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BACKGROUND: Successful gametogenesis requires the establishment of an appropriate epigenetic state in developing germ cells. Nevertheless, an association between abnormal spermatogenesis and epigenetic disturbances in germline-specific genes remains to be demonstrated. METHODS: In this study, the DNA methylation pattern of the promoter CpG island (CGI) of two germline regulator genes--DAZL and DAZ, was characterized by bisulphite genomic sequencing in quality-fractioned ejaculated sperm populations from normozoospermic (NZ) and oligoasthenoteratozoospermic (OAT) men. RESULTS: OAT patients display increased methylation defects in the DAZL promoter CGI when compared with NZ controls. Such differences are recorded when analyzing sperm fractions enriched either in normal or defective germ cells (Ptextless 0.001 in both cases). Significant differences in DNA methylation profiles are also observable when comparing the qualitatively distinct germ cell fractions inside the NZ and OAT groups (P= 0.003 and P= 0.007, respectively). Contrastingly, the unmethylation pattern of the DAZ promoter CGI remains correctly established in all experimental groups. CONCLUSIONS: An association between disrupted DNA methylation of a key spermatogenesis gene and abnormal human sperm is described here for the first time. These results suggest that incorrect epigenetic marks in germline genes may be correlated with male gametogenic defects.

Killer cell lectin-like receptor F1 (KLRF1) is an activating C-type lectin-like receptor expressed on human NK cells and subsets of T cells. In this study, we show that activation-induced C-type lectin (AICL) is a unique KLRF1 ligand expressed on tumor cell lines of hematopoietic and non-hematopoietic origins. We screened a panel of human tumor cell lines using the KLRF1 reporter cells and found that several tumor lines expressed KLRF1 ligands. We characterized a putative KLRF1 ligand expressed on the U937 cell line. The molecular mass for the deglycosylated ligand was 28 kDa under non-reducing condition and 17 kDa under reducing condition, suggesting that the KLRF1 ligand is a homodimer. By expression cloning from a U937 cDNA library, we identified AICL as a KLRF1 ligand. We generated mAbs against AICL to identify the KLRF1 ligands on non-hematopoietic tumor lines. The anti-AICL mAbs stained the tumor lines that express the KLRF1 ligands and importantly the interaction of KLRF1 with the KLRF1 ligand on non-hematopoietic tumors was completely blocked by the two anti-AICL mAbs. Moreover, NK cell degranulation triggered by AICL-expressing targets was partially inhibited by the anti-AICL mAb. Finally, we demonstrate that AICL is expressed in human primary liver cancers. These results suggest that AICL is expressed on tumor cells of non-hematopoietic origins and raise the possibility that AICL may contribute to NK cell surveillance of tumor cells.

RATIONALE: Chitin is a ubiquitous polysaccharide in fungi, insects, allergens, and parasites that is released at sites of infection. Its role in the generation of tissue inflammation, however, is not fully understood. OBJECTIVES: We hypothesized that chitin is an important adjuvant for adaptive immunity. METHODS: Mice were injected with a solution of ovalbumin and chitin. MEASUREMENTS AND MAIN RESULTS: We used in vivo and ex vivo/in vitro approaches to characterize the ability of chitin fragments to foster adaptive immune responses against ovalbumin and compared these responses to those induced by aluminum hydroxide (alum). In vivo, ovalbumin challenge caused an eosinophil-rich pulmonary inflammatory response, Th2 cytokine elaboration, IgE induction, and mucus metaplasia in mice that had been sensitized with ovalbumin plus chitin or ovalbumin plus alum. Toll-like receptor-2, MyD88, and IL-17A played critical roles in the chitin-induced responses, and MyD88 and IL-17A played critical roles in the alum-induced responses. In vitro, CD4(+) T cells from mice sensitized with ovalbumin plus chitin were incubated with ovalbumin-stimulated bone marrow-derived dendritic cells. In these experiments, CD4(+) T-cell proliferation, IL-5, IL-13, IFN-γ, and IL-17A production were appreciated. Toll-like receptor-2, MyD88, and IL-17A played critical roles in these in vitro adjuvant properties of chitin. TLR-2 was required for cell proliferation, whereas IL-17 and TLR-2 were required for cytokine elaboration. IL-17A also inhibited the generation of adaptive Th1 responses. CONCLUSIONS: These studies demonstrate that chitin is a potent multifaceted adjuvant that induces adaptive Th2, Th1, and Th17 immune responses. They also demonstrate that the adjuvant properties of chitin are mediated by a pathway(s) that involves and is regulated by TLR-2, MyD88, and IL-17A.

OBJECTIVE: To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS: Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS: MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS: MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.

RATIONALE: Fibrocytes are progenitor cells characterized by the simultaneous expression of mesenchymal, monocyte, and hematopoietic stem cell markers. We previously documented their presence in lungs of patients with idiopathic pulmonary fibrosis. However, the mechanisms involved in their migration, subsequent homing, and local role remain unclear. Matrix metalloproteinases (MMPs) facilitate cell migration and have been implicated in the pathogenesis of pulmonary fibrosis. OBJECTIVES: To evaluate the expression and role of matrix metalloproteinases in human fibrocytes. METHODS: Fibrocytes were purified from CD14(+) monocytes and cultured for 8 days; purity of fibrocyte cultures was 95% or greater as determined by flow cytometry. Conditioned media and total RNA were collected and the expression of MMP-1, MMP-2, MMP-7, MMP-8, and MMP-9 was evaluated by real-time polymerase chain reaction. Protein synthesis was examined using a Multiplex assay, Western blot, fluorescent immunocytochemistry, and confocal microscopy. MMP-2 and MMP-9 enzymatic activities were evaluated by gelatin zymography. Migration was assessed using collagen I-coated Boyden chambers. Stromal cell-derived factor-1α and platelet-derived growth factor-B were used as chemoattractant with or without a specific MMP-8 inhibitor. MEASUREMENTS AND MAIN RESULTS: Fibrocytes showed gene and protein expression of MMP-2, MMP-9, MMP-8, and MMP-7. MMP-2 and MMP-9 enzymatic activities were also demonstrated by gelatin zymography. Likewise, we found colocalization of MMP-8 and MMP-7 with type I collagen in fibrocytes. Fibrocyte migration toward platelet-derived growth factor-B or Stromal cell-derived factor-1α in collagen I-coated Boyden chambers was significantly reduced by a specific MMP-8 inhibitor. CONCLUSIONS: Our findings reveal that fibrocytes express a variety of MMPs and that MMP-8 actively participates in the process of fibrocyte migration.

