenomegaly and bone marrow fibrosis, while these were rarely observed in patients under 20 years old, suggesting that these manifestations are associated with aging. Finally, the life expectancy of family members with thrombocytosis was significantly shorter than that of members without thrombocytosis (P=0.003). Conclusions Patients with familial thrombocytosis caused by a MPL(Ser505Asn) mutation have a high risk of thrombosis and, with aging, develop splenomegaly and bone marrow fibrosis, significantly affecting their life expectancy.
Subject(s)
Amino Acid Substitution/genetics , Primary Myelofibrosis/genetics , Receptors, Thrombopoietin/genetics , Splenomegaly/genetics , Thrombocytosis/genetics , Thrombosis/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Asparagine/genetics , Child , Child, Preschool , Disease Progression , Female , Follow-Up Studies , Humans , Infant , Male , Middle Aged , Pedigree , Primary Myelofibrosis/mortality , Risk Factors , Serine/genetics , Splenomegaly/mortality , Thrombocytosis/complications , Thrombocytosis/mortality , Thrombopoietin/genetics , Thrombopoietin/metabolism , Thrombosis/mortality , Young AdultABSTRACT
B cells have acquired an important role in the pathogenesis of rheumatoid arthritis (RA) since B cell depletion allowed to rescue patients poorly responders to TNFalpha blockers. This study focused on the involvement of ZAP-70 as a bio-marker of B cells immune activation in RA. ZAP-70 expression in synovial fluid (SF) B cells obtained from RA patients was increased compared to SF B cells of osteoarthritis (OA) patients. Moreover we found that ZAP-70 positive/CD38 positive and ZAP-70 positive/CD5 positive B cells were enriched in SF. The analysis of B cell apoptosis in vitro showed that the percentage of ZAP-70 negative B cells spontaneously undergoing apoptosis was significantly higher than ZAP-70 positive B cells. The ZAP-70 positive B cell ratio (SF/peripheral blood (PB)) showed a positive correlation with SF autoantibody levels and with local levels of BAFF and IL6. ZAP-70 positive B cells seem to define a subset characterized by increased survival and high relationship with local inflammation and autoimmunity.
Subject(s)
Arthritis, Rheumatoid/immunology , B-Lymphocytes/immunology , Synovial Fluid/immunology , ZAP-70 Protein-Tyrosine Kinase/biosynthesis , Antigens, CD/biosynthesis , Antigens, CD/immunology , Apoptosis/immunology , Arthritis, Rheumatoid/enzymology , Arthritis, Rheumatoid/pathology , Autoantibodies/immunology , B-Cell Activating Factor/blood , B-Cell Activating Factor/immunology , B-Lymphocytes/enzymology , B-Lymphocytes/pathology , Female , Flow Cytometry , Gene Expression , Humans , Immunohistochemistry , Interleukin-6/blood , Interleukin-6/immunology , Male , Middle Aged , Osteoarthritis/immunology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Synovial Fluid/cytology , ZAP-70 Protein-Tyrosine Kinase/genetics , ZAP-70 Protein-Tyrosine Kinase/immunologyABSTRACT
Alterations of the transcription factors CCAAT/enhancer binding protein alpha (CEBPA) and PU.1 have been described in acute myeloid leukemia (AML). We studied CEBPA and PU.1 mRNA levels by real-time RT-PCR in 109 primary AML samples, compared with normal bone marrow and peripheral blood cells. Low PU.1 levels were observed in monoblastic leukemias, while low CEBPA levels were associated with leukopenia at diagnosis and lack of expression of differentiation antigens CD33 and CD11c. We conclude that down-regulation of CEBPA and PU.1 is not a general feature of primary AML, but appears to be restricted to distinct AML subtypes.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute/genetics , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Adolescent , Adult , Aged , Antigens, CD/genetics , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/metabolism , Blotting, Western , CCAAT-Enhancer-Binding Proteins/metabolism , CD11c Antigen/genetics , CD11c Antigen/metabolism , Female , Flow Cytometry , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Leukopenia/genetics , Leukopenia/metabolism , Leukopenia/pathology , Male , Middle Aged , Proto-Oncogene Proteins/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialic Acid Binding Ig-like Lectin 3 , Trans-Activators/metabolismABSTRACT
