ALBERT

Description: Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α–induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α–induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α–induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

Description: Leukemia often results in severe anemia, which may significantly contributes to mortality and morbidity of the patients. However, the mechanisms underlying the insufficient erythropoiesis in leukemia have been poorly understood. In this study, with our recently established non-irradiated MLL-AF9 acute myeloid leukemia (AML) murine model (Cheng H et al, Blood 2015), we observed a significant decrease in hemoglobin and red blood cells (RBCs) of Peripheral blood (PB) in the leukemic mice (n=6 per group, p=0.0122 vs p=0.0003). The absolute numbers of the erythroblasts at different stages (Pro Es, Ery.A, Ery.B, Ery.C) in bone marrow (BM) were also reduced. Consistently, by gene set enrichment analysis (GSEA) of microarray data of LKS+ cells (GSE52506 ) from leukemic mice, we found significant down-regulation of erythroid differentiation related genes such as GATA1, FOG-1, LMO2 and KLF1. These genes were more significantly inhibited in megakaryocytic-erythroid progenitors (MEPs) and Pro Es from the leukemic mice. Notably, the MEPs were the most reduced subset among all the committed hematopoietic progenitor cells (HPCs) during leukemia progression (90% decrease compared to control, p = 0.0007). MEPs were gradually accumulated in the G0 phase (from 22% to 70%, p

Description: Although results from preclinical studies in animal models have proven the concept for use of anti–cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibodies in cancer immunotherapy, 2 major obstacles have hindered their successful application for human cancer therapy. First, the lack of in vitro correlates of the antitumor effect of the antibodies makes it difficult to screen for the most efficacious antibody by in vitro analysis. Second, significant autoimmune side effects have been observed in a recent clinical trial. In order to address these 2 issues, we have generated human CTLA4 gene knock-in mice and used them to compare a panel of anti–human CTLA-4 antibodies for their ability to induce tumor rejection and autoimmunity. Surprisingly, while all antibodies induced protection against cancer and demonstrated some autoimmune side effects, the antibody that induced the strongest protection also induced the least autoimmune side effects. These results demonstrate that autoimmune disease does not quantitatively correlate with cancer immunity. Our approach may be generally applicable to the development of human therapeutic antibodies.

Description: MiRNAs are a new class of small RNAs, of 19–23 nucleotides that were discovered less than two decades ago. These tiny RNAs can negatively regulate genes at the post-transcriptional level by either triggering translational repression or direct cleavage of mRNAs. It has become evident that miRNAs are involved in hematopoiesis and that the aberrant expression of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to characterize the miRNA profile of naïve, germinal center and memory B cells sorted from tonsils and review expression of selected miRNAs in tonsils and in B cell malignancies by miRNA in situ hybridization (ISH). Quantitative (q)RT-PCR profiling revealed that several miRNAs were elevated in germinal center B cells, including miR-17–5p, miR-106a and miR-181b. miR-150 was one of the most abundant miRNAs in all subsets, but the expression level was more than 10 fold lower in germinal center B cell as compared to the other two subsets. MiRNA ISH on tonsillar tissue sections confirmed findings from the profiling work, and at the same time depicted differences in staining intensities within germinal centers. According to miRNA ISH, expression levels of miR-17-5p, miR-106a, and miR-181b were indeed higher in germinal center B cells as compared to naïve and memory B cells in the mantle zone. Surprisingly, we also observed gradual decrease of miR-17-5p, miR-106a, and miR-181b staining from dark to light zone in the germinal centers. Moreover, miRNA ISH with a probe for miR-150 demonstrated an interesting staining pattern in lymph node tissue sections. Naïve and memory B cells located in the mantle zone showed a higher miR-150 expression as compared to most of the cells in the germinal centers. However, within the germinal centers a minority of cells showed a much stronger cytoplasmic staining in part of the blasts located specifically in the dark zone. This indicated that part of the centroblasts have a high expression level of miR-150. The level of miR-150 was surprisingly low in 22 B cell lymphoma cell lines, irrespective of germinal center or non germinal center B cell origin. This seemingly negative association of miR-150 with proliferation suggests a role in B cell growth/death. We observed an inverse expression pattern of miR-150 and Survivin in the germinal centers by miRNA ISH and immunohistochemistry. Moreover, induction of miR-150 using synthetic mature miR-150 duplex resulted in reduced Survivin expression levels. Our results suggested that aside the experimentally proven target c-Myb, Survivin may also be regulated by miR-150. In conclusion, we have revealed a unique miRNA profile of naïve, germinal center and memory B cells sorted from normal tonsils and the results were confirmed by miRNA ISH. Within the germinal centers a marked difference was observed between the light zone and the dark zone.

