MicroRNAs and induced pluripotent stem cells for human disease mouse modeling.

Human disease animal models are absolutely invaluable tools for our understanding of mechanisms involved in both physiological and pathological processes. By studying various genetic abnormalities in these organisms we can get a better insight into potential candidate genes responsible for human disease development. To this point a mouse represents one of the most used and convenient species for human disease modeling. Hundreds if not thousands of inbred, congenic, and transgenic mouse models have been created and are now extensively utilized in the research labs worldwide. Importantly, pluripotent stem cells play a significant role in developing new genetically engineered mice with the desired human disease-like phenotype. Induced pluripotent stem (iPS) cells which represent reprogramming of somatic cells into pluripotent stem cells represent a significant advancement in research armament. The novel application of microRNA manipulation both in the generation of iPS cells and subsequent lineage-directed differentiation is discussed. Potential applications of induced pluripotent stem cell--a relatively new type of pluripotent stem cells--for human disease modeling by employing human iPS cells derived from normal and diseased somatic cells and iPS cells derived from mouse models of human disease may lead to uncovering of disease mechanisms and novel therapies.

Antitumor activity of type I and type III interferons in BNL hepatoma model.

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Mutation of the IFNAR-1 receptor binding site of human IFN-alpha2 generates type I IFN competitive antagonists.

Type I interferons (IFNs) are multifunctional cytokines that activate cellular responses by binding a common receptor consisting of two subunits, IFNAR-1 and IFNAR-2. Although the binding of IFNs to IFNAR-2 is well characterized, the binding to the lower affinity IFNAR-1 remains less well understood. Previous reports identified a region of human IFN-alpha2 on the B and C helices ("site 1A": N65, L80, Y85, Y89) that plays a key role in binding IFNAR-1 and contributes strongly to differential activation by various type I IFNs. The current studies demonstrate that residues on the D helix are also involved in IFNAR-1 binding. In particular, residue 120 (Arg in IFN-alpha2; Lys in IFN-alpha2/alpha1) appears to be a "hot-spot" residue: substitution by alanine significantly decreased biological activity, and the charge-reversal mutation of residue 120 to Glu caused drastic loss of antiviral and antiproliferative activity for both IFN-alpha2 and IFN-alpha2/alpha1. Mutations in residues of helix D maintained their affinity for IFNAR-2 but had decreased affinity for IFNAR-1. Single-site or multiple-site mutants in the IFNAR-1 binding site that had little or no detectable in vitro biological activity were capable of blocking in vitro antiviral and antiproliferative activity of native IFN-alpha2; i.e., they are type I IFN antagonists. These prototype IFN antagonists can be developed further for possible therapeutic use in systemic lupus erythematosus, and analogous molecules can be designed for use in animal models.

Characterization of the mouse IFN-lambda ligand-receptor system: IFN-lambdas exhibit antitumor activity against B16 melanoma.

Recently discovered type III IFNs (IFN-lambda) exert their antiviral and immunomodulatory activities through a unique receptor complex composed of IFN-lambdaR1 and interleukin-10 receptor 2. To further study type III IFNs, we cloned and characterized mouse IFN-lambda ligand-receptor system. We showed that, similar to their human orthologues, mIFN-lambda2 and mIFN-lambda3 signal through the IFN-lambda receptor complex, activate IFN stimulated gene factor 3, and are capable of inducing antiviral protection and MHC class I antigen expression in several cell types including B16 melanoma cells. We then used the murine B16 melanoma model to investigate the potential antitumor activities of IFN-lambdas. We developed B16 cells constitutively expressing murine IFN-lambda2 (B16.IFN-lambda2 cells) and evaluated their tumorigenicity in syngeneic C57BL/6 mice. Although constitutive expression of mIFN-lambda2 in melanoma cells did not affect their proliferation in vitro, the growth of B16.IFN-lambda2 cells, when injected s.c. into mice, was either retarded or completely prevented. We found that rejection of the modified tumor cells correlated with their level of IFN-lambda2 expression. We then developed IFN-lambda-resistant B16.IFN-lambda2 cells (B16.IFN-lambda2Res cells) and showed that their tumorigenicity was also highly impaired or completely abolished similar to B16.IFN-lambda2 cells, suggesting that IFN-lambdas engage host mechanisms to inhibit melanoma growth. These in vivo experiments show the antitumor activities of IFN-lambdas and suggest their strong therapeutic potential.

Chronic lymphocytic leukaemia genetics overview.

Although the familial aspect of chronic lymphocytic leukaemia (CLL) has been appreciated for decades, it is only with the recent confluence of improved molecular and gene technologies and world-wide collaborative networks that accelerated progress has become apparent. In this summary we highlight selected themes in the genetics of CLL emphasizing the opportunities and challenges of this malignancy.

