ALBERT

Description: NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by calcineurin, NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. CLL is a clonal disorder of mature B cells characterized by the expression of CD19, CD23 and CD5. With respect to prognosis, it constitutes a heterogeneous disease with some patients exhibiting an indolent course for many years and others progressing rapidly and requiring early treatment. Expression of CD38 and ZAP70 define a subgroup of patients with enhanced responsiveness to stimulation of the B cell receptor (BCR) complex and more aggessive disease. In contrast, another subset of CLL patients with more indolent course is characterized by an anergic B cell phenotype refering to B cell unresponsiveness to IgM ligation and essential lack of phosphotyrosine induction and calcium flux. Here, we analyzed the role of NFAT2 in the pathogenesis of B-CLL and in anergy induction in CLL cells. For this purpose, we generated mice with a conditional NFAT2 knock out allele (NFAT2fl/fl). In order to achieve NFAT2 deletion limited to the B cell lineage, we bred NFAT2fl/fl mice to CD19-Cre mice. To investigate the role of NFAT2 in the pathogenesis of CLL we made use of the Eµ-TCL1 transgenic mouse model in which the TCL1 oncogene is expressed under the control of the Eµ enhancer. TCL1 transgenic mice develop a human-like CLL at the age of approximately 14 wks to which the animals eventually succumb at an average age of 10 months. To analyze the role of NFAT2 in CLL, we generated mice (n=10) whose B cells exhibited a specific deletion of this transcription factor in addition to their transgenic expression of the TCL1 oncogene (TCL1 CD19-Cre NFAT2fl/fl). TCL1 transgenic mice without an NFAT2 deletion served as controls (n=10). To identify novel NFAT2 target genes in CLL cells, we performed a comparative gene expression analysis on CLL cells with intact NFAT2 expression and on CLL cells with NFAT2 deletion using affymetrix microarrays. Mice with NFAT2 knock out exhibited a significantly more aggressive disease course with accelerated accumulation of CD5+CD19+ CLL cells and a significantly reduced life expectancy (200 vs. 325 days) as compared to control animals. Flow cytometric analysis at distinct time points showed a pronounced infiltration by CD5+ B cells of the peritoneal cavity, spleen, lymph nodes, liver and bone marrow which was significantly stronger in the NFAT2 ko cohort. Most of the CD5+ B cells in TCL1+NFAT2 ko mice showed high expression of ZAP70 and CD38, whereas TCL1 transgenic mice only demonstrated very few CD5+ B cells with concomitant expression of ZAP70 and CD38. To investigate the effects of an NFAT2 ko on proliferation and apoptosis of CD5+CD19+ CLL cells, we performed in vivo BrdU incorporation assays with subsequent flow cytometric analysis. Interestingly, we could show that CLL cells isolated from spleens, bone marrow and peripheral blood from mice with an NFAT ko exhibited significantly higher rates of proliferation than control animals. To identify NFAT2 target genes resonsible for the observed alterations in the disease phenotype, we subsequently peformed a gene expression analysis with CD5+CD19+ CLL cells from TCL1+NFAT2 ko mice with CLL cells from TCL1+ mice serving as controls. Here, we detected a significantly altered expression of 22 genes associated with B cell anergy in the TCL1+NFAT2 ko cohort. The vast majority of these genes was expressed significantly less in the absence of NFAT2 with Lck, Pacsin1, Hspa14 and CD166 constituting the strongest hits with up to 10fold reduced gene expression. Downregulation of the identified target genes was subsequently confirmed using RT-PCR and Western Blotting. In summary, our data provide strong evidence that NFAT2 is a critical regulator of CD38 and ZAP70 expression and substantially controls cell cycle progression in CLL cells. In addition, we could show that NFAT2 controls the expression of several anergy-associated genes and that its absence prevents the acquisition of an anergic phenotype by the CLL cells correlating with a significantly more aggressive course of the disease. Taken together, our data demonstrate that NFAT2 plays an essential role in the pathogenesis of CLL and implicate this transcription factor as a potential target in its treatment. Disclosures: No relevant conflicts of interest to declare.