Description: MiRNAs are small regulatory RNAs which control gene expression at the post-transcriptional level. Tumor cells of Hodgkin lymphoma (HL), the so-called Hodgkin Reed-Sternberg (HRS) cells, originate from defective germinal center B cells. HRS cells are characterized by their loss of B cell phenotype, large multi-nucleated cell size, a disturbed cell cycle and resistance to undergo apoptosis. The aim of our study is to investigate the role of deregulated miRNA expression to the pathogenesis of HL. To identify genes which are regulated by miRNAs in a high throughput manner, an approach called Ribonucleoprotein ImmunoPrecipitation – microarray (RIP-CHIP) was applied. As a first step, the AGO2 protein, which is part of the RISC complex, is immunoprecipitated (IP) with an AGO2 specific antibody. RNA isolation of the IP fraction followed by microarray analysis thus leads to the identification of miRNA targets from the whole transcriptome. With this approach, 1255 genes were found to be commonly regulated by miRNAs in both L1236 and L428 HL cell lines. Genes known to be absent or down regulated in HRS cells, like CDKN1A, CDKN1B, and PRDM1, were included in this gene list. Gene ontology analysis of these candidate miRNA target genes using Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed a significant enrichment in KEGG pathways termed p53 signaling pathway and cell cycle. TargetScan prediction and seed sequence search at the 3′UTRs of these genes both indicated that about half of these genes were targets of the highly abundant miRNAs found in Hodgkin lymphoma cell lines. 10 randomly selected target genes were analyzed using luciferase reporter assays. For all genes, targeting by the predicted miRNA was confirmed using specific antisense Locked Nucleic Acids (LNA) inhibitors. In conclusion, we demonstrated an approach for large scale identification of endogenous miRNA targets without prior manipulation of the cells. HL specific target genes that may contribute to the characteristic phenotype of HRS cells and to the malignant transformation of germinal center B cells were identified.

Description: Key Points Two SNPs in PTX3 and CLEC7a previously associated with development of proven or probable invasive aspergillosis were validated. Thirteen SNPs in 9 genes were associated at P ≤ .05 with development of IA using a different genetic model than the original study.

Description: The natural history of de novo cardiac amyloidosis is poorly described, a limitation that makes clinical decision-making difficult given the growing number of therapies for light-chain (AL)-amyloidosis (AL). We identified all patients presenting to our center from 3/99 to 8/07 at, or within 4 months of, diagnosis with symptomatic cardiac amyloidosis, and analyzed the baseline and post-treatment factors influencing survival for those with AL. During that period, 34.5% (157/455) of amyloid patients were diagnosed with de novo cardiac amyloidosis: 112 men and 45 women with a median age of 62 (31–83). Heart biopsies were obtained and were positive in 39% of men (44/112) and 31% of women (14/45). AL was diagnosed in 86% of patients (n=135) and hereditary (n=7) and senile cardiac (n=15) in the rest. Eight patients underwent heart transplant for hereditary (2 men) and AL (5 men, 1 woman). AL was diagnosed in 81% of men and 98% of women (p=0.005, chi square). We analyzed survival from diagnosis based on intention-to-treat. Ninety percent of patients with AL (n=122) received chemotherapy. Hematologic responses were scored as complete (CR), partial (PR, 〉 50% reduction) or non. Patients received IV melphalan with stem cell transplant (SCT, n=45), oral melphalan and dexamethasone (MDex, n=42) or other regimens (n = 35). Selection for SCT was based on age and extent of organ disease. Thirty-eight patients (28%) subsequently received second- and third-line therapies. The median survival for all patients was 10 months (range, 1 to 94). Baseline predictors of survival included age, gender and troponin I. Patients ≤ 60 years old had a median survival of 22 and those 〉 60 of 8 months (p=0.007). Women had a median survival of 24 and men of 8 months (p=0.02). Patients with troponin I ≤ 0.10 lived a median of 21 and those with levels 〉 0.10 of 9 months (p=0.01). Median ages at diagnosis of the 91 men and 44 women with AL were 59 (31–83) and 63 (38–82) respectively (p=0.19), and there were no differences in baseline albumin, alkaline phosphatase, CRP, brain natriuretic peptide, troponin I or clonal free light chains. There was a significant difference in serum creatinine (medians of 1.35 and 1.00 in men and women, p 〈 0.01). Overall, 60% of patients responded to chemotherapy: 55% of men and 74% of women (p=0.04, chi-square). Responders lived a median of 40 and non-responders 7 months (p

Description: MicroRNAs (miRNAs) are 19–25 nucleotide long RNA molecules derived from precursor genes that inhibit the expression of target genes by binding to their 3′ UTR region. Expression of miRNAs is often tissue specific and miRNA profiling has shown specific miRNA expression patterns in both B-cell development and lymphomagenesis. Hodgkin lymphoma is derived from pre-apoptotic germinal center B-cells, although a general loss of B cell phenotype is noted. Using quantitative RT-PCR and miRNA microarray, we determined the miRNA profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas (NHL). The two methods showed a very good correlation for the expression levels of the individual miRNAs. Using a large panel of cell lines, we confirmed differential expression between HL and other B-cell lymphoma derived cell lines for 27 miRNAs. The HL specific miRNAs included miR-155, miR-21 and miR-106b seed family members miR-17-5p, miR-20a, miR-93, miR-106a and miR- 106b. Next, we performed target gene validation of predicted target genes for miR-17-5p, which is highly expressed in HL. Using luciferase reporter assays with stabilized anti-sense miR17-5p oligonucleotides, we showed that GPR137B, RAB12 and RBJ are likely miR-17-5p target genes in two different HL cell lines. Previous publications indicated that miR-106b seed family members negatively regulate the cyclin-dependent kinase inhibitor 1A (p21/CIP1) resulting in cell cycle arrest at G1. Consistent with these findings, we show that the miR-106b family members are highly expressed in L428, whereas p21 is not. However, inhibition of the miR-106b seed family members in L428 does not result in elevated p21 protein expression. Furthermore, there is no cell cycle arrest, growth reduction or increase in cell death and apoptosis after inhibition of the miR-106b seed family members. Thus, we conclude that blocking of the miR-106b seed family members does not necessarily lead to indiction of p21 protein. This suggests an additional regulatory layer of p21 expression in L428 cells.