OBJECTIVES: We hypothesized that the expansion of unusual T lymphocytes, CD4+CD28null T cells, might represent a key pathogenetic mechanism of recurrent instability. BACKGROUND: Clinical presentation of acute coronary syndromes (ACS) is variable. Some patients have recurrent episodes of instability, despite optimal treatment, whereas others have a single acute event in their life. The CD4+CD28null T cells, with a functional profile that favors vascular injury, have recently been found both in peripheral blood and in unstable coronary plaques of patients with ACS. METHODS: Peripheral blood T cells from 120 consecutive unstable angina (UA) patients were analyzed for the distribution of T-cell subsets by flow cytometry. Patients were subgrouped according to the occurrence of prior (during the 24 months before the study enrollment) and subsequent (during the 24 months of follow-up) acute coronary events. For 51 patients, the index event was the first ever (G1); 30 patients had prior events (G2); and 39 patients had further events at follow-up (death, myocardial infarction, or UA) or both before and after the index event (G3). RESULTS: The CD4+CD28null T-cell frequency was higher in G3 than in G2 and G1 (median 9.5% [range 2.4% to 48.0%] vs. 5.1% [range 0.4% to 27.8%] and 2.3% [range 0.2% to 22.8%], respectively; p < 0.001). The expansion of these unusual T lymphocytes was higher in patients with elevated C-reactive protein levels, and it was reduced by statin therapy. On multivariate logistic regression analysis, CD4+CD28null T-cell frequency was an independent predictor of future acute coronary events (odds ratio 3.01, 95% confidence interval 1.1 to 8.25; p = 0.023). CONCLUSIONS: A perturbation of T-cell repertoire is strongly associated with the recurrence of acute coronary events, conceivably playing a key pathogenetic role.
Subject(s)
Angina, Unstable/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Lymphocytes, Null/immunology , Myocardial Infarction/immunology , T-Lymphocyte Subsets/immunology , Aged , C-Reactive Protein/analysis , CD28 Antigens/immunology , CD4 Lymphocyte Count , Female , Flow Cytometry , Humans , Logistic Models , Male , Middle Aged , Phenotype , Prognosis , Prospective Studies , Recurrence , Risk Assessment , Syndrome , T-Lymphocyte Subsets/cytology , Troponin T/analysisABSTRACT
The clinical heterogeneity that characterizes chronic lymphocytic leukemia (CLL) poses critical questions concerning the identification of high risk patients. Unmutated IgV(H) genes, CD38 and ZAP-70 expression have emerged as the most useful tools in identifying aggressive CLL. The simultaneous expression of ZAP-70 and CD38 in 157 patients with CLL has been evaluated. Fifty-seven patients (36%) were positive for ZAP-70 and 46 patients (29%) were positive for CD38. Both molecules were highly correlated and predictive of the clinical course of the disease. According to the simultaneous evaluation of ZAP-70 and CD38, patients were divided into three groups. In 81 patients (52%), there was a negative concordance of both molecules (ZAP-70(-)/CD38(-)); in 27 patients (17%) there was a positive concordance (ZAP-70(+)/CD38(+)); in 49 patients (31%) there was a discordant expression (ZAP-70(+)/CD38(-) and ZAP-70(-)/CD38(+)). A comparison of the clinical and laboratory data showed in ZAP-70(+)/CD38(+) patients a significantly higher bone marrow and peripheral blood lymphocytosis, lower hemoglobin levels, more advanced clinical stage, and higher number of unmutated IgV(H) status with respect to the other two groups. Furthermore, ZAP-70(+)/CD38(+) patients displayed a much shorter treatment-free interval (median 12 months vs 42 months in discordant patients and not reached in ZAP-70(-)CD38(-) patients). These results prove that the concomitant evaluation of ZAP-70 and CD38 expression allows the separation of CLL patients in prognostic subgroups and suggest that their simultaneous assessment should become an integral component of the CLL diagnostic grid.
