Meetha P. Gould

Pretreatment with Recombinant Flt3 Ligand Partially Protects against Progressive Cutaneous Leishmaniasis in Susceptible BALB/c Mice

ABSTRACT Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-γ)-producing Th1-type CD4+ T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 μg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-γ and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.

Distinct Modulatory Effects of LPS and CpG on IL-18-Dependent IFN-γ Synthesis

Innate cellular production of IFN-γ is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-γ production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-γ synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-γ in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-γ without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-γ in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-γ when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-γ production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-γ, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-γ production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.

An optimization framework for unsupervised identification of rare copy number variation from SNP array data

Copy number variants (CNVs) have roles in human disease, and DNA microarrays are important tools for identifying them. In this paper, we frame CNV identification as an objective function optimization problem. We apply our method to data from hundreds of samples, and demonstrate its ability to detect CNVs at a high level of sensitivity without sacrificing specificity. Its performance compares favorably with currently available methods and it reveals previously unreported gains and losses.

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.

In vivo and In vitro Regulation of Type I IFN Synthesis by Synergistic Effects of CD40 and Type II IFN

During cognate interaction with CD40 ligand (CD154)-expressing T cells, Ag-presenting accessory cells are activated for increased cytokine synthetic and costimulatory function. We examined whether CD40 modulates in vivo innate immune function over time, hypothesizing that distinct cytokine responses evolve to delayed microbial exposure. C3H/HeN mice pretreated with activating anti-CD40 Ab (FGK45) produced 10-fold more serum IFN-γ and IL-12 p70 to delayed, but not synchronous, challenge with LPS. A novel finding was that LPS-induced IFN-α increased by 20-fold in mice pretreated for 24 h, but not 6 h or less, with anti-CD40. Anti-CD40-pretreated C57BL/6 RAG-2−/− mice similarly increased IFN-α responses to delayed LPS challenge, confirming mediation by innate immunity. Type I IFNR- and IFN-γ-deficient mice treated with anti-CD40 failed to expand serum IFN-α responses to LPS challenge. Combined pretreatment with anti-CD40 and anti-IFN-γ mAb showed that IFN-γ produced after anti-CD40 pretreatment, but before LPS challenge, was necessary for IFN-α synthetic enhancement. Anti-CD40 also increased polyinosinic-polycytidylic acid (poly(I:C))-inducible IFN-α by 5-fold in an IFN-γ-dependent fashion, but did not significantly increase IFN-α production to CpG or Pam3Cys challenges. Poly(IC)-stimulated splenocytes from anti-CD40-pretreated mice produced 4-fold more IFN-α than controls and production associated with CD11c+ cells. Finally, rIFN-γ and anti-CD40 combined synergistically to increase poly(IC)-inducible IFN-α synthetic capacity in bone marrow dendritic cells. We conclude that innate immune production of IFN-α is cooperatively regulated by CD40 and IFN-γ acting on dendritic cells, suggesting a unique mechanism by which innate immune function evolves in response to specific adaptive immune signals.

DB2: a probabilistic approach for accurate detection of tandem duplication breakpoints using paired-end reads

BackgroundWith the advent of paired-end high throughput sequencing, it is now possible to identify various types of structural variation on a genome-wide scale. Although many methods have been proposed for structural variation detection, most do not provide precise boundaries for identified variants. In this paper, we propose a new method, Distribution Based detection of Duplication Boundaries (DB2), for accurate detection of tandem duplication breakpoints, an important class of structural variation, with high precision and recall.ResultsOur computational experiments on simulated data show that DB2 outperforms state-of-the-art methods in terms of finding breakpoints of tandem duplications, with a higher positive predictive value (precision) in calling the duplications’ presence. In particular, DB2’s prediction of tandem duplications is correct 99% of the time even for very noisy data, while narrowing down the space of possible breakpoints within a margin of 15 to 20 bps on the average. Most of the existing methods provide boundaries in ranges that extend to hundreds of bases with lower precision values. Our method is also highly robust to varying properties of the sequencing library and to the sizes of the tandem duplications, as shown by its stable precision, recall and mean boundary mismatch performance. We demonstrate our method’s efficacy using both simulated paired-end reads, and those generated from a melanoma sample and two ovarian cancer samples. Newly discovered tandem duplications are validated using PCR and Sanger sequencing.ConclusionsOur method, DB2, uses discordantly aligned reads, taking into account the distribution of fragment length to predict tandem duplications along with their breakpoints on a donor genome. The proposed method fine tunes the breakpoint calls by applying a novel probabilistic framework that incorporates the empirical fragment length distribution to score each feasible breakpoint. DB2 is implemented in Java programming language and is freely available at http://mendel.gene.cwru.edu/laframboiselab/software.php.