Description: The Global Consortium for the Classification of Fungi and fungus-like taxa is an international initiative of more than 550 mycologists to develop an electronic structure for the classification of these organisms. The members of the Consortium originate from 55 countries/regions worldwide, from a wide range of disciplines, and include senior, mid-career and early-career mycologists and plant pathologists. The Consortium will publish a biannual update of the Outline of Fungi and funguslike taxa, to act as an international scheme for other scientists. Notes on all newly published taxa at or above the level of species will be prepared and published online on the Outline of Fungi website (https://www.outlineoffungi.org/), and these will be finally published in the biannual edition of the Outline of Fungi and fungus-like taxa. Comments on recent important taxonomic opinions on controversial topics will be included in the biannual outline. For example, ‘to promote a more stable taxonomy in Fusarium given the divergences over its generic delimitation’, or ‘are there too many genera in the Boletales?’ and even more importantly, ‘what should be done with the tremendously diverse ‘dark fungal taxa?’ There are undeniable differences in mycologists’ perceptions and opinions regarding species classification as well as the establishment of new species. Given the pluralistic nature of fungal taxonomy and its implications for species concepts and the nature of species, this consortium aims to provide a platform to better refine and stabilise fungal classification, taking into consideration views from different parties. In the future, a confidential voting system will be set up to gauge the opinions of all mycologists in the Consortium on important topics. The results of such surveys will be presented to the International Commission on the Taxonomy of Fungi (ICTF) and the Nomenclature Committee for Fungi (NCF) with opinions and percentages of votes for and against. Criticisms based on scientific evidence with regards to nomenclature, classifications, and taxonomic concepts will be welcomed, and any recommendations on specific taxonomic issues will also be encouraged; however, we will encourage professionally and ethically responsible criticisms of others’ work. This biannual ongoing project will provide an outlet for advances in various topics of fungal classification, nomenclature, and taxonomic concepts and lead to a community-agreed classification scheme for the fungi and fungus-like taxa. Interested parties should contact the lead author if they would like to be involved in future outlines.

Description: Axial backmixing is an important issue in extraction columns, since it heavily affects mass transfer performance. A miniaturized, stirred-pulsed column was investigated regarding its backmixing characteristics in the continuous phase using the system water/n-butyl acetate. Residence time distributions were determined through pulse experiments with potassium chloride, which was detected via electrical conductivity. To perform the conductivity measurement without distortion of the two-phase flow, electrodes with an annular design were manufactured, which precisely line up with the inner column wall. This design is a promising alternative for using in small-scale tubular devices, where commercial electrodes do not fit, and in applications, where interference with the flow must be avoided.

Description: Twenty-one (5.7%) of 368 cases of acute lymphoblastic leukemia (ALL), studied fully for karyotype and immunophenotype, had breakpoints in the q23 region of chromosome 11. This abnormality resulted from reciprocal translocation in 17 cases [with chromosomes 4 (n = 5), 10 (n = 2), and variable chromosomes (n = 10)], from deletions in three cases, and from a duplication in one case. The 17 children with 11q23 translocations had higher leukocyte counts (P less than .01) and were more likely to be black (P less than .01) and younger (P = .08) as compared with each of the following non-11q23 translocation groups: t(1;19), t(9;22), random translocations, and cases without translocations. Event-free survival at 3 years for the 11q23 translocation group did not differ significantly from that of the t(1;19), t(9;22), or random translocation groups. Leukemic cells from ten of the 21 patients with an 11q23 structural chromosomal abnormality had an immunophenotype indicative of B-lineage ALL (HLA-DR+, CD19+, CD2-, CD3-); this was confirmed by the presence of rearranged immunoglobulin heavy-chain genes in seven cases. In eight of these ten B-lineage cases, the blasts were negative for expression of the CD10 antigen, indicating a primitive stage of B-cell development. Four cases were classified as T- cell ALL, and seven others were characterized by blasts that failed to react with our panel of lineage-associated monoclonal antibodies (MoAbs). Myeloid antigens were expressed by leukemic cells in three of the cases that were tested. The initial clinical features associated with translocations involving the 11q23 chromosomal region may define a distinct subtype of ALL. Whether the constellation of findings relates to a breakpoint at 11q23 per se or to the specific translocation will require further study.

Description: Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL- 103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, - 105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.

Description: Twenty-one (5.7%) of 368 cases of acute lymphoblastic leukemia (ALL), studied fully for karyotype and immunophenotype, had breakpoints in the q23 region of chromosome 11. This abnormality resulted from reciprocal translocation in 17 cases [with chromosomes 4 (n = 5), 10 (n = 2), and variable chromosomes (n = 10)], from deletions in three cases, and from a duplication in one case. The 17 children with 11q23 translocations had higher leukocyte counts (P less than .01) and were more likely to be black (P less than .01) and younger (P = .08) as compared with each of the following non-11q23 translocation groups: t(1;19), t(9;22), random translocations, and cases without translocations. Event-free survival at 3 years for the 11q23 translocation group did not differ significantly from that of the t(1;19), t(9;22), or random translocation groups. Leukemic cells from ten of the 21 patients with an 11q23 structural chromosomal abnormality had an immunophenotype indicative of B-lineage ALL (HLA-DR+, CD19+, CD2-, CD3-); this was confirmed by the presence of rearranged immunoglobulin heavy-chain genes in seven cases. In eight of these ten B-lineage cases, the blasts were negative for expression of the CD10 antigen, indicating a primitive stage of B-cell development. Four cases were classified as T- cell ALL, and seven others were characterized by blasts that failed to react with our panel of lineage-associated monoclonal antibodies (MoAbs). Myeloid antigens were expressed by leukemic cells in three of the cases that were tested. The initial clinical features associated with translocations involving the 11q23 chromosomal region may define a distinct subtype of ALL. Whether the constellation of findings relates to a breakpoint at 11q23 per se or to the specific translocation will require further study.

Description: Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL- 103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, - 105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.