Subject(s)
ADP-ribosyl Cyclase 1/analysis , Leukemia, Lymphocytic, Chronic, B-Cell/diagnosis , ZAP-70 Protein-Tyrosine Kinase/analysis , ADP-ribosyl Cyclase 1/genetics , Adult , Aged , Aged, 80 and over , Cohort Studies , Female , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Leukemic , Humans , Immunoglobulin Heavy Chains/analysis , Immunoglobulin Heavy Chains/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Male , Middle Aged , Predictive Value of Tests , Prognosis , ZAP-70 Protein-Tyrosine Kinase/geneticsSubject(s)
Diagnostic Errors , Killer Cells, Natural/pathology , Lymphoma, Non-Hodgkin/diagnosis , Pseudolymphoma/diagnosis , Skin Neoplasms/diagnosis , Aged , Antigens, CD/analysis , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor , Bleomycin/administration & dosage , CD4 Antigens/analysis , CD56 Antigen/analysis , Cyclophosphamide/administration & dosage , Diagnosis, Differential , Disease Progression , Doxorubicin/administration & dosage , HLA-DR Antigens/analysis , Humans , Lymph Nodes/diagnostic imaging , Lymphoma, Non-Hodgkin/chemistry , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/pathology , Lymphoma, T-Cell, Cutaneous/diagnosis , Male , Neoplasm Proteins/analysis , Prednisone/administration & dosage , Proto-Oncogene Proteins c-bcl-2/analysis , Radiography , Skin Neoplasms/chemistry , Skin Neoplasms/drug therapy , Vincristine/administration & dosageABSTRACT
OBJECTIVE: A non-invasive prenatal determination of the fetal RhD status might be useful for the management of pregnancies in RhD-negative women whose partners are RhD positive. METHODS: Maternal peripheral blood of 32 RhD-negative women (17-24 weeks of gestation) was collected, and circulating fetal cells were enriched by CD71 mini-magnetic activated cell sorting. The RhD status of the fetuses was assessed using multiparametric flow cytometry, and results were compared to those of reverse transcriptase (RT)-polymerase chain reaction (PCR), or PCR, which acted as control. Flow-cytometric study of fetal cells employed monoclonal antibodies directed against CD71, glycophorin A (GPA) and RhD antigens. RESULTS: The median percentage of CD71- and RhD-positive cells was 0.83% (range 0.14-6.44%), and that of CD71 and GPA-positive cells was 10.07% (range 0.52-45.84%). Flow-cytometric analysis correlated with RT-PCR results of RNA obtained from whole maternal blood. In 1 case, an incorrect result was due to the failure of the amplification of the specific RhD band on RNA extracted from the CD71-positive fraction. In two instances, we observed false-positive results for RhD in PCR of DNA obtained from maternal plasma. CONCLUSION: Based on our results, flow-cytometric analysis might be proposed as a clinical tool for the non-invasive prenatal determination of the fetal RhD status independently of fetal gender.
Subject(s)
Blood Grouping and Crossmatching/methods , Flow Cytometry , Prenatal Diagnosis , Reverse Transcriptase Polymerase Chain Reaction , Rh-Hr Blood-Group System/blood , Antibodies, Monoclonal , Antigens, CD/analysis , Erythroblasts/chemistry , Female , Fetal Blood/cytology , Gestational Age , Glycophorins/analysis , Humans , Pregnancy , Receptors, Transferrin/analysis , Rh-Hr Blood-Group System/classification , Rh-Hr Blood-Group System/geneticsSubject(s)
Anemia/blood , Anemia/complications , Antigens, CD34/blood , Coronary Disease/blood , Coronary Disease/etiology , Renal Insufficiency/blood , Renal Insufficiency/complications , Coronary Artery Disease/epidemiology , Coronary Artery Disease/etiology , Coronary Disease/epidemiology , Female , Glomerular Filtration Rate , Humans , Male , Middle Aged , Risk FactorsABSTRACT