Description: The cancer-testis antigens (CTA) are highly immunogenic antigens expressed in various tumors but not in normal tissues (except during gametogenesis), making them an attractive target for cancer immunotherapy. Expression of CTAs such as MAGE-A3, MAGE-C1 (CT7), MAGE-C2 (CT10), NY-ESO1 and the SSX antigens has been previously reported in multiple myeloma (MM). To date, however, these reports have included a heterogeneous group of newly diagnosed and relapsed/refractory patients, all in different stages of treatment. Therefore, the extent and prognostic significance of CTA expression, and of de novo immune responses against CTA in newly-diagnosed MM patients are not known. We now report on both CTA expression and antibody responses in MM patients at diagnosis and on their prognostic significance. From 8/00-11/04, we treated 67 newly-diagnosed, symptomatic patients with a thalidomide, doxorubicin, and dexamethasone-based induction regimen. (Brit J Haematol2006;132:155). Median age was 58; 54% were ISS stage I, 28% ISS II, and 18% ISS III. Nine of 63 tested (14%) had deletion 13q by FISH, while 24% had soft tissue involvement by MM. Responses to induction therapy included 10 (15%) CR, 16 (24%) VGPR, 26 (39%) PR, 6 (8%) stable or progressive disease, and 9 (13%) inevaluable. Post-induction 54 underwent autoSCT and 9 also underwent alloSCT.. Median overall survival (OS) has not been reached with 61% alive at median follow up of 65 months. Cryopreserved pre-treatment bone marrow plasma cells were used to assess CTA expression by RT-PCR. Pre- and post-treatment sera were used to assess antibody (Ab) responses against CTA proteins by ELISA. Fifty-two patients had sufficient RNA for PCR, and 46 had baseline serum for ELISA. OS of these groups did not differ significantly from the entire cohort. At least 1 CTA was expressed in 77% of cases, including MAGE-A3 (52%), SSX1 (40%), CT7 (29%), CT10 (25%), NY-ESO1 (21%), and SSX5 (17%). Three or more CTA were expressed in 29% of cases. Individually MAGE-A3 or NY-ESO1 expression at diagnosis conferred a poorer prognosis (MAGE-A3: median OS 66 mos. vs. not reached, p=0.02 by log-rank; NY-ESO1: median OS 65 mos. vs. not reached, p=0.09). These poorer outcomes were independent of ISS stage, presence of del 13q, or response to induction therapy. No other CTA was associated with an OS difference, nor was the total number of CTA expressed prognostically significant. Baseline Ab responses, all at titers 〉 1:1600, were noted to NY-ESO1 in 6/46 (13%) patients, 5 of whom also had Ab to the NY-ESO1 homologue LAGE-1. Ab responses were also noted to CT7 (n=2), CT10 (n=1) and SSX4 (n=1). No Ab responses were noted to MAGE-A3. The effect of induction therapy on antibody titers was inconsistent, with increases, decreases, and no changes seen. Interestingly, 2 of the 6 NY-ESO1 Ab+ patients had no NY-ESO1 expression in bone marrow plasma cells. Both, however, had extensive soft tissue (ST) plasmacytomas, suggesting another source of NY-ESO1 antigen. Presence of NY-ESO1 Ab correlated significantly with baseline ST involvement, with 67% of Ab+ patients having ST disease compared with 20% of Ab− patients (p=0.05). NY-ESO1 Ab+ patients also had significantly poorer OS (med 21 mos. vs. not reached, p=0.009), independent of other prognostic factors. In sum, CTA expression is frequent in newly diagnosed MM patients, and expression of MAGE-A3 or NY-ESO1 is associated with worse long-term survival. Spontaneous antibody responses against NY-ESO1 are seen in untreated patients, and are associated with ST involvement and poorer survival. Further exploration of biologic differences between CTA+ and CTA-MM, as well as immunotherapeutic strategies which target these antigens, are warranted.

Description: Introduction: B cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen. B-CLL behaves like leukemia and is found mostly in the blood. The expression of ZAP-70 and mutation status of the immunoglobulin heavy-chain gene (IgH) can serve as prognostic markers in B-CLL. ZAP-70 positive cases usually present with unmutated IgH genes and have a bad prognosis, whereas ZAP-70 negative cases mostly present with mutated IgH genes and have good prognosis. Gene expression studies of B-CLL indicated that the profile of IgH mutated and unmutated cases are similar to normal memory B cells. Several studies showed that miRNAs play important roles in pathogenesis of B-CLL and some miRNAs correlate with the prognosis in B-CLL. B-SLL is considered to be the same disease entity as CLL, but this variant is found mostly in bone marrow and the lymphatic system. The most aggressive type of B-SLL is characterized by neoplastic cells that are more responsive to B-cell receptor signaling and are characterized by proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. Until now, only a few reports have been published about the ZAP-70 expression and IgH mutation status and no data are available about the microRNA expression profile. Methods: 33 B-SLL cases were retrieved from the pathology files. ZAP-70 expression was analyzed by using immunohistochemistry. IgH mutation status was determined using PCR followed by direct sequencing. Cases with homology of ≥98% with germline sequences were considered as unmutated and cases with homologies