The standard screening test for the recognition of autoimmune diseases is the proof of autoantibodies in serum of patients by indirect immunofluorescence (IIF) based on HEp-2 cells. Manual evaluation of this test is very subjective, slow, and there are no objective parameters as guidelines available. Interlaboratory tests showed occasionally large deviations in the test evaluation resulting in a high variance of results. The aim of this project is fast, objective, safe, and economical automatic analysis of HEp-2 IIF patterns. Images of IIF patterns were completely and automatically captured using an inverse motorized fluorescence microscope. Thereby, device-specific parameters were controlled automatically, too. For fast analysis of IIF patterns new algorithms of image processing were developed. Artifacts were recognized and excluded from analysis by the developed software. Analysis of more than 80,000 images clearly demonstrated full automatization and fast processing of IIF patterns. Additionally serum-specific fluorescence could be easily distinguished from background. Even very weak but positive patterns can be recognized and used for diagnosis. A detailed separation into different basic patterns is possible. Objective, fast, and disease-related economical analysis of HEp-2 immunofluorescence patterns is feasible. The implemented software algorithms allowed a mathematical way of describing IIF patterns and can therefore be a useful tool for the needed standardization process.

This is the first report showing that an epitope-specific ex vivo modulation of an allogeneic hematopoietic stem cell graft by the anti-human CD4 antibody MAX.16H5 IgG1 simultaneously facilitates the anti-tumor capacity of the graft (Graft-versus-leukemia effect, GvL) and the long-term suppression of the deleterious side effect Graft-versus-host-disease (GvHD). To distinguish and consolidate GvL from GvHD, the anti-human CD4 antibody MAX16.H5 IgG1 was tested in murine GvHD and tumor models. The survival rate was significantly increased in recipients receiving a MAX.16H5 IgG1 short-term (2 h) pre-incubated graft even when tumor cells were co-transplanted or when recipient mice were treated by MAX.16H5 IgG1 before transplantation. After engraftment, regulatory T-cells are generated only supporting the GvL effect. It was also possible to transfer the immune tolerance from GvHD-free recipient chimeras into third party recipient mice without the need of reapplication of MAX.16H5 IgG1 anti-human CD4 antibodies. These findings are also benefical for patients with leukemia when no matched related or unrelated donor is available and provides a safer allogeneic HSCT, which is more effective against leukemia. It also facilitates allogeneic (stem) cell transplantations for other indications (e.g., autoimmune-disorders).

NOD.Cg-Prkdcscid IL-2rg tm1Wjl /SzJ (NSG) mice are a valuable tool for studying Graftversus-Host-Disease (GvHD) induced by human immune cells. We used a model of acute GvHD by transfer of human peripheral blood mononuclear cells (PBMCs) into NSG mice. The severity of GvHD was reflected by weight loss and was associated with engraftment of human cells and the expansion of leukocytes, particularly granulocytes and monocytes. Pre-treatment of PBMCs with the anti-human CD4 antibody MAX.16H5 IgG1 or IgG4 attenuated GvHD. The transplantation of 2 3 10 7 PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 3 10 7 PBMCs pre-incubated with MAX.16H5 IgG1 or IgG4, GvHD development was reduced and survival was increased. Immune reconstitution was measured by flow cytometry and confirmed for human leukocytes (CD45), CD3 /CD41 T helper cells. Human B cells (CD19) and monocytes (CD14) could not be detected. Histopathological analysis (TUNEL assay) of the gut of recipient animals showed significantly less apoptotic crypt cells in animals receiving a MAX.16H5 IgG1 pre-incubated graft. These findings indicate that preincubation of an allogeneic graft with an anti-human CD4 antibody may decrease the frequency and severity of GvHD after hematopoietic stem cell transplantation (HSCT) and the need of conventional immunosuppressive drugs. Moreover, this approach most probably provides a safer HSCT that must be confirmed in appropriate clinical trials in the future. V C 2016 International Society for Advancement of Cytometry

Transcription factor C/EBPα is a master regulator of myelopoiesis and its inactivation is associated with acute myeloid leukemia. Deregulation of C/EBPα by microRNAs during granulopoiesis or acute myeloid leukemia development has not been studied. Here we show that oncogenic miR-182 is a strong regulator of C/EBPα. Moreover, we identify a regulatory loop between C/EBPα and miR-182. While C/EBPα blocks miR-182 expression by direct promoter binding during myeloid differentiation, enforced expression of miR-182 reduces C/EBPα protein level and impairs granulopoiesis in vitro and in vivo. In addition, miR-182 expression is highly elevated particularly in acute myeloid leukemia patients with C-terminal CEBPA mutations, thereby depicting a mechanism by which C/EBPα blocks miR-182 expression. Furthermore, we present miR-182 expression as a prognostic marker in cytogenetically high-risk acute myeloid leukemia patients. Our data demonstrate the importance of a controlled balance between C/EBPα and miR-182 for the maintenance of healthy granulopoiesis.