BACKGROUND AND OBJECTIVES: The protein tyrosine kinase ZAP-70 has recently emerged as a major prognostic indicator in chronic lymphocytic leukemia (CLL). ZAP-70 is structurally and functionally homologous to Syk, a key mediator of B-cell receptor signaling. We therefore evaluated ZAP-70 expression in CLL B cells using Syk as an intracellular standard. DESIGN AND METHODS: The relative amounts of ZAP-70 and Syk were determined in purified B cells from 92 CLL patients using a novel reverse transcriptase/polymerase chain reaction (RT-PCR) procedure that co-amplifies both transcripts with equal efficiency. The ZAP-70/Syk mRNA ratio was correlated with VH gene mutation status, median treatment-free survival and FACS analysis of ZAP-70 expression. RESULTS: ZAP-70 was expressed in the majority of cases with unmutated VH genes (88%), but also at lower levels in a substantial fraction of cases with mutated VH genes (44%). High levels of ZAP-70, defined as ZAP-70/Syk mRNA ratios above 0.25, were observed mainly in cases with unmutated VH genes and correlated with short treatment-free survival. In contrast, no difference was observed in the median treatment-free survival between patients with low ZAP-70/Syk ratios (0.05-0.25) and patients with no or negligible ZAP-70 expression (ZAP-70/Syk<0.05). In 73 cases ZAP-70 expression was investigated by RT/PCR and FACS analysis; concordance with VH gene mutation status was 86% and 71%, respectively. INTERPRETATION AND CONCLUSIONS: ZAP-70 is frequently expressed in CLL B cells, but only high levels correlate with unmutated VH gene status and progressive disease. Expression of ZAP-70 can be accurately assessed by analysis of the ZAP-70/Syk mRNA ratio, thus providing an alternative to FACS analysis.
Subject(s)
Genes, Immunoglobulin Heavy Chain/genetics , Intracellular Signaling Peptides and Proteins/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation , Protein-Tyrosine Kinases/genetics , RNA, Messenger/genetics , ZAP-70 Protein-Tyrosine Kinase/genetics , Adult , Aged , Aged, 80 and over , Base Sequence , Disease Progression , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/enzymology , Male , Middle Aged , Molecular Sequence Data , Syk KinaseABSTRACT
OBJECTIVE: To determine in the same blood sample the concentrations of cyclosporin A (CsA) and the degree of CsA-induced lymphocyte inhibition; to establish a relationship between these parameters; and to investigate the factor(s) influencing such a putative relationship. METHODS: Ten patients with a diagnosis of Crohn's disease (n=7) or ulcerative colitis (n=3) were enrolled in the study. The patients, who had never been immunosuppressed, were treated with microemulsion CsA twice daily by the oral route; at steady-state, blood samples were collected 0, 0.5, 1, 2, 3, 5, 7 and 12 h after the morning dose. CsA blood levels were measured by means of radioimmunoassay. The percentages of lymphocytes during the S-phase were assessed by flow-cytometry on the same blood specimens, only for samples collected at 0 h and 2 h. RESULTS: An inverse relationship emerged between CsA blood concentrations and the percentage of lymphocytes during the S-phase: the latter was maximal before the beginning of treatment and minimal in association with peak CsA levels. Furthermore, a highly significant correlation was found between trough CsA levels and the age of the patient, since the percentage of inhibited lymphocytes increases with age.
Subject(s)
Colitis, Ulcerative/blood , Cyclosporine/blood , Inflammatory Bowel Diseases/blood , Lymphocytes/drug effects , Administration, Oral , Adult , Cell Division/drug effects , Cellular Senescence/drug effects , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/drug therapy , Cyclosporine/pharmacology , Cyclosporine/therapeutic use , Drug Administration Schedule , Emulsions , Female , Humans , Inflammatory Bowel Diseases/diagnosis , Inflammatory Bowel Diseases/drug therapy , Lymphocyte Activation/drug effects , Lymphocytes/cytology , Male , Middle AgedABSTRACT
In this study, we investigated whether hematopoietic stem cells (HSC) and progenitors present in human cord blood can express nerve growth factor (NGF)-specific receptors, TrkA and p75. Our results showed a marked expression of TrkA and NGF in cord blood CD34(+) cells. A gradient of TrkA and NGF expression exists and is highest in cord blood CD34(+) cells, reduced in cord blood mononuclear cells (MNC) and minimal in mononuclear cells isolated from adult peripheral blood. Our findings suggest that NGF may play a role in the differentiation of hematopoietic progenitors and indicate a different requirement for NGF by immune cells, depending on their state of maturity.