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Paiva B.,Hospital Universitario Of Salamanca | Paiva B.,University of Salamanca | Vidriales M.-B.,Hospital Universitario Of Salamanca | Vidriales M.-B.,University of Salamanca | And 21 more authors.
Blood | Year: 2011

The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients withAL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL. © 2011 by The American Society of Hematology.


Paiva B.,Hospital Universitario Of Salamanca | Montes M.C.,Hospital Universitario Of Salamanca | Garcia-Sanz R.,Hospital Universitario Of Salamanca | Ocio E.M.,Hospital Universitario Of Salamanca | And 19 more authors.
Leukemia | Year: 2014

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenström's Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P<.001), with only 1% of IgM-MGUS patients showing >10% B cells or 100% light-chain-isotype-positive B-cells (P<.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P=0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P=0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstrom's phenotype (CD22+dim/ CD25+/CD27+/IgM+) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenström's clone, as well as for the differential diagnosis, risk of progression and survival in WM. © 2014 Macmillan Publishers Limited.


Paiva B.,University of Navarra | Corchete L.A.,Hospital Universitario Of Salamanca | Vidriales M.-B.,Hospital Universitario Of Salamanca | Garcia-Sanz R.,Hospital Universitario Of Salamanca | And 24 more authors.
Blood | Year: 2015

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25-CD22+ B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25+CD22+low) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone. © 2015 by The American Society of Hematology.


PubMed | Complejo Hospitalario Of Leon, Hospital General Of Segovia, Hospital Virgen Of La Concha, Hospital Nuestra Senora Of Sonsoles and 8 more.
Type: Journal Article | Journal: Blood | Year: 2015

Although information about the molecular pathogenesis of Waldenstrm macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenstrm B-cell clone was highly different than that of normal CD25(-)CD22(+) B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenstrm B-cell clone was compared with its normal phenotypic (CD25(+)CD22(+low)) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenstrm clone.


PubMed | University of Salamanca, Hospital Santos Reyes, Complejo Hospitalario Of Leon, Hospital General Of Segovia and 9 more.
Type: Journal Article | Journal: Leukemia | Year: 2014

Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenstrms Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (P<.001), with only 1% of IgM-MGUS patients showing >10% B cells or 100% light-chain-isotype-positive B-cells (P<.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P=0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P=0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstroms phenotype (CD22(+dim) / CD25+ /CD27+ / IgM+) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenstrms clone, as well as for the differential diagnosis, risk of progression and survival in WM.


Guerra-Laso J.M.,Hospital Of Leon | Gonzalez-Garcia S.,Hospital Of Leon | Gonzalez-Cortes C.,Hospital Of Leon | Diez-Tascon C.,Hospital Of Leon | And 3 more authors.
Age | Year: 2013

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-?, but not IL-1?, IL-6 or TNF-?, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth. © American Aging Association 2012.


Perez-Persona E.,Hospital Universitario Of Salamanca | Mateo G.,Hospital Universitario Of Salamanca | Garcia-Sanz R.,Hospital Universitario Of Salamanca | Garcia-Sanz R.,University of Salamanca | And 15 more authors.
British Journal of Haematology | Year: 2010

The present study explored the impact of two novel criteria; having >95% abnormal plasma cells by flow cytometry at diagnosis and the evolving subtype of the disease, as predictors of progression in 61 smouldering multiple myeloma (SMM) and 311 monoclonal gammopathy of unknown significance (MGUS) patients. Although both criteria were of prognostic value, the risk of progression was better identified by immunophenotyping [Hazard Ratio (HR) 6·2 and 17·2 for SMM and MGUS, respectively] than evolving subtype, which had independent prognostic value only in MGUS (HR 3·6). Immunophenotyping discriminated the different risk of progression within the evolving and non-evolving subgroups of SMM (P = 0·01) and MGUS (P < 0·001). © 2009 Blackwell Publishing Ltd.


Jimenez C.,University of Salamanca | Sebastian E.,University of Salamanca | Chillon M.C.,University of Salamanca | Chillon M.C.,Instituto Biosanitario Of Salamanca Ibsal | And 25 more authors.
Leukemia | Year: 2013

We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10 -3). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27++FMC7++), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival. © 2013 Macmillan Publishers Limited. All rights reserved.


Lopez-Medrano R.,Hospital Comarcal Del Bierzo | Guerra-Laso J.M.,Complejo Asistencial Universitario Of Leon | Lopez-Fidalgo E.,Complejo Asistencial Universitario Of Leon Caule | Diez-Tascon C.,Complejo Asistencial Universitario Of Leon Caule | And 6 more authors.
Innate Immunity | Year: 2016

The whole blood model for infection has proven useful to analyze the immunological response to Mycobacterium tuberculosis, because it exerts a significant antimicrobial activity. Although this activity has been generally assumed to be cellular, we have found that the leukocyte fraction of blood from healthy volunteers did not kill the bacilli. We have discovered that plasma was responsible for a large proportion, but not all, of the antimicrobial activity. Furthermore, infected monocytes controlled the mycobacterial multiplication when cultivated in the presence of plasma. Intriguingly, serum from the same donors did not share this activity, although it was able to eliminate the non-pathogenic Mycobacterium gordonae. To identify the remaining components that participate in the antimycobacterial activity we fractionated blood in leukocytes, plasma, erythrocytes and platelets, and analyzed the bactericidal power of each fraction and their combinations using a factorial design. We found that erythrocytes, but not platelets, participated and showed by flow cytometry that mycobacteria physically associated with erythrocytes. We propose that in exposed healthy individuals that show 'early clearance' of the mycobacteria, the innate response is predominantly humoral, probably through the effect of antimicrobial peptides and proteins. © SAGE Publications.

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