Overview of monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) has been the subject of more intensive investigation for the last 10 years. The increased presence of MBL in unaffected, first-degree relatives with familial chronic lymphocytic leukaemia (CLL) suggest that it is surrogate marker for early disease. In normal population studies, MBL is found to be increased in ageing subjects. Consensus criteria for the diagnosis of MBL have been proposed. The differential diagnosis has been further clarified and the prevalence of MBL is most prominent in the elderly. The aetiology of MBL is unknown but probably involves immune mechanism of senescence or altered response. Environmental health studies suggest that exposure to certain toxins may lead to MBL but further work is needed. MBL is a precursor to CLL but may also regress, remain stable or progress to clinical CLL.

B-cell repertoire and clonal analysis in unaffected first degree relatives in familial chronic lymphocytic leukaemia kindred.

Monoclonal B cell lymphocytosis (MBL) was detected in four unaffected first-degree relatives (FDR) in a familial chronic lymphocytic leukaemia (CLL) kindred. The proband remains untreated and two male siblings have died. The four unaffected siblings have been followed for a five-year period. All four FDR developed a kappa(+)CD5(+) MBL detected by flow cytometry. Poymerase chain reaction (PCR) for IGHV rearrangement showed evidence of oligoclonality in three of these individuals. Single cell PCR of flow cytometric sorted kappa(+) cells combined with Ig kappa light chain gene sequencing revealed further evidence of monoclonality in two of these individuals. Three of these individuals all showed evidence of hyper-somatic mutations. The B-cell repertoire in unaffected FDR in familial CLL offers a new area to investigate the interface between the immune system and lymphoid neoplasm.

Murine models of chronic lymphocytic leukaemia: role of microRNA-16 in the New Zealand Black mouse model.

Mouse models are valuable tools in the study of human chronic lymphocytic leukaemia (CLL). The New Zealand Black (NZB) strain is a naturally occurring model of late-onset CLL characterized by B-cell hyperproliferation and autoimmunity early in life, followed by progression to CLL. Other genetically engineered models of CLL that have been developed include (NZB x NZW) F1 mice engineered to express IL5, mice expressing human TCL1A, and mice overexpressing both BCL2 and a tumour necrosis factor receptor-associated factor. The applicability to human CLL varies with each model, suggesting that CLL is a multifactorial disease. Our work with the de novo NZB model has revealed many similarities to the human situation, particularly familial CLL. In NZB, the malignant clones express CD5, zap-70, and have chromosomal instability and germline Ig sequence. We also identified a point mutation in the 3'-flanking sequence of Mirn16-1, which resulted in decreased levels of the microRNA, miR-16 in lymphoid tissue. Exogenous restoration of miR-16 to an NZB malignant B-1 cell line resulted in cell cycle alterations, suggesting that the altered expression of Mirn15a/16-1 is an important molecular lesion in CLL. Future studies utilizing the NZB mouse could ascertain the role of environmental triggers, such as low dose radiation and organic chemicals in the augmentation of a pre-existing propensity to develop CLL.

Alterations in the mir-15a/16-1 Loci Impairs Its Processing and Augments B-1 Expansion in De Novo Mouse Model of Chronic Lymphocytic Leukemia (CLL).

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to pre-miR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (NmiR+/-) and DBA congenic mice (DmiR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16-1 loci with DBA congenic mice (DmiR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (NmiR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (DmiR-/-) relative to wild-type (DmiR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.

Concerted action of IFN-α and IFN-λ induces local NK cell immunity and halts cancer growth.

Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. No significant improvement has been reported with currently available systemic therapies. IFN-α has been tested in both clinic and animal models and only moderate benefits have been observed. In animal models, similar modest antitumor efficacy has also been reported for IFN-λ, a new type of IFN that acts through its own receptor complex. In the present study, the antitumor efficacy of the combination of IFN-α and IFN-λ was tested in the BNL mouse hepatoma model. This study was accomplished by using either engineered tumor cells (IFN-α/IFN-λ gene therapy) or by directly injecting tumor-bearing mice with IFN-α/IFN-λ. Both approaches demonstrated that IFN-α/IFN-λ combination therapy was more efficacious than IFN monotherapy based on either IFN-α or IFN-λ. In complement to tumor surgery, IFN-α/IFN-λ combination induced complete tumor remission. Highest antitumor efficacy has been obtained following local administration of IFN-α/IFN-λ combination at the tumor site that was associated with strong NK cells tumor infiltration. This supports the use of IFN-α/IFN-λ combination as a new cancer immunotherapy for stimulating antitumor response after cancer surgery.

Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia.

In both human chronic lymphocytic leukemia (CLL) and the New Zealand Black (NZB) murine model of CLL, decreased levels of microRNAs miR-15a/16 play an important role in the disease. Here we investigate the effects of this microRNA on early steps of B cell development and the capacity of miR-15a-deficient hematopoietic stem cells (HSC) and B1 progenitor cells (B1P) to reproduce CLL-like phenotype both in vitro and in vivo. Our results demonstrate that both miR-15a deficient HSC and B1P cells are capable of repopulating irradiated recipients and produce higher numbers of B1 cells than sources with normal miR-15a/16 levels. Furthermore, induced pluripotent stem (iPS) cells derived for the first time from NZB mice, provided insights into the B cell differentiation roadblock inherent in this strain. In addition, exogenously delivered miR-15a into the NZB derived B cell line provided valuable clues into novel targets such as Mmp10 and Mt2. Our data supports the hypothesis that miR-15a/16 deficient stem cells and B1Ps experience a maturation blockage, which contributes to B1 cells bias in development. This work will help understand the role of miR-15a in early events of CLL and points to B1P cells as potential cells of origin for this incurable disease.

Agent Orange Exposure and Monoclonal Gammopathy of Undetermined Significance: An Operation Ranch Hand Veteran Cohort Study.

IMPORTANCE: Multiple myeloma has been classified as exhibiting "limited or suggestive evidence" of an association with exposure to herbicides in Vietnam War veterans. Occupational studies have shown that other pesticides (ie, insecticides, herbicides, fungicides) are associated with excess risk of multiple myeloma and its precursor state, monoclonal gammopathy of undetermined significance (MGUS); however, to our knowledge, no studies have uncovered such an association in Vietnam War veterans. OBJECTIVE: To examine the relationship between MGUS and exposure to Agent Orange, including its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in Vietnam War veterans. DESIGN, SETTING, AND PARTICIPANTS: This was a prospective cohort study conducted in 2013 to 2014, testing for MGUS in serum specimens collected and stored in 2002 by the Air Force Health Study (AFHS). The relevant exposure data collected by the AFHS was also used. We tested all specimens in 2013 without knowledge of the exposure status. The AFHS included former US Air Force personnel who participated in Operation Ranch Hand (Ranch Hand veterans) and other US Air Force personnel who had similar duties in Southeast Asia during the same time period (1962 to 1971) but were not involved in herbicide spray missions (comparison veterans). Agent Orange was used by the US Air Force personnel who conducted aerial spray missions of herbicides (Operation Ranch Hand) in Vietnam from 1962 to 1971. We included 479 Ranch Hand veterans and 479 comparison veterans who participated in the 2002 follow-up examination of AFHS. EXPOSURES: Agent Orange and TCDD. Serum TCDD levels were measured in 1987, 1992, 1997, and 2002. MAIN OUTCOMES AND MEASURES: Risk of MGUS measured by prevalence, odds ratios (ORs), and 95% CIs. RESULTS: The 479 Ranch Hand veterans and 479 comparison veterans had similar demographic and lifestyle characteristics and medical histories. The crude prevalence of overall MGUS was 7.1% (34 of 479) in Ranch Hand veterans and 3.1% (15 of 479) in comparison veterans. This translated into a 2.4-fold increased risk for MGUS in Ranch Hand veterans than comparison veterans after adjusting for age, race, BMI in 2002, and the change in BMI between 2002 and the time of blood draw for TCDD measurement (adjusted OR, 2.37; 95% CI, 1.27-4.44; P=.007). CONCLUSIONS AND RELEVANCE: Operation Ranch Hand veterans have a significantly increased risk of MGUS, supporting an association between Agent Orange exposure and multiple myeloma.

RNA interference of IL-10 in leukemic B-1 cells.

