IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Stem cell factor is implicated in microenvironmental interactions and cellular dynamics of chronic lymphocytic leukemia.

The inflammatory cytokine stem cell factor (SCF, ligand of c-kit receptor) has been implicated as a pro-oncogenic driver and an adverse prognosticator in several human cancers. Increased SCF levels have recently been reported in a small series of patients with chronic lymphocytic leukemia (CLL), however its precise role in CLL pathophysiology remains elusive. In this study, CLL cells were found to express predominantly the membrane isoform of SCF, which is known to elicit a more robust activation of the c-kit receptor. SCF was significantly overexpressed in CLL cells compared to healthy tonsillar B cells and it correlated with adverse prognostic biomarkers, shorter time-to-first treatment and shorter overall survival. Activation of immune receptors and long-term cell-cell interactions with the mesenchymal stroma led to an elevation of SCF primarily in CLL cases with an adverse prognosis. Contrariwise, suppression of oxidative stress and the BTK inhibitor ibrutinib lowered SCF levels. Interestingly, SCF significantly correlated with mitochondrial dynamics and hypoxia-inducible factor-1a which have previously been linked with clinical aggressiveness in CLL. SCF was able to elicit direct biological effects in CLL cells, affecting redox homeostasis and cell proliferation. Overall, the aberrantly expressed SCF in CLL cells emerges as a key response regulator to microenvironmental stimuli while correlating with poor prognosis. On these grounds, specific targeting of this inflammatory molecule could serve as a novel therapeutic approach in CLL.

The Effects of Photosensitizing Dyes Fagopyrin and Hypericin on Planktonic Growth and Multicellular Life in Budding Yeast.

Naphthodianthrones such as fagopyrin and hypericin found mainly in buckwheat (Fagopyrum spp.) and St. John's wort (SJW) (Hypericum perforatum L.) are natural photosensitizers inside the cell. The effect of photosensitizers was studied under dark conditions on growth, morphogenesis and induction of death in Saccharomyces cerevisiae. Fagopyrin and hypericin induced a biphasic and triphasic dose response in cellular growth, respectively, over a 10-fold concentration change. In fagopyrin-treated cells, disruptions in the normal cell cycle progression were evident by microscopy. DAPI staining revealed several cells that underwent premature mitosis without budding, a striking morphological abnormality. Flow Cytometric (FC) analysis using a concentration of 100 µM showed reduced cell viability by 41% in fagopyrin-treated cells and by 15% in hypericin-treated cells. FC revealed the development of a secondary population of G1 cells in photosensitizer-treated cultures characterized by small size and dense structures. Further, we show that fagopyrin and the closely related hypericin altered the shape and the associated fluorescence of biofilm-like structures. Colonies grown on solid medium containing photosensitizer had restricted growth, while cell-to-cell adherence within the colony was also affected. In conclusion, the photosensitizers under dark conditions affected culture growth, caused toxicity, and disrupted multicellular growth, albeit with different efficiencies.

Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production.

BACKGROUND: Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system. RESULTS: Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation. CONCLUSION: Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.

Hematopoietic stem cell mobilization for gene therapy of adult patients with severe ß-thalassemia: results of clinical trials using G-CSF or plerixafor in splenectomized and nonsplenectomized subjects.

The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

The Calcitriol/Vitamin D Receptor System Regulates Key Immune Signaling Pathways in Chronic Lymphocytic Leukemia.

It has been proposed that vitamin D may play a role in prevention and treatment of cancer while epidemiological studies have linked vitamin D insufficiency to adverse disease outcomes in various B cell malignancies, including chronic lymphocytic leukemia (CLL). In this study, we sought to obtain deeper biological insight into the role of vitamin D and its receptor (VDR) in the pathophysiology of CLL. To this end, we performed expression analysis of the vitamin D pathway molecules; complemented by RNA-Sequencing analysis in primary CLL cells that were treated in vitro with calcitriol, the biologically active form of vitamin D. In addition, we examined calcitriol effects ex vivo in CLL cells cultured in the presence of microenvironmental signals, namely anti-IgM/CD40L, or co-cultured with the supportive HS-5 cells; and, CLL cells from patients under ibrutinib treatment. Our study reports that the calcitriol/VDR system is functional in CLL regulating signaling pathways critical for cell survival and proliferation, including the TLR and PI3K/AKT pathways. Moreover, calcitriol action is likely independent of the microenvironmental signals in CLL, since it was not significantly affected when combined with anti-IgM/CD40L or in the context of the co-culture system. This finding was also supported by our finding of preserved calcitriol signaling capacity in CLL patients under ibrutinib treatment. Overall, our results indicate a relevant biological role for vitamin D in CLL pathophysiology and allude to the potential clinical utility of vitamin D supplementation in patients with CLL.

Novel mutation of the PRNP gene of a clinical CJD case.

BACKGROUND: Transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases, are thought to be caused by an abnormal isoform of a naturally occurring protein known as cellular prion protein, PrPC. The abnormal form of prion protein, PrPSc accumulates in the brain of affected individuals. Both isoforms are encoded by the same prion protein gene (PRNP), and the structural changes occur post-translationally. Certain mutations in the PRNP gene result in genetic TSEs or increased susceptibility to TSEs. CASE PRESENTATION: A 70 year old woman was admitted to the hospital with severe confusion and inability to walk. Relatives recognized memory loss, gait and behavioral disturbances over a six month period prior to hospitalization. Neurological examination revealed Creutzfeldt-Jakob disease (CJD) related symptoms such as incontinence, Babinski sign and myoclonus. EEG showed periodic sharp waves typical of sporadic CJD and cerebrospinal fluid analysis (CSF) was positive for the presence of the 14-3-3-protein. As the disease progressed the patient developed akinetic mutism and died in the tenth month after onset of the disease symptoms. Unfortunately, no autopsy material was available. PRNP sequencing showed the occurrence of a point mutation on one allele at codon 193, which is altered from ACC, coding for a threonine, to ATC, encoding an isoleucine (T193I). CONCLUSION: Here we report a novel mutation of the PRNP gene found in an elderly female patient resulting in heterozygosity for isoleucine and threonine at codon 193, in which normally homozygosity for threonine is expected (T193). The patient presented typical clinical symptoms of CJD. EEG findings and the presence of the 14-3-3 protein in the CSF, contributed to CJD diagnosis, allowing the classification of this case as a probable CJD according to the World Health Organization (WHO) accepted criteria.

The histone methyltransferase EZH2 as a novel prosurvival factor in clinically aggressive chronic lymphocytic leukemia.

The histone methyltransferase EZH2 induces gene repression through trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 overexpression has been reported in many types of cancer and associated with poor prognosis. Here we investigated the expression and functionality of EZH2 in chronic lymphocytic leukemia (CLL). Aggressive cases with unmutated IGHV genes (U-CLL) displayed significantly higher EZH2 expression compared to indolent CLL cases with mutated IGHV genes (M-CLL); furthermore, in U-CLL EZH2 expression was upregulated with disease progression. Within U-CLL, EZH2high cases harbored significantly fewer (p = 0.033) TP53 gene abnormalities compared to EZH2low cases. EZH2high cases displayed high H3K27me3 levels and increased viability suggesting that EZH2 is functional and likely confers a survival advantage to CLL cells. This argument was further supported by siRNA-mediated downmodulation of EZH2 which resulted in increased apoptosis. Notably, at the intraclonal level, cell proliferation was significantly associated with EZH2 expression. Treatment of primary CLL cells with EZH2 inhibitors induced downregulation of H3K27me3 levels leading to increased cell apoptosis. In conclusion, EZH2 is overexpressed in adverse-prognosis CLL and associated with increased cell survival and proliferation. Pharmacologic inhibition of EZH2 catalytic activity promotes apoptosis, highlighting EZH2 as a novel potential therapeutic target for specific subgroups of patients with CLL.

Characterization of in vitro expanded bone marrow-derived mesenchymal stem cells isolated from experimental autoimmune encephalomyelitis mice.

Extensive experimental studies indicate that autologous bone marrow mesenchymal stem cells (BMSCs) are able to ameliorate experimental autoimmune encephalomyelitis (EAE) and potentially multiple sclerosis. However, the impact that the inflammatory environment present in EAE may have on the biological properties of BMSCs expanded in vitro for transplantation is yet to be clarified. It was investigated whether BMSCs isolated from EAE-induced C57bl6/J mice and expanded in vitro preserve the properties of BMSCs isolated from healthy donors (BMSCs-control). The mesenchymal origin, the differentiation potential, and the transcriptional expression profile of six histone-modifying genes were studied in both groups of BMSCs. BMSCs-EAE exhibited distinct morphology and larger size compared to BMSCs-control, higher degree of proliferation and apoptosis, differences in the adipogenesis and the osteogenesis induction, and differential expression of stromal markers and markers of progenitor and mature neuronal/glial cells. Moreover, BMSCs-EAE exhibited different expression patterns on a number of histone-modifying genes compared to controls. We recorded manifold differences, both phenotypical and functional, of in vitro expanded BMSCs-EAE in comparison to their healthy donor-derived counterparts that may be attributed to the inflammatory environment they originated from. Whether our findings may be of any clinical relevance needs to be clarified in future studies, in vivo.

Variable behavior and complications of autologous bone marrow mesenchymal stem cells transplanted in experimental autoimmune encephalomyelitis.

Autologous bone marrow stromal cells (BMSCs) offer significant practical advantages for potential clinical applications in multiple sclerosis (MS). Based on recent experimental data, a number of clinical trials have been designed for the intravenous (IV) and/or intrathecal (ITH) administration of BMSCs in MS patients. Delivery of BMSCs in the cerebrospinal fluid via intracerebroventricular (ICV) transplantation is a useful tool to identify mechanisms underlying the migration and function of these cells. In the current study, BMSCs were ICV administered in severe and mild EAE, as well as naive animals; neural precursor cells (NPCs) served as cellular controls. Our data indicated that ICV-transplanted BMSCs significantly ameliorated mild though not severe EAE. Moreover, BMSCs exerted significant anti-inflammatory effect on spinal cord with concomitant reduced axonopathy only in the mild EAE model. BMSCs migrated into the brain parenchyma and, depending on their cellular density, within brain parenchyma formed cellular masses characterized by focal inflammation, demyelination, axonal loss and increased collagen-fibronectin deposition. These masses were present in 64% of ICV BMASC-transplanted severe EAE animals whereas neither BMSCs transplanted in mild EAE cases nor the NPCs exhibited similar behavior. BMSCs possibly exerted their fibrogenic effect via both paracrine and autocrine manner, at least partly due to up-regulation of connective tissue growth factor (CTGF) under the trigger of TGFb1. Our findings are of substantial relevance for clinical trials in MS, particularly regarding the possibility that ICV transplanted BMSCs entering the inflamed central nervous system may exhibit - under conditions - a local pathology of yet unknown consequences.

Titanium dioxide photocatalytic inactivation of prions.

Prions are postulated to be the infectious agents of a family of transmissible, fatal, neurodegenerative disorders affecting both humans and animals. The possibility of prion transmission constitutes a public-health risk that confronts regulatory authorities everywhere. The main problem in handling prions is the fact that they are extremely resistant to standard decontamination methods. Thus, the use of harsh and expensive practices to destroy prions is inevitable. The development of applicable and efficient prion-inactivation practices is still highly important for the prevention of accidental transmission. In the search for effective and environmentally friendly methods to eliminate organic compounds and bacteria, much attention has been focused on the so-called advanced oxidation processes. These are based on the formation of hydroxyl radicals, which are known to possess a high reductive potential. This study tested the potential of titanium dioxide, an inexpensive and completely inert reagent, to inactivate prions in a heterogeneous photocatalytic process. Initial in vitro experiments were followed by a bioassay with the scrapie strain 263K in Syrian hamsters. The results obtained from this study indicate that titanium dioxide photocatalytic treatment of scrapie-infected brain homogenates reduces infectivity titres significantly.