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Cell migration is a key aspect of the development of the immune system and mediating an immune response. There is extensive and continual redistribution of cells to different anatomic sites throughout the body. These trafficking patterns control immune function, tissue regeneration, and host responses to insult. The ability to monitor the fate and function of cells, therefore, is imperative to both understanding the role of specific cells in disease processes and to devising rational therapeutic strategies. Determining the fate of immune cells and understanding the functional changes associated with migration and proliferation require effective means of obtaining in vivo measurements in the context of intact organ systems. A variety of imaging methods are available to provide structural information, such as X-ray CT and MRI, but only recently new tools have been developed that reveal cellular and molecular changes as they occur within living animals. We have pioneered one of these techniques that is based on the observations that light passes through mammalian tissues, and that luciferases can serve as internal biological sources of light in the living body. This method, called in vivo bioluminescence imaging, is a rapid and noninvasive functional imaging method that employs light-emitting reporters and external photon detection to follow biological processes in living animals in real time. This imaging strategy enables the studies of trafficking patterns for a variety of cell types in live animal models of human biology and disease. Using this approach we have elucidated the spatiotemporal trafficking patterns of lymphocytes within the body. In models of autoimmune disease we have used the migration of "pathogenic" immune cells to diseased tissues as a means to locally deliver and express therapeutic proteins. Similarly, we have determined the tempo of NK-T cell migration to neoplastic lesions and measured their life span in vivo. Using bioluminescence imaging individual groups of animals can be followed over time significantly reducing the number of animals per experiment, and improving the statistical significance of a study since changes in a given population can be studied over time. Such rapid assays that reveal cell fates in vivo will increase our basic understanding of the molecular signals that control these migratory pathways and will substantially speed up the development and evaluation of therapies.

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

Little is known about adoptive transfer of allogeneic ex vivo expanded dendritic cells (eDCs). We investigated the trafficking pattern of eDCs in mice after allogeneic bone marrow transplantation by using bioluminescence imaging. eDCs were expanded from bone marrow precursors in the presence of GM-CSF, interleukin-4, and Flt3L and retrovirally transduced to express luciferase (luc) and green fluorescence protein (gfp). Flow cytometry showed polyclonal DC populations after expansion that consisted of CD11c+CD11b+ and CD11c-CD11b+ cells that co-expressed CD40, CD80, CD86, and MHCII. eDCs were functional in mixed lymphocyte reactions and produced tumor necrosis factor-alpha on phytohemagglutinin stimulation. The eDCs were then injected intravenously into BALB/c recipient mice that had received allogeneic bone marrow transplantation 6 weeks previously. On day 1 after transfer, eDCs were detected by bioluminescence imaging throughout the lungs and spleen. In the later course, signals were observed throughout thymus, lower abdomen, and spleen throughout a period of more than 42 days. Immunofluorescence microscopy confirmed CD11c positivity on the gfp+ donor cells, which localized in T-cell zones of mesenteric lymph nodes, Peyer's patches, spleen, and thymus. These findings are important for adoptive immunotherapies because they indicate that eDCs migrate efficiently in vivo and are capable of surviving long term.

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.

Acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) are thought to arise from malignant hematopoietic progenitor cells representing early and undifferentiated stem cell clones. In CML there is evidence for a progenitor cell subset free of leukemic clones, depending on the course of the disease. Additionally, it has been suggested that in AML, the early stem cell compartment (CD34 /CD90+ and CD90 − population, thus making CD90 an inappropriate marker to distinguish benign from malignant cells in these leukemias.

