AID overexpression leads to aggressive murine CLL and nonimmunoglobulin mutations that mirror human neoplasms.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eµ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Somatic hypermutation profiles in stereotyped IGHV4-34 receptors from South American chronic lymphocytic leukemia patients.

Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

Characterization, identification and virulence of Metarhizium species from Cuba to control the sweet potato weevil, Cylas formicarius Fabricius (Coleoptera: Brentidae).

AIMS: Entomopathogenic Metarhizium fungi are widely recognized for their biological control potential. In Cuba, several fungus-based bio-insecticides have been developed and are produced as part of integrated pest management (IPM) programmes for economically relevant agricultural pests. Screening of fungal isolates from the INISAV strain collection was used for the development of bio-insecticides against important pest insects as, for example the sweet potato weevil, Cylas formicarius. METHODS AND RESULTS: Six fungal isolates from Cuba were microscopically, morphologically and molecular-taxonomically characterized using marker sequences ef1a, rpb1 and rpb2, and the 5TEF region of the ef1a gene. Five isolates were assigned to the species Metarhizium anisopliae sensu stricto and one isolate to Metarhizium robertsii. The pathogenic potential was evaluated against adults of C. formicarius, and growth and conidial production on different nutritional media were determined. Metarhizium anisopliae strain LBM-267 displayed pronounced virulence against the sweet potato weevil and abundant conidia production on several culture media. CONCLUSIONS: Entomopathogenic fungal isolates from Cuba were assigned to the taxonomic species M. anisopliae sensu stricto and M. robertsii. Virulence assessment with respect to C. formicarius led to the identification of two M. anisopliae isolates holding biocontrol potential. Isolate LBM-11 has previously been developed into the bio-insecticide METASAVE-11 that is widely used to control several species of plant pathogenic weevils, Lepidoptera and thrips in Cuba. Isolate LBM-267 has not been employed previously but is as virulent against C. formicarius as LBM-11; its growth and conidial production capacities on different nutritional media will likely facilitate economically feasible bio-insecticide development. SIGNIFICANCE AND IMPACT OF THE STUDY: Metarhizium anisopliae isolate LBM-267 has been selected as a promising candidate for biocontrol of the sweet potato weevil, an economically important agricultural pest in Cuba, and for further R&D activities within the framework of the Biological Control Program of Cuba.

A method of treatment for nonunion after fractures using mesenchymal stromal cells loaded on collagen microspheres and incorporated into platelet-rich plasma clots.

PURPOSE: There is evidence showing that mesenchymal stromal cells (MSC) may constitute a potential therapeutic strategy to induce bone regeneration. In this work, we investigate the capacity of autologous bone marrow (BM) MSC loaded on collagen microspheres (CM) and included into autologous platelet-rich plasma (PRP) clots (MSC/CM/PRP) to induce bone formation in patients with nonunion lesions. METHODS: MSC were isolated from BM cells of patients with nonunion lesions. Phenotypical (marker expression) and functional studies (osteogenic differentiation) were performed. MSC were seeded on CM and included into autologous PRP clot (MSC/CM/PRP). The capacity of MSC/CM/PRP to induce bone formation was evaluated in three patients diagnosed with nonunion. RESULTS: MSC loaded on CM/PRP clots maintain their biological functions, in vitro. After three months, post-MSC transplantation, all patients showed evidence of osteogenesis at the site of nonunion. After one year, all patients showed a complete healing of the nonunion. CONCLUSIONS: Our results support the use of autologous MSC transplanted as MSC/CM/PRP for the treatment of nonunion fractures. Future studies incorporating a larger number of patients may confirm the results obtained in this work.

Bone marrow stromal mesenchymal cells induce down regulation of CD20 expression on B-CLL: implications for rituximab resistance in CLL.

Although the majority of B cells express surface CD20 in chronic lymphocytic leukaemia (B-CLL), only ∼50% of patients respond to treatment with rituximab. Decreased CD20 expression on these tumour B cells could be responsible for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. At the bone marrow level, mesenchymal stromal cells (MSC) may regulate and support the survival of malignant cells, such as B-CLL cells. In this study, we investigated whether MSC may regulate the CD20 expression on B-CLL. For this purpose, B cells from CLL patients were isolated and co-cultured on MSC. B-CLL cells were collected from B-CLL/MSC co-cultures and examined for their expression of CD20. We demonstrate decreased CD20 expression in B-CLL cells after 2 weeks of co-culture with MSC, under contact and non-contact conditions, which was associated with a decreased susceptibility to rituximab. Additionally, B cells co-cultured with MSCs show an increase in CD59 expression. Our findings strongly suggest that the interaction between B-CLL cells and MSC may play a major role in the resistance to rituximab-induced apoptosis of B-CLL cells.

