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The serological complement-dependent cytotoxicity crossmatch (CDC-XM) permits routine identification of anti-donor alloantibodies in the sera of allotransplant recipients. However, in a small group of recipients, antibodies below the threshold of detection may still be responsible for hyperacute rejection. For the same reason, approximately 20% of recipients develop acute rejection episodes. The flow cytometry crossmatch (FCXM) was designed to address these problems, but because of the presence of clinically insignificant antibodies (linked, non-lytic), the FCXM appears to be too sensitive yielding false-positive results. We compared FCXM with its modified version assessing cell viability (cytolytic flow cytometry crossmatch; cFCXM) using sera from previously sensitised kidney recipients. The presence of alloantibodies was detected using the Luminex platform. The cFCXM proved to be of greater sensitivity than CDC-XM, which was additionally confirmed with bead-based Luminex techniques. The cFCXM was also superior to FCXM because it distinguished lytic from non-lytic antibodies. The cFCXM was superior to assess donor specificity, sensitivity, and detection of clinically relevant lytic antibodies.

The antitumor activity of LPS was first described by Dr. William Coley. However, its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs), regulatory T cells, myeloid-derived suppressor cells, and CD8(+) regulatory T cells. In contrast, high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs, as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs, which were immunosuppressive after the low-dose LPS, and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration, whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion, our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice.

Fibroblast growth factor (FGF) signaling plays an important role in embryonic stem cells and adult tissue homeostasis, but the function of FGFs in mammary gland stem cells is less well defined. Both FGFR1 and FGFR2 are expressed in basal and luminal mammary epithelial cells (MECs), suggesting that together they might play a role in mammary gland development and stem cell dynamics. Previous studies have demonstrated that the deletion of FGFR2 resulted only in transient developmental defects in branching morphogenesis. Using a conditional deletion strategy, we investigated the consequences of FGFR1 deletion alone and then the simultaneous deletion of both FGFR1 and FGFR2 in the mammary epithelium. FGFR1 deletion using a keratin 14 promoter-driven Cre-recombinase resulted in an early, yet transient delay in development. However, no reduction in functional outgrowth potential was observed following limiting dilution transplantation analysis. In contrast, a significant reduction in outgrowth potential was observed upon the deletion of both FGFR1 and FGFR2 in MECs using adenovirus-Cre. Additionally, using a fluorescent reporter mouse model to monitor Cre-mediated recombination, we observed a competitive disadvantage following transplantation of both FGFR1/R2-null MECs, most prominently in the basal epithelial cells. This correlated with the complete loss of the mammary stem cell repopulating population in the FGFR1/R2-attenuated epithelium. FGFR1/R2-null MECs were partially rescued in chimeric outgrowths containing wild-type MECs, suggesting the potential importance of paracrine mechanisms involved in the maintenance of the basal epithelial stem cells. These studies document the requirement for functional FGFR signaling in mammary stem cells during development.

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans.

HIV elite controllers (EC) are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment. Adaptive immunity and host genetic factors, although implicated, do not entirely explain this phenomenon. On the other hand, plasmacytoid dendritic cells (pDCs) are the principal type I interferon (IFN) producers in response to viral infection, and it is unknown whether pDCs are involved in the control of HIV infection in EC. In our study, we analyzed peripheral pDC levels and IFN-α production by peripheral blood mononuclear cells (PBMCs) in EC compared to other groups of HIV-infected patients, the ability of pDCs to reduce HIV production in vitro, and the mechanisms potentially involved. We showed preserved pDC counts and IFN-α production in EC. We also observed a higher capacity of pDCs from EC to reduce HIV production and to induce T cell apoptosis, whereas pDCs from viremic patients barely responded without previous Toll-like receptor 9 (TLR-9) stimulus. The preserved functionality of pDCs from EC to reduce viral production may be one of the mechanisms involved in the control of HIV viremia in these subjects. These results demonstrate the importance of innate immunity in HIV pathogenesis, and an understanding of pDC mechanisms would be helpful for the design of new therapies.

Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (ptextless9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity.

IL-27, which is produced by activated APCs, bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed, proinflammatory and anti-inflammatory activities are attributed to IL-27, and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however, the molecular mechanism behind this phenomenon is not understood. In this study, we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6, TNF-α, MIP-1α, and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming, we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore, IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge, this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics. The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest (1), followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis (2-4). Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup. This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits the replication of human immunodeficiency virus (HIV) by deaminating cytidine residues to uridine. This causes guanosine-to-adenosine hypermutation in the opposite strand and results in inactivation of the virus. HIV counteracts A3G through the activity of viral infectivity factor (Vif), which promotes degradation of A3G. We report that viral protein R (Vpr), which interacts with a uracil glycosylase, also counteracted A3G by diminishing the incorporation of uridine. However, this process resulted in activation of the DNA-damage–response pathway and the expression of natural killer (NK) cell–activating ligands. Our results show that pathogen-induced deamination of cytidine and the DNA-damage response to virus-mediated repair of the incorporation of uridine enhance the recognition of HIV-infected cells by NK cells.

How HIV controllers (HICs) maintain undetectable viremia without therapy is unknown. The strong CD8(+) T-cell HIV suppressive capacity found in many, but not all, HICs may contribute to long-lasting viral control. However, other earlier defense mechanisms may be involved. Here, we examined intrinsic HIC cell resistance to HIV-1 infection. After in vitro challenge, monocyte-derived macrophages and anti-CD3-activated CD4(+) T cells from HICs showed low HIV-1 susceptibility. CD4 T-cell resistance was independent of HIV-1 coreceptors and affected also SIVmac infection. CD4(+) T cells from HICs expressed ex vivo higher levels of p21(Waf1/Cip1), which has been involved in the control of HIV-1 replication, than cells from control subjects. However, HIV restriction in anti-CD3-activated CD4(+) T cells and macrophages was not associated with p21 expression. Restriction inhibited accumulation of reverse transcripts, leading to reduction of HIV-1 integrated proviruses. The block could be overcome by high viral inocula, suggesting the action of a saturable mechanism. Importantly, cell-associated HIV-1 DNA load was extremely low in HICs and correlated with CD4(+) T-cell permissiveness to infection. These results point to a contribution of intrinsic cell resistance to the control of infection and the containment of viral reservoir in HICs.

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort, which, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes, which resulted in downregulation. Among the upregulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

Pseudo-Pelger-Huët anomaly (PPHA) has been documented in association with transplant medications and other drugs. This iatrogenic neutrophilic dysplasia is reversible with cessation or adjustment of medications but is frequently confused with myelodysplastic syndrome (MDS) based on the conventional concept that PPHA is a marker for dysplasia. We investigated the clinicopathologic features in iatrogenic PPHA and compared them with MDS-related PPHA. The 13 cases studied included 5 bone marrow/stem cell transplantations, 3 solid organ transplantations, 1 autoimmune disease, 3 chronic lymphocytic leukemias, and 1 breast carcinoma. For 12 cases, there was follow-up evaluation, and all demonstrated at least transient normalization of neutrophilic segmentation. All 9 cases of MDS demonstrated at least 2 of the following pathologic abnormalities on bone marrow biopsy: hypercellularity (8/9), morphologic dysplasia (8/9), clonal cytogenetic abnormality (7/9), and increased blasts (3/9), whereas these abnormalities were typically absent in iatrogenic PPHA. Iatrogenic PPHA displayed a higher proportion of circulating PPHA cells than in MDS (mean, 47.4%; SD, 31.6% vs mean, 12.3%; SD, 9.8; P textless .01). A diagnostic algorithm is proposed in which isolated PPHA is indicative of transient or benign PPHA unless proven otherwise.