RNA interference, or RNAi, is designed to work by Watson-Crick base pairing and to result in a posttranscriptional block in protein synthesis. Antiapoptotic proteins are a major focus of cancer therapy and make attractive targets for RNAi. An IL-10 RNAi sequence was designed in accordance with Tuschl rules and was modeled to a hairpin configuration. In chronic lymphocytic leukemia (CLL), the most common leukemia in the Western world, the failure to undergo apoptosis may be responsible for the accumulation of malignant B-1 cells. Interleukin-10, despite controversy, has been shown to have antiapoptotic properties, and increased endogenous IL-10 production has been found in CLL by several labs. A malignant B-1 cell line, LNC, derived from an NZB mouse (a murine model for CLL) was utilized as a target for IL-10 RNAi. Our earlier studies of antisense IL-10 resulted in antiproliferative and proapoptotic effects. The cytotoxic effects of IL-10 RNAi were dose- and time-dependent, with an optimal dose 10-fold lower than that of antisense IL-10. IL-10 RNAi lowered IL-10 protein as measured by ELISA. 2 micro M IL-10 RNAi initiated a G2/M block and a decrease in the message for cdc25C, the M-phase inducer phosphatase. IL-10 RNAi efficiently induced apoptosis. Bcl7C, a member of the antiapoptotic Bcl family, was significantly down-regulated. IL-10 modulating Bcl7C expression represents a novel mechanism in the evasion of apoptosis. This approach, by itself or in conjunction with current therapies, merits consideration in similar B-cell malignancies.

Investigation of promoter polymorphisms in the tumor necrosis factor-alpha and interleukin-10 genes in liver transplant patients.

BACKGROUND: Cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 play significant roles in the inflammatory and immune responses that mediate allograft rejection. The presence of a G-->A polymorphism at position -308 in the promoter region of the TNF-alpha gene increased its transcription 6- to 7-fold. A similar polymorphism at position -1082 of the IL-10 promoter results in decreased production of IL-10 protein. In this study we have determined whether the single nucleotide polymorphisms in the promoter regions of the TNF-alpha and IL-10 genes can predict the outcome of the allograft in liver recipients. METHODS: DNA was extracted from whole blood of liver recipients. The genotype of the patients was determined by polymerase chain reaction using sequence-specific primers. The level of TNF-alpha and IL-10 protein was measured by ELISA after stimulation of peripheral blood mononuclear cells with concanavalin A. RESULTS: There was significant correlation between acute cellular rejection and the presence of the -308A polymorphism (P<0.001), with 8 of 13 patients with the TNF-alpha polymorphism having evidence of acute rejection. Cell stimulation studies revealed that the level of TNF-alpha protein produced by patients with liver rejection was significantly higher than for patients without rejection (P=0.001). There were no strong associations between the presence of the IL-10 polymorphisms and rejection (P=0.71). CONCLUSIONS: This study adds to the understanding of the role of cytokine polymorphisms in liver transplants. The data suggest that cytokine promoter polymorphisms may be a risk factor associated with allograft rejection in the liver.

Reversible mitochondrial DNA accumulation in nuclei of pluripotent stem cells.

According to the endosymbiotic hypothesis, the precursor of mitochondria invaded the precursor of eukaryotic cells, a process that began roughly 2 billion years ago. Since then, the majority of the genetic material translocated from the mitochondria to the nucleus, where now almost all mitochondrial proteins are expressed. Only a tiny amount of DNA remained in the mitochondria, known as mitochondrial DNA (mtDNA). In this study, we report that the transfer of mtDNA fragments to the nucleus of pluripotent stem cells is still ongoing. We show by in situ hybridization and agarose DNA two-dimensional gel technique that induced pluripotent stem (iPS) cells contain high levels of mtDNA in the nucleus. We found that a large proportion of the accumulated mtDNA sequences appear to be extrachromosomal. Accumulation of mtDNA in the nucleus is present not only in the iPS cells, but also in embryonic stem (ES) cells. However upon differentiation, the level of mtDNA in the nuclei of iPS and ES cells is substantially reduced. This reversible accumulation of mtDNA in the nucleus supports the notion that the nuclear copy number of mtDNA sequences may provide a novel mechanism by which chromosomal DNA is dynamically regulated in pluripotent stem cells.

MicroRNAs in Acute Myeloid Leukemia and Other Blood Disorders.

Common blood disorders include hematopoietic cell malignancies or leukemias and plasma cell dyscrasia, all of which have associated microRNA abnormalities. In this paper, we discuss several leukemias including acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) and identify altered microRNAs and their targets. Immune disorders with altered blood levels of antibodies include autoimmune disorders, such as systemic lupus erythematosus (SLE) with associated anti-self-autoantibodies and immunoglobulin A nephropathy (IgAN) also have related microRNA abnormalities. The alterations in microRNAs may serve as therapeutic targets in these blood disorders.

Role of microRNA-15a in autoantibody production in interferon-augmented murine model of lupus.

MicroRNAs (miRNAs) are involved in the regulation of immunity via targeting of mRNA encoding immune response elements. In this report, alterations in the expression of microRNAs as autoantibody levels increase was investigated. The (NZB×NZW)F1 or B/W mouse model of systemic lupus erythematosus (SLE) naturally has increased autoantibodies with aging. IFNα (type I IFN) accelerates B/W disease, however, the effects of a related IFN, IFNλ, which is a type III IFN, is largely unknown. The purpose of the study was to investigate the relationship between IFN-accelerated disease, microRNAs, immunoregulatory B cell subsets and autoantibody production in the autoimmune-prone environment in vivo. B/W mice received osmotic pumps to chronically deliver IFNα and IFNλ for up to 16 weeks. Urine protein level was monitored weekly by urine strips and proteinuria was used as the disease marker. Splenic cells were taken for flow analysis of B cell subsets and levels of microRNAs determined. Plasma were analyzed for autoantibodies and microRNA levels. As a result of treatment, IFNα accelerated proteinuria in a dose dependent manner, while IFNλ single treatment did not show a significant effect, but greatly enhanced low dose IFNα effects in the combination treatment. Both the splenic cellular and plasma miR-15a were elevated in diseased compared to pre-diseased mice as well as autoantibody levels. Autoantibodies and miR-15a levels were significantly correlated. The immunosuppressive B subpopulation, B-10, was reduced following IFNα treatment. In addition in diseased mice, B-10 versus B-2 ratios were reduced in IFN-treated B/W compared to the control PBS treated group. In all B/W the miR-15a was highly expressed in the B-10 subset and this increased with disease development, suggesting that miR-15a has a specific negative effect on the B-10 subpopulation. In conclusion, our data support the involvement of elevated miR-15a in autoimmune disease development in B/W mice and suggest that decreasing this microRNA might be beneficial in B/W mice.

The New Zealand black mouse as a model for the development and progression of chronic lymphocytic leukemia.

BACKGROUND: Similar to a subset of human patients who progress from monoclonal B lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL), New Zealand Black (NZB) mice have an age-associated progression to CLL. The murine disease is linked to a genetic abnormality in microRNA mir-15a/16-1 locus, resulting in decreased mature miR-15a/16. METHODS: Spleens of aging NZB were analyzed for the presence of B-1 cells via flow cytometry and for the presence of a side population (SP) via the ability of cells to exclude Hoechst 33342 dye. The SP was assayed for the presence of hyperdiploid B-1 clones and for the ability to differentiate into B-1 cells in vitro and transfer disease in vivo. In addition, enhanced apoptosis of chemoresistant NZB B-1 cells was examined by restoring miR-16 levels in nutlin-treated cells. RESULTS: Aging NZB mice develop a B-1 expansion and clonal development that evolves from MBL into CLL. An expansion in SP is also seen. Although the SP did contain increased cells with stem cell markers, they lacked malignant B-1 cells and did not transfer disease in vivo. Similar to B-1 cells, splenic NZB SP also has decreased miR-15a/16 when compared with C57Bl/6. Exogenous addition of miR-15a/16 to NZB B-1 cells resulted in increased sensitivity to nutlin. CONCLUSION: NZB serve as an excellent model for studying the development and progression of age-associated CLL. NZB SP cells do not seem to contain cancer stem cells, but rather the B-1 stem cell. NZB B-1 chemoresistance may be related to reduced miR-15a/16 expression.

Prevalence of monoclonal B-cell lymphocytosis: a systematic review.

BACKGROUND: Individuals with monoclonal B-cell lymphocytosis (MBL) have been identified in clinic outpatients, in unaffected relatives of patients with chronic lymphocytic leukemia (CLL), and in general populations. MBL and its relationship with CLL have been actively investigated over the last decade. This report systematically reviews the prevalence of MBL in the context of the populations studied and the evolution of laboratory methods used to define MBL. METHODS: To identify published studies that have assessed the prevalence of MBL, we systematically searched the MEDLINE databases and consulted with members of the International MBL Study Group. We reviewed the 10 articles that were identified by this process. We abstracted information on study populations, laboratory tests, criteria for designating MBL, and the reported frequencies. RESULTS: Three of the ten studies were published in 2009, three between 2007 and 2008, and four between 2002 and 2004. Reported prevalences varied widely, ranging from 0.12 to 18.2%. This variability was clearly associated with both the laboratory methods and the populations studied. MBL was more common among older individuals and kindred of persons with CLL. The most common MBL subtype was CLL-like MBL. CONCLUSIONS: Large population-based studies of MBL that employ standardized laboratory methods with a consensus case definition are needed to assess prevalence and establish risk factors. These studies should include prospective follow-up of MBL cases to determine the relationship between MBL and CLL. Data from original studies should be reported in sufficient detail to allow future synthesis of information from multiple studies, such as meta-analysis.

Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity.

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G(1) in non-New Zealand Black B cell and New Zealand Black-derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G(1) accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G(1)/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3' untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease.