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Many forms of adoptive T cell therapy are on the verge of being translated to the clinic. To gain further insight in their immunomodulating functions and to optimize future clinical trials it is essential to develop techniques to study their homing capacity. CD4+ T cells were labeled using [(111)In]oxine, and the radioactive uptake was determined in vitro before intravenous injection in immunodeficient mice. In vivo biodistribution of [(111)In]oxine-labeled cells or tracer alone was subsequently measured by μSPECT/CT and organ distribution. CD4+ T cells incorporated [(111)In]oxine with higher labeling yield using Ringer-Acetate compared to 0.9% NaCl. Cellular viability after labeling with [(111)In]oxine was not compromised using less than 0.4 MBq/million cells. After intravenous infusion CD4+ T cells preferentially homed to the liver (p<0.01) and spleen (p<0.05). This study presents a protocol for labeling of T cells by [(111)In]oxine with preserved viability and in vivo tracking by SPECT for up to 8days, which can easily be translated to clinical cell therapy trials., (© 2013.)

Spermatogonial stem cells are unique cells of testes that can restore fertility upon transplantation into recipient testes. However, use of suitable markers for enrichment of these cells have important potential application. THY1, is an established conserved marker of spermatogonial stem cells in bovine, rodents, and primates, but there is no information available in goats. After three rounds of enzymatic digestion of prepubertal goat testicular tissues, undifferentiated spermatogonia positive for THY1 were isolated by magnetic-activated cell sorting and were used for immunocytochemistry, real-time polymerase chain reaction analysis for gene expression, protein expression, and transplantation into recipient mice. Immunocytochemical analyses showed that significantly higher percentage of THY1(+) cells were positive for PLZF and VASA when compared with unselected population. This result for PLZF was further confirmed at the protein level. Real-time polymerase chain reaction analysis revealed that expression of THY1, PLZF, VASA, BCL6B, and UCHL1 as SCCs characteristic genes in THY1(+) cells was significantly higher than in the initial population. Finally, transplantation of PKH26-labeled cells revealed that THY1(+) cells had higher capacity for colony formation when compared with unselected cells. In conclusion, the results provide indications that THY1 surface marker can be reliably used for enrichment of undifferentiated spermatogonial in the goats., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: The spectrum of TNF-α-producing cells in patients with psoriasis, as well as their fate during treatment with TNF-α antagonists, is not clearly defined., Objective: We sought to analyze the effects of anti-TNF-α treatment on TNF-α(+) cells in the skin and blood of patients with psoriasis., Methods: Lesional psoriatic skin was analyzed by means of immunohistologic staining and quantitative RT-PCR, and peripheral blood cells were phenotypically characterized by means of multicolor immunofluorescence labeling., Results: By using a tyramide-based signal amplification system, TNF-α was detected in dermal CD45(+)HLA-DR(+) leukocytes consisting of CD11c(+) dendritic cells and CD163(+) macrophages. In peripheral blood we observed an increase in the TNF-α-producing myeloid subsets of CD14(-) 6-sulfo-LacNac(+) dendritic cells and CD14(+)CD16(+) "intermediate" monocytes compared with healthy control subjects. Strikingly, we did not find detectable levels of TNF-α in other cells, including keratinocytes or T cells, making these cell types unlikely targets of TNF-α blockers. Up to 48 hours after the intravenous administration of the TNF-α antagonist infliximab, we encountered no overt changes in numbers of TNF-α(+) cells or signs of apoptosis in lesional psoriatic skin. Yet we observed a rapid decrease in IL-12p40, IL-1β, CCL20, and IL12RB1 mRNA levels. Consistently, TNF-α blockade during in vitro stimulation of 6-sulfo-LacNac DCs resulted in decreased production of IL-12 and IL-23 but not IL-6. In a mixed leukocyte reaction infliximab led to significantly decreased proliferation rates of T cells independent of the Fc antibody fragment., Conclusion: The decrease in tissue inflammation during anti-TNF-α therapy is not due to immediate killing of TNF-α-producing cells but rather results from a rapid downregulation of the pathogenic IL-12/IL-23-driven immune response., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

The olfactory epithelial layer contains multipotent horizontal basal cells (HBCs) that differentiate into olfactory sensory neurons. Here, we show that rat HBCs express oligodendrocyte progenitor cell (OPC) and astrocyte markers. We generated olfactory sphere (OS) cells in cultures that were derived from adult rat olfactory mucosa. Fluorescence-activated cell sorting and immunofluorescence analyses showed that OS cells also express OPC and astrocyte markers. Interestingly, OS cells underwent oligodendrocyte differentiation in vitro. To study oligodendrocyte differentiation in vivo, OS cells were transplanted into injured rat spinal cords. The transplanted cells integrated into host tissue and differentiated into oligodendrocytes. When transected saphenous nerve ends were encased in collagen-containing silicone tubes with or without OS cells, the transplanted OS cells differentiated into Schwann cells. Our data provide new insights into of the stemness of OS cells., (© 2013.)

Interferons are made by cells in response to appropriate stimuli such as viruses, bacteria, parasites or tumor cells and are released into the surrounding medium. They then bind to receptors on target cells to allow for communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors. IFN-α is produced by leukocytes and is mainly involved in innate immune response against viral or bacterial infections and for tumor control. The aim of this work is to explore the detailed modulation of IFN-α on phenotypic and functional maturation inside and outside murine bone marrow derived dendritic cells (BMDCs). The maturity of BMDCs post treatment with IFN-α was evaluated with conventional light microscope and transmission electron microscopy (TEM) for morphology changes; flow cytometry (FCM) for changes of surface molecules on BMDCs; cytochemistry, acid phosphatase activity (ACP) test, and FITC-dextran bio-assay for biochemistry analysis and enzyme-linked immunosorbent assay (ELISA) for cytokine production by BMDCs. We have shown that IFN-α 1) up-regulates the expression of MHC II, CD40, CD83, CD80 and CD86 molecules on BMDCs; 2) down-regulates the rates of pinocytosis and phagocytosis by BMDCs as evidenced by the results of decreased ACP, and FITC-dextran bio-assay; 3) enhances the ability of BMDCs to drive T cell function; and 4) induces higher levels of IL-12 and TNF-α secreted by BMDCs. Therefore, we conclude that IFN-α can efficiently promote the maturation of BMDCs through detailed modulation inside and outside BMDCs. Our study has provided more detailed data on changes of BMDCs modulated by IFN-α, and rationale on future application of IFN-α for enhancing host immunity and potent adjuvant administration in the design of DC-based vaccines., (© 2013.)

IFN-γ is a cytokine that plays crucial role in innate and adaptive immunity against viral and intracellular bacterial infections and for tumor control. IFN-γ is also a key activator of macrophages [1,2]. In the present study, we studied detailed modulation of IFN-γ on phenotypic and functional maturation of murine bone marrow derived dendritic cells (BMDCs). Phenotypic and functional maturation of BMDCs was evaluated by light microscope, flow cytometry(FCM), transmission electron microscopy (TEM), cytochemistry method, acid phosphatase activity(ACP), FITC-dextran bio-assay and enzyme linked immunosorbent assay (ELISA). We elucidated that IFN-γ up-regulated the expression of MHC II, CD40, CD80, CD83 and CD86 molecules on BMDCs, down-regulated the activity of pinocytosis and phagocytosis by BMDCs, and induced higher levels of IL-12 and TNF-α secreted by BMDCs. It is therefore confirmed that IFN-γ can effectively promote the maturation of BMDCs. Our study provides more evidence and rationale on future application of IFN-γ for enhancing host immunity., (© 2013.)

Treatment of liver disease, caused by hepatotoxins, viral infections, alcohol ingestion, or autoimmune conditions, remains challenging and costly. The liver has a powerful capacity to repair and regenerate, thus a thorough understanding of this tightly orchestrated process will undoubtedly improve clinical means of restoring liver function after injury. Using a murine model of acute liver injury caused by overdose of acetaminophen (APAP), our studies demonstrated that the combined absence of liver resident macrophages (Kupffer cells, KCs), and infiltrating macrophages (IMs) resulted in a marked delay in liver repair, even though the initiation and extent of peak liver injury was not impacted. This delay was not due to impaired hepatocyte proliferation but rather prolonged vascular leakage, which is caused by APAP-induced liver sinusoidal endothelial cell (LSEC) injury. We also found that KCs and IMs express an array of angiogenic factors and induce LSEC proliferation and migration. Our mechanistic studies suggest that hypoxia-inducible factor (HIF) may be involved in regulating the angiogenic effect of hepatic macrophages (Macs), as we found that APAP challenge resulted in hypoxia and stabilization of HIF in the liver and hepatic Macs. Together, these data indicate an important role for hepatic Macs in liver blood vessel repair, thereby contributing to tissue recovery from acute injury., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells (DC). In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells (Adh-DC) and magnetic activated cell sorting- dendritic cells (MACS-DC), and their effects on phagocytic activity of differentiated immature DCs (immDCs)., Materials and Methods: : In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate (FITC)-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups., Results: We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity (MFI) of phagocytic cells was higher in MACS-DC (p<0.05)., Conclusion: : We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs.

The cytometric enumeration and characterization of antigen-specific lymphocytes, as introduced about 15 years ago, has contributed significantly to our understanding of adaptive immune responses in health and disease. Despite the development of several technologies, allowing to directly or indirectly analyze many aspects of lymphocyte specificity and function, several unresolved issues remain, due to the low frequency of certain antigen-specific lymphocyte subsets and the complexity of T cell antigen recognition. This is especially true for CD4(+) conventional as well as regulatory T cells, which bring major contributions to immune protection and pathology. Here we review the current technologies for the analysis of antigen specific T cells within the physiologic T cell repertoire and with a special focus on recent technologies addressing the analysis of rare antigen-specific T cell populations including naive and regulatory T cells., (Copyright © 2013 International Society for Advancement of Cytometry.)

The aim of this study is to show the effect of donor-specific transfusion (DST) in inducing immunological tolerance mediated by regulatory T cells (Treg) and indoleamine 2,3-dioxygenase (IDO). Skin grafts from H2(d) Balb/c were transplanted into H2(k) C3H/He 7days after the infusion of donor splenocytes, isolated each immune cell populations. Graft survival prolonged in recipients who received splenocytes, MHC class II(+) CD90(-) cells and CD3(-)CD19(-) cells (p<0.001, p<0.05 and p<0.01, respectively). CD11b(+) cell infusion resulted in prolongation of graft survival when compared to CD11c(+) cell infusion (p<0.01). Foxp3(+)CD4(+)CD25(+) T cells were increased after the transplant in recipients infused with CD11b(+) cells (p<0.05). The mixed lymphocyte reaction showed donor-specificity (p<0.001). High IDO expression was observed in CD11b(+) cell infusion group. Graft survival with DST using IDO antagonist (1MT) were not prolonged. In conclusion, DST allows induction of donor-specific tolerance which involves Foxp3(+)CD4(+)CD25(+) T cells and IDO expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Study Background: DEFB4/103A encoding β-defensin 2 and 3, respectively, inhibit CXCR4-tropic (X4) viruses in vitro. We determined whether DEFB4/103A Copy Number Variation (CNV) influences time-to-X4 and time-to-AIDS outcomes., Methods: We utilized samples from a previously published Multicenter AIDS Cohort Study (MACS), which provides longitudinal account of viral tropism in relation to the full spectrum of rates of disease progression. Using traditional models for time-to-event analysis, we investigated association between DEFB4/103A CNV and the two outcomes, and interaction between DEFB4/103A CNV and disease progression groups, Fast and Slow., Results: Time-to-X4 and time-to-AIDS were weakly correlated. There was a stronger relationship between these two outcomes for the fast progressors. DEFB4/103A CNV was associated with time-to-AIDS, but not time-to-X4. The association between higher DEFB4/103A CNV and time-to-AIDS was more pronounced for the slow progressors., Conclusion: DEFB4/103A CNV was associated with time-to-AIDS in a disease progression group-specific manner in the MACS cohort. Our findings may contribute to enhancing current understanding of how genetic predisposition influences AIDS progression.