Expression pattern of protease activated receptors in lymphoid cells.

Protease-activated receptors (PARs) are a subfamily of four G-protein-coupled receptors mediating multiple functions. PARs expression was studied in subpopulations of human lymphocytes. Our results indicate that natural killer cells expressed mRNA for PAR1, PAR2 and PAR3, CD4+ T cells expressed PAR1 and PAR2, while γδ and CD8+ T cells only expressed PAR1. PAR4 was absent at mRNA level and B cells did not express any PAR. Analyses of the cell surface PARs expression by flow cytometry were consistent with the mRNA data and also between different donors. PAR1 is the most abundant member of the PAR family present in lymphocytes.

HPLC-ESI-IT-MS/MS analysis and biological activity of triterpene glycosides from the Colombian marine sponge Ectyoplasia ferox.

The marine sponge Ectyoplasia ferox produces antipredatory and allelopathic triterpenoid glycosides as part of its chemical defense repertoire against predators, competitors, and fouling organisms. These molecules are responsible for the pharmacological potential found in the glycosides present in this species. In order to observe the glycochemical diversity present in E. ferox, a liquid chromatography coupled to a tandem mass spectrometry approach to analyse a complex polar fraction of this marine sponge was performed. This gave valuable information for about twenty-five compounds three of which have been previously reported and another three which were found to be composed of known aglycones. Furthermore, a group of four urabosides, sharing two uncommon substitutions with carboxyl groups at C-4 on the terpenoid core, were identified by a characteristic fragmentation pattern. The oxidized aglycones present in this group of saponins can promote instability, making the purification process difficult. Cytotoxicity, cell cycle modulation, a cell cloning efficiency assay, as well as its hemolytic activity were evaluated. The cytotoxic activity was about IC50 40 µg/mL on Jurkat and CHO-k1 cell lines without exhibiting hemolysis. Discussion on this bioactivity suggests the scanning of other biological models would be worthwhile.

Ulososides and urabosides--triterpenoid saponins from the Caribbean marine sponge Ectyoplasia ferox.

Three new triterpene glycosides, named ulososide F (1), urabosides A (2) and B (3), together with the previously reported ulososide A (4), were isolated from the Caribbean marine sponge Ectyoplasia ferox. Their structures were elucidated using extensive interpretation of 1D and 2D-NMR data, as well as HRESIMS. The aglycon of all compounds is a rare 30-norlonastane and the sugar residues were identified after acid hydrolysis and GC analyses. Cytotoxicities of the isolated compounds were evaluated against Jurkat and CHO cell lines by a MTT in vitro assay as well as a hemolysis assay. Unexpectedly, all these saponin derivatives showed very low activity in our bioassays.

TGF-ß/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression.

Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-ß/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21-Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-ß modulation and proposes an alternative strategy to explore in CLL therapy.

CD16 cross-linking induces increased expression of CD56 and production of IL-12 in peripheral NK cells.

Human NK cells are classified into two populations according to the intensity of CD56 surface expression, as well as possession of CD16, FcRIII. CD56(dim)CD16(bright) make up 90% circulating NK cells, whereas CD56(bright)CD16(-/dim) comprises the remaining 10%. Here we report that peripheral NK cells upon CD16 cross-linking up-regulates the expression of activating markers and receptors such as CD25, CD69, NKp44, NKp30, CD40L and the intensity of CD56 expression. Additionally, co-culturing immature DCs with CD16 activated NK cells was found to significantly increase the expression of maturation markers on DCs. These results suggest that CD16 cross-linking on resting peripheral blood NK cells triggers the activation of these cells and induces the appearance of CD56(bright) NK cells. The latter were found capable of producing pro-inflammatory cytokines, IFN-gamma and TNF-alpha and notably IL-12.

Centered reduced moments and associate density functions applied to alkaline comet assay.

The single cell gel electrophoresis assay is a sensitive, rapid, and visual technique for deoxyribonucleic acid (DNA) strand-break detection in individual mammalian cells, whose application has significantly increased in the past few years. The cells are embedded in agarose on glass slides followed by lyses of the cell membrane. Thereafter, damaged DNA strands are electrophoresed away from the nucleus towards the anode giving the appearance of a comet tail. Nowadays, charge coupled device cameras are attached at optical microscopes for recording the images of the cells, and digital image processing is applied for obtaining quantitative descriptors. However, the conventional software is usually expensive, inflexible and, in many cases, can only provide low-order descriptors based in image segmentation, determination of centers of mass, and Euclidean distances. Associated density functions and centered reduced moments offer an effective and flexible alternative for quantitative analysis of the comet cells. We will show how the position of the center of mass, the lengths and orientation of the main semiaxes, and the eccentricity of such images can be accurately determined by this method.

Unexpectedly high frequency of European parentage in Venezuelan patients with chronic lymphocytic leukemia.

There is insufficient information on the characteristics of chronic lymphocytic leukemia (CLL) in Latin American patients. Immunoglobulin variable-region heavy-chain (IGVH) gene usage and mutation status and prognostic factors were investigated in patients resident in Venezuela. The most frequently used IGVH family genes were: VH3 > VH1 > VH4 > VH5, with a high incidence of IGVH1.69 and IGVH3.21 genes, and 55.2% of IGVH genes were mutated. Analysis of HCDR3 (third complementarity-determining region of the heavy chain) revealed that 24% of Venezuelan HCDR3s belonged to a CLL stereotyped HCDR3. Results for prognostic factors were similar to those reported previously for Caucasian populations. Interestingly, we found an over-representation of people of European extraction among Venezuelan patients with CLL, suggesting the possibility of a higher frequency of susceptibility genes for CLL in Europeans in comparison with Latin American mestizos.

Different competitive capacities of Stat4- and Stat6-deficient CD4+ T cells during lymphophenia-driven proliferation.

The outcome of an immune response relies on the competitive capacities acquired through differentiation of CD4(+) T cells into Th1 or Th2 effector cells. Because Stat4 and Stat6 proteins are implicated in the Th1 vs Th2 generation and maintenance, respectively, we compare in this study the kinetics of Stat4(-/-) and Stat6(-/-) CD4(+) T cells during competitive bone marrow reconstitution and lymphopenia-driven proliferation. After bone marrow transplantation, both populations reconstitute the peripheral T cell pools equally well. After transfer into lymphopenic hosts, wild-type and Stat6(-/-) CD4(+) T cells show a proliferation advantage, which is early associated with the expression of an active phospho-Stat4 and the down-regulation of Stat6. Despite these differences, Stat4- and Stat6-deficient T cells reach similar steady state numbers. However, when both Stat4(-/-) and Stat6(-/-) CD4(+) T cells are coinjected into the same hosts, the Stat6(-/-) cells become dominant and out-compete Stat4(-/-) cells. These findings suggest that cell activation, through the Stat4 pathway and the down-regulation of Stat6, confers to pro-Th1 T cells a slight proliferation advantage that in a competitive situation has major late repercussions, because it modifies the final homeostatic equilibrium of the populations and favors the establishment of Th1 CD4(+) T cell dominance.

CD8 T cell sensory adaptation dependent on TCR avidity for self-antigens.

Adaptation of the T cell activation threshold may be one mechanism to control autoreactivity. To investigate its occurrence in vivo, we engineered a transgenic mouse model with increased TCR-dependent excitability by expressing a Zap70 gain-of-function mutant (ZAP-YEEI) in postselection CD8 thymocytes and T cells. Increased basal phosphorylation of the Zap70 substrate linker for activation of T cells was detected in ZAP-YEEI-bearing CD8 T cells. However, these cells were not activated, but had reduced levels of TCR and CD5. Moreover, they produced lower cytokine amounts and showed faster dephosphorylation of linker for activation of T cells and ERK upon activation. Normal TCR levels and cytokine production were restored by culturing cells in the absence of TCR/spMHC interaction, demonstrating dynamic tuning of peripheral T cell responses. The effect of avidity for self-ligand(s) on this sensory adaptation was studied by expressing ZAP-YEEI in P14 or HY TCR transgenic backgrounds. Unexpectedly, double-transgenic animals expressed ZAP-YEEI prematurely in double-positive thymocytes, but no overt alteration of selection processes was observed. Instead, modifications of TCR and CD5 expression due to ZAP-YEEI suggested that signal tuning occurred during thymic maturation. Importantly, although P14 x ZAP-YEEI peripheral CD8 T cells were reduced in number and showed lower Ag-induced cytokine production and limited lymphopenia-driven proliferation, the peripheral survival/expansion and Ag responsiveness of HY x ZAP-YEEI cells were enhanced. Our data provide support for central and peripheral sensory T cell adaptation induced as a function of TCR avidity for self-ligands and signaling level. This may contribute to buffer excessive autoreactivity while optimizing TCR repertoire usage.