Background: Because different spectral sensitivities of human eye and image sensor lead to different perception of fluorescence signals, data generation is an important step in image analysis, because following work steps depend on it. Methods: We developed a method to determine image parameters allowing an objective appraisal of quality of image data as well as a separation of object and background.

Aims: We established a real-time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. Methods and Results: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was >/= 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross-reaction to human DNA or Aspergillus species could be observed. Conclusions: The established real-time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. Significance and Impact of the Study: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously

], the data showing the reconstitution of huCD4, HLA-DR, CD8, H2Kb, and muCD4 in the control animals that received 2 3 10 6 syngeneic bone marrow cells (Supporting Information Fig. S2-S4) were first published in Figure 5 of the reference 22:Fricke S, Fricke C, Oelkrug C, Hilger N, Sch€ onfelder U, Kamprad M, Lehmann J, Boltze J, Emmrich F, Sack U. Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis. Cell Mol Life Sci 2010;671231:4095-4106.The source article was not properly referenced in the legends of Figures S2, S3, and S4. This corrigendum is to correct this referencing error.In addition, the legend for Figure S3 was further modified by moving "(Day 0)" from the end of the second sentence explaining panel C to the beginning of the sentence, immediately following the words "After irradiation." The human CD4 molecule (only expressed on host T cells) decreases from initial 36.3% 6 3.66% to 8.20% 6 7.52% in co-transplanted animals and 23.23% 6 3.3% to 4.18% 6 0.43% in the bone marrow controls, respectively (Day 61). The controls in panels (A) and (B) were previously reported by us elsewhere (22). B: HLA-DR3 molecule (expressed on host APCs) decreases from initial 22.50% 6 6.73% to 0.18% 6 0.05% in co-transplanted animals and 20.98% 6 4.05% to 0.23% 6 0.05% in the bone marrow controls, respectively (Day 61). C: Flow cytometric dot plots of human CD4 (green) and HLA-DR3 (orange), and humanCD42/HLA-DR32 (red) on lymphocytes after syngeneic transplantation. Fig. S3: Transplantation of C57Bl/6 wild-type in C57Bl/6-TTG mice. The expression of murine CD31/CD81 on host cells after syngeneic transplantation. A: The expression of murine CD31/CD81 decreases after transplantation in the co-transplanted animals and in the bone marrow controls (13.35% 6 1.74% [Day 22] to 6.45% 6 3.33% [Day 19] vs.12.43% 6 2.46% [Day 22] to 4.48% 6 2.07% [Day 19]. The controls in panels (A) and (C) were previously reported by us elsewhere (22). After 61 days the initial CD8 levels were not reached. B: Flow cytometric dot plots of murine CD3 and murine CD8 on lymphocytes after syngeneic transplantation of BM or BM1Tregs. Murine CD31/CD81 are dark green events, murine CD31 are light green and pink events, and murine CD32 are black events. C: Murine MHC-H2Kb (from C57Bl/6) expression after syngeneic transplantation. The murine MHC-H2Kb was detectable at any time point during the experiment (over 95%). After irradiation (Day 0), the MHCH2Kb expression was 96.15% 6 3.5% in co-transplanted animals and 95.35% 6 2.96% in the bone marrow controls. D: Flow cytometric dot plots of murine MHC-H2Kb on lymphocytes after syngeneic transplantation of BM or BM1Tregs. All dots > 10 3 counts represent MHC-H2Kb1 cells, dots < 10 3 MHC-H2Kb2 cells.

BackgroundNon adherent bone marrow derived cells (NA-BMCs) have recently been described to give rise to multiple mesenchymal phenotypes and have an impact in tissue regeneration. Therefore, the effects of murine bone marrow derived NA-BMCs were investigated with regard to engraftment capacities in allogeneic and syngeneic stem cell transplantation using transgenic, human CD4+, murine CD4−/−, HLA-DR3+ mice.Methodology/Principal FindingsBone marrow cells were harvested from C57Bl/6 and Balb/c wild-type mice, expanded to NA-BMCs for 4 days and characterized by flow cytometry before transplantation in lethally irradiated recipient mice. Chimerism was detected using flow cytometry for MHC-I (H-2D[b], H-2K[d]), mu/huCD4, and huHLA-DR3). Culturing of bone marrow cells in a dexamethasone containing DMEM medium induced expansion of non adherent cells expressing CD11b, CD45, and CD90. Analysis of the CD45+ showed depletion of CD4+, CD8+, CD19+, and CD117+ cells. Expanded syngeneic and allogeneic NA-BMCs were transplanted into triple transgenic mice. Syngeneic NA-BMCs protected 83% of mice from death (n = 8, CD4+ donor chimerism of 5.8±2.4% [day 40], P<.001). Allogeneic NA-BMCs preserved 62.5% (n = 8) of mice from death without detectable hematopoietic donor chimerism. Transplantation of syngeneic bone marrow cells preserved 100%, transplantation of allogeneic bone marrow cells 33% of mice from death.Conclusions/SignificanceNA-BMCs triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls. These findings may be of relevance in the refinement of strategies in the treatment of hematological malignancies.