Mark Daniels

Novel APC-like properties of human NK cells directly regulate T cell activation

Initiation of the adaptive immune response is dependent on the priming of naive T cells by APCs. Proteomic analysis of unactivated and activated human NK cell membrane-enriched fractions demonstrated that activated NK cells can efficiently stimulate T cells, since they upregulate MHC class II molecules and multiple ligands for TCR costimulatory molecules. Furthermore, by manipulating antigen administration, we show that NK cells possess multiple independent unique pathways for antigen uptake. These results highlight NK cell-mediated cytotoxicity and specific ligand recognition by cell surface-activating receptors on NK cells as unique mechanisms for antigen capturing and presentation. In addition, we analyzed the T cell-activating potential of human NK cells derived from different clinical conditions, such as inflamed tonsils and noninfected and CMV-infected uterine decidual samples, and from transporter-associated processing antigen 2-deficient patients. This in vivo analysis revealed that proinflammatory, but not immune-suppressive, microenvironmental requirements can selectively dictate upregulation of T cell-activating molecules on NK cells. Taken together, these observations offer new and unexpected insights into the direct interactions between NK and T cells and suggest novel APC-like activating functions for human NK cells.

A methylated oligonucleotide inhibits IGF2 expression and enhances survival in a model of hepatocellular carcinoma

IGF-II is a mitogenic peptide that has been implicated in hepatocellular oncogenesis. Since the silencing of gene expression is frequently associated with cytosine methylation at cytosine-guanine (CpG) dinucleotides, we designed a methylated oligonucleotide (MON1) complementary to a region encompassing IGF2 promoter P4 in an attempt to induce DNA methylation at that locus and diminish IGF2 mRNA levels. MON1 specifically inhibited IGF2 mRNA accumulation in vitro, whereas an oligonucleotide (ON1) with the same sequence but with nonmethylated cytosines had no effect on IGF2 mRNA abundance. MON1 treatment led to the specific induction of de novo DNA methylation in the region of IGF2 promoter hP4. Cells from a human hepatocellular carcinoma (HCC) cell line, Hep 3B, were implanted into the livers of nude mice, resulting in the growth of large tumors. Animals treated with MON1 had markedly prolonged survival as compared with those animals treated with saline or a truncated methylated oligonucleotide that did not alter IGF2 mRNA levels in vitro. This study demonstrates that a methylated sense oligonucleotide can be used to induce epigenetic changes in the IGF2 gene and that inhibition of IGF2 mRNA accumulation may lead to enhanced survival in a model of HCC.

Bone mineral density in pediatric transplant recipients1

Background. Reduced bone mass and fragility fractures are known complications after transplantation in adults. Far less is known about the skeletal effects of transplantation in children and adolescents. Methods. This cross-sectional study examined the skeletal status of children (ages 9–18 years) who were at least 1 year post-cardiac (n=13), post-renal (n=8), or post-bone marrow (BMT; n=15) transplantation. Bone mass at total hip, femoral neck, spine (L2-4), and whole body (WB) was determined by dual energy x-ray absorptiometry and compared with age, sex, and ethnic-specific reference data. Standard deviations (z-scores) were calculated for both areal bone mineral density (BMD) and estimated volumetric bone density (bone mineral apparent density [BMAD]). Results. Cardiac transplant patients had significantly lower BMD z-scores compared with the reference population at all skeletal sites. BMT recipients had significantly reduced BMD z-scores at total hip, spine, and WB. Kidney transplant patients had a significantly reduced WB BMD z-score only. Spine BMAD z-scores remained significantly reduced in cardiac and BMT subjects. Three of 36 patients had radiographic evidence of spinal fracture after transplantation. No correlation between steroid dosage and any measure of bone mass was observed. Conclusions. Cardiac and BMT recipients had reduced BMD at multiple skeletal sites, and renal transplant recipients had reduced WB BMD for age. Deficits in spine bone density persisted after correcting for small bone size using BMAD. Low bone density and the occurrence of vertebral fractures indicate that cardiac, renal, and bone marrow transplantation in children is associated with reduced bone health.

Sample Preparation of Formalin-Fixed Paraffin-Embedded (FFPE) Tissue for Proteomic Analyses

A sample preparation procedure has been developed to prepare Formalin-Fixed Paraffin-Embedded (FFPE) tissues for proteomic profiling. Protocols have been established for processing FFPE sections on slides, including deparaffinization, rehydration, protein extraction, and enzymatic digestion, all using in-house developed and optimized buffers and techniques. These methods are practical and allow for parallel sample processing. The efficiency of protein extraction from FFPE tissue is comparable to the results from fresh or frozen tissue. The prepared samples can be directly coupled to a wide range of proteomic technologies, including isobaric labeling and on-line multidimensional chromatography. Comprehensive proteomic profiling of prepared FFPE tissue was performed using a fully automated tandem multidimensional liquid chromatography (LC) system coupled with on-line electrospray ionization (ESI) tandem mass spectrometry (MS): LC/LC-MS/MS. Routinely around 4,000 unique proteins of high confidence could be identified from 100 μg of extracted protein. The method presented here will be valuable for others who plan to analyze FFPE tissues.