Summary:Skin involvement due to a systemic infection with Aspergillus species in the course of allogeneic hematopoietic cell transplantation is extremely rare. We report the clinical course of two patients with hematologic malignancies who underwent allogeneic hematopoietic cell transplantation (HCT) and developed disseminated skin involvement as the first clinical symptom of a proven systemic Aspergillus infection. Bone Marrow Transplantation (2001) 27, 753-755. Keywords: cutaneous Aspergillus infection; HCT; BMT; STX; skin infection Aspergillus species are increasingly recognized as major fungal pathogens in severely immunosuppressed or neutropenic patients, particularly after allogeneic hematopoietic cell transplantation (HCT). 1 The risk of developing systemic Aspergillus infection increases with the duration of immunosuppression. The most common manifestation of an Aspergillus infection in these patients is invasive pulmonary aspergillosis characterized by hyphal invasion and destruction of pulmonary tissue. 2,3 Hematogenous dissemination as a secondary event occurs in 20-50% of these patients and most commonly involves the central nervous system and the gastrointestinal tract. 4 The overall mortality is up to 82% and reflects the clinical importance of Aspergillus infection in immunosuppressed or neutropenic patients. 5 Solitary extrapulmonary Aspergillus infection occurs most frequently in the maxillary sinuses 6,7 and colonization of the nasal sinuses can lead to endogenous spread to the lungs causing invasive pulmonary aspergillosis. The skin is a rare site of infection, 8 and cutaneous infections are most often found at the entry site of Hickman catheters. 4,7,9,10 Cutaneous aspergillosis is a poorly described entity Case report No. 1A 50-year-old male with Philadelphia-positive chronic myeloid leukemia in accelerated phase received a non-T cell-depleted bone marrow graft (1.2 × 10 8 MNC/kg BW) from his HLA-identical, MLC-negative brother. He was conditioned with busulfan (10 mg/kg), thiotepa (750 mg/kg) and cyclophosphamide (120 mg/kg) and received methotrexate and cyclosporine as GVHD prophylaxis. On day −1 twice daily prophylactic aerosol inhalation of 10 mg amphotericin B was started. On day +2 he developed fever of unknown origin and was empirically treated with a combination of cefotaxim, piperacillin and vancomycin. Because there was no resolution of the fever, intravenous (i.v.) amphotericin B was started at a dose of 0.8 mg/kg/day on day +4. On day +9 a round warm red swelling resembling a small furuncle appeared on the right thigh. This lesion grew rapidly and was extremely painful. On day +13 this lesion measured approximately 5 cm in diameter and in the centre a circular area of necrosis, 2 cm in diameter, had developed (Figure 1). Until that time microbiological examination of multiple blood cultures had yielded no positive result. Necrotic tissue from the lesion on the right thigh was sent for microbiological culture and histological examination. Both microbiological and histological exa...

Stimulating lymphocytes with Ifn-γ, anti-CD3, and interleukin-2 promotes the proliferation of a cell population coexpressing T-lymphocyte surface antigens such as CD3, CD8a, and CD25 as well as natural killer cell markers such as NK1.1, CD49, and CD69. These cells, referred to as cytokine-induced killer cells (CIKs), display cytotoxic activity against tumour cells, even without prior antigen presentation, and offer a new cell-based approach to the treatment of malignant diseases. Because CIKs are limited in vivo, strategies to optimize in vitro culture yield are required. In the last 10 years, mesenchymal stem cells (MSCs) have gathered considerable attention. Aside from their uses in tissue engineering and as support in haematopoietic stem cell transplantations, MSCs show notable immunomodulatory characteristics, providing further possibilities for therapeutic applications. In this study, we investigated the influence of murine MSCs on proliferation, phenotype, vitality, and cytotoxicity of murine CIKs in a coculture system. We found that CIKs in coculture proliferated within 7 days, with an average growth factor of 18.84, whereas controls grew with an average factor of 3.7 in the same period. Furthermore, higher vitality was noted in cocultured CIKs than in controls. Cell phenotype was unaffected by coculture with MSCs and, notably, coculture did not impact cytotoxicity against the tumour cells analysed. The findings suggest that cell–cell contact is primarily responsible for these effects. Humoral interactions play only a minor role. Furthermore, no phenotypical MSCs were detected after coculture for 4 h, suggesting the occurrence of immune reactions between CIKs and MSCs. Further investigations with DiD-labelled MSCs revealed that the observed disappearance of MSCs appears not to be due to differentiation processes.