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless, many details of the immunopathogenesis of TRALI, particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast, 34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia, severe pulmonary edema, and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8(+) T lymphocytes or CD4(+) T cells but not CD19(+) B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia, lung damage, and mortality, suggesting that T lymphocytes were responsible for the protective effect. Taken together, these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions.

Caveolin 1 (Cav-1) is a major protein of a specific membrane lipid raft known as caveolae. Cav-1 interacts with the gp41 of the human immunodeficiency virus (HIV) envelope, but the role of Cav-1 in HIV replication and pathogenesis is not known. In this report, we demonstrate that HIV infection in primary human monocyte-derived macrophages (MDMs), THP-1 macrophages, and U87-CD4 cells results in a dramatic upregulation of Cav-1 expression mediated by HIV Tat. The activity of p53 is essential for Tat-induced Cav-1 expression, as our findings show enhanced phosphorylation of serine residues at amino acid positions 15 and 46 in the presence of Tat with a resulting Cav-1 upregulation. Furthermore, inhibition of p38 mitogen-activated protein kinase (MAPK) blocked phosphorylation of p53 in the presence of Tat. Infection studies of Cav-1-overexpressing cells reveal a significant reduction of HIV production. Taken together, these results suggest that HIV infection enhances the expression of Cav-1, which subsequently causes virus reduction, suggesting that Cav-1 may contribute to persistent infection in macrophages.

Rheumatoid arthritis (RA) is closely associated with HLA-DRB1 alleles that code a five-amino acid sequence motif in positions 70-74 of the HLA-DRbeta-chain, called the shared epitope (SE). The mechanistic basis of SE-RA association is unknown. We recently found that the SE functions as an allele-specific signal-transducing ligand that activates an NO-mediated pathway in other cells. To better understand the role of the SE in the immune system, we examined its effect on T cell polarization in mice. In CD11c(+)CD8(+) dendritic cells (DCs), the SE inhibited the enzymatic activity of indoleamine 2,3 dioxygenase, a key enzyme in immune tolerance and T cell regulation, whereas in CD11c(+)CD8(-) DCs, the ligand activated robust production of IL-6. When SE-activated DCs were cocultured with CD4(+) T cells, the differentiation of Foxp3(+) T regulatory cells was suppressed, whereas Th17 cells were expanded. The polarizing effects could be seen with SE(+) synthetic peptides, but even more so when the SE was in its natural tridimensional conformation as part of HLA-DR tetrameric proteins. In vivo administration of the SE ligand resulted in a greater abundance of Th17 cells in the draining lymph nodes and increased IL-17 production by splenocytes. Thus, we conclude that the SE acts as a potent immune-stimulatory ligand that can polarize T cell differentiation toward Th17 cells, a T cell subset that was recently implicated in the pathogenesis of autoimmune diseases, including RA.

Imatinib mesylate (IM) induces clinical remissions in chronic-phase chronic myeloid leukemia (CML) patients but IM resistance remains a problem. We recently identified several features of CML CD34(+) stem/progenitor cells expected to confer resistance to BCR-ABL-targeted therapeutics. From a study of 25 initially chronic-phase patients, we now demonstrate that some, but not all, of these parameters correlate with subsequent clinical response to IM therapy. CD34(+) cells from the 14 IM nonresponders demonstrated greater resistance to IM than the 11 IM responders in colony-forming cell assays in vitro (P textless .001) and direct sequencing of cloned transcripts from CD34(+) cells further revealed a higher incidence of BCR-ABL kinase domain mutations in the IM nonresponders (10%-40% vs 0%-20% in IM responders, P textless .003). In contrast, CD34(+) cells from IM nonresponders and IM responders were not distinguished by differences in BCR-ABL or transporter gene expression. Interestingly, one BCR-ABL mutation (V304D), predicted to destabilize the interaction between p210(BCR-ABL) and IM, was detectable in 14 of 20 patients. T315I mutant CD34(+) cells found before IM treatment in 2 of 20 patients examined were preferentially amplified after IM treatment. Thus, 2 properties of pretreatment CML stem/progenitor cells correlate with subsequent response to IM therapy. Prospective assessment of these properties may allow improved patient management.

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive, negative regulator of membrane RAGE activation, inhibiting ligand binding. However, some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment, survival, and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE, the recombinant protein was intratracheally administered to mice, which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore, sRAGE binding to cells increased during maturation, which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation, we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages, sRAGE treatment activated Akt, Erk, and NF-kappaB, and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation.

The B cell-activating factor of the TNF family receptor (BAFF-R), encoded by the TNFRSF13C gene, is critically important for transitional B cell survival to maturity. Thus, ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however, there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression, although characterized in murine B cells, has not yet been reported in human B lymphopoiesis. In this study, we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e., the TNFRSF13C promoter). To accomplish this, we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites, which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology, these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally, chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region, and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA.