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Purpose An unphysiologic accumulation of fat cells in many parts of the body including abdomen and joints results in increased production of pro-inflammatory cytokines which have adverse effects on serum lipids, glucose and on joint cartilage. The special extract of Cimicifuga racemosa CR BNO 1055 was shown to reduce the size of the abdominal fat depot. It was therefore tempting to test whether this extract, its saponin and its unpolar and polar fractions S- and R-fraction respectively (no quotation) also reduce fat depots and fat cell accumulation in a fat depot located in the lower hind leg (called paratibial fat depot = PFD), in joint fat pads (in the knee joint this is called Hoffa's fat pad) that occur in response to ovariectomy and whether this was accompanied by reduced serum lipids, glucose and improved cartilage features in the knee joint. Methods Rats ( n = 10/group) were ovariectomized (ovx) and fed with CR BNO 1055, S- or R-fraction containing food (average intake 8.2, or 2.05 or 7.07 mg/day/animal) for 4 weeks. Ovx rats kept under no additive-containing food served as controls. The sizes of the PFD, of Hoffa's fat pad and of the cartilage thickness of the knee joints were determined by quantitative computer tomography and histomorphometrically. In the serum cholesterol, leptin and glucose levels were measured. Results High load with fat tissue in the PFD and in the knee joints was present in the ovx rats. Treatment with CR BNO 1055 and its S-fraction reduced fat load of both, Hoffa's fat pad and of the PFD significantly and this resulted in reduced body weight which was significant under CR BNO 1055. Fat load in the PFD correlated significantly with the height of serum leptin and cholesterol. The fat load in the knee joint correlated inversely with the size of knee cartilage tissue. Conclusions High fat load of the body increases following ovx and this causes increased serum leptin, cholesterol and glucose levels. Following ovx the size of Hoffa's fat pad increases also significantly and this has adverse effects on knee cartilage tissue. Therefore, increased fat tissue in joints appears to belong to the Metabolic Syndrome. This effect can be largely prevented by CR BNO 1005 and its S- but not by its R-fraction. Hence, the saponins in CR BNO 1055 may be useful in preventing the Metabolic Syndrome and osteoarthritis.

20-hydroxyecdysone has numerous favorable effects on a variety of organs, including the skin, where it improves wound healing. It is devoid of estrogenic and androgenic effects. Therefore, application of 20-hydroxyecdysone might be a new approach to improve skin conditions in postmenopausal women, and this was investigated in ovariectomized (OVX) rats.After ovariectomy, rats received Ecd (18, 57, or 116 mg/animal/day) or 17β-estradiol (E₂)-3-benzoate (60 μg/kg body weight) in food for 12 weeks, and skin samples were evaluated histologically to quantify two dermal layers, the subcutaneous fat and muscle layers.Epidermal thickness was lowest in the OVX animals, slightly higher in the E₂-treated animals, and significantly higher in the Ecd-treated animals. Dermal thickness was lowest in the intact and E₂-treated animals and highest in the Ecd-treated animals. The subcutaneous fat layer was thickest in the OVX animals, thinner in the intact animals, and intermediate in the Ecd-treated animals. The muscle layer was smallest in the OVX and intact animals and significantly larger in the E₂- and Ecd-treated animals. The number of proliferating cell nuclear antigen antibody-positive cells was lowest in OVX controls and significantly higher in all other groups.The Ecd-induced increases in epidermal and dermal thickness are suggestive of functional changes of the skin. The decreased amounts of subcutaneous fat in the E₂- and Ecd-treated animals point to either a fat catabolic or an antianabolic effect. The ovariectomy-induced decrease in subcutaneous musculature was prevented by Ecd but not by E₂. The stimulatory effects of Ecd on epidermal and dermal thickness and the muscle-increasing effects in the skin of OVX rats may indicate functional changes of the skin.

Endothelial progenitor cells (EPCs) protect kidneys from acute ischemic damage. The aim of this study was to identify “treatment parameters” that optimize an EPC-based therapy of acute ischemic renal failure. Male C57BL/6N mice underwent unilateral nephrectomy with simultaneous contralateral renal artery clamping for 30, 35, and 40 min. Tagged murine EPCs were systemically injected at the time of reperfusion. In some experiments, EPCs were pretreated with the Epac (exchange protein directly activated by cAMP-1) activator 8-pCPT-2′- O-Me-cAMP (Epac-1 Ac) and the integrin binding antagonist cyclic Arg-Gly-Asp peptide (cRGD). Injections of 106 EPCs after 30 and 35 min of renal ischemia protected animals from acute renal failure. The same effect occurred with 0.5×106 EPCs after a 35-min period of ischemia. If ischemia lasted for 40 min, 0.5×106 cells mice did not prevent acute renal failure. To analyze whether EPC integrin receptor activation would modify the cells' renoprotective activity, EPCs were pretreated with Epac-1 Ac. Such animals did not develop acute renal failure, even if ischemia lasted for 40 min. This effect was negated if the cells were pretreated with both Epac-1 Ac and cRGD. In kidneys from those animals medullopapillary EPCs were significantly accumulated. In vitro Epac-1 Ac preactivation of EPCs did not increase the overall expression intensity but induced a redistribution of β1-integrins toward the cell membranes. We conclude that EPC pretreatment with the integrin receptor activator 8-pCPT-2′- O-Me-cAMP augments the anti-ischemic potential of the cells.

Objective.In Wegener’s granulomatosis (WG), vasculitic lesions are characterized by prominent infiltration of polymorphonuclear neutrophils (PMN) and T cells, but underlying pathogenic mechanisms remain to be defined. We analyzed the expression and functional role of the C-class chemokine lymphotactin, XCL1, in WG.Methods.Sera and peripheral blood mononuclear cells (PBMC) were obtained from 16 patients with WG and healthy controls. Serum XCL1 concentrations were measured by ELISA. PBMC were subjected to flow cytometry for activation markers and immunophenotype of XCL1+ T cells. Renal biopsies were analyzed by double-label immunohistochemistry.In vitrostimulation of PMN with XCL1 was performed to study its function.Results.Flow cytometry demonstrated coexpression of the activation markers CD25, CD69, and HLA-DR in a significantly higher proportion of T cells in WG patients in comparison to controls. XCL1 was found to be mainly expressed in CD4+CD28− T cells in WG patients. In renal biopsies, the presence of XCL1 was only detected within interstitial CD4+ and CD8+ T cells. Functional studies demonstrated a significant enhancement of IL-8 production in isolated PMN afterin vitrostimulation with XCL1. There were no significant differences in XCL1 serum concentrations between WG patients and controls (p = 0.88).Conclusion.Our data indicated increased expression of XCL1 in CD4+ and CD8+ T cells in WG. Considering its function as a lymphocyte-specific chemoattractant, XCL1 might be a key modulator of T cell recruitment in WG. Functional studies further suggest that XCL1 may support vascular inflammation by induction of release of interleukin 8 in PMN.

Objective: The aim of this study was to establish a preclinical mouse model to study metastases of paediatric rhabdomyosarcoma at the macroscopic and cellular levels, with different imaging methods. Experimental Design: The alveolar rhabdomyosarcoma cell line Rh30 was stably transfected with the red fluorescent protein (DsRed2) then was xenotransplanted (intravenous injection [n = 8], and footpad injection [n = 8]) into nude mice (NMRI nu/nu). Macroscopic imaging of metastases was performed using DsRed2‐fluorescence and flat‐panel volumetric computed tomography scan. In a further series of animals (n = 8), in vivo cell trafficking of rhabdomyosarcoma cells using cellular imaging with an Olympus OV100 variable‐magnification small‐animal imaging system was used. Results: Metastases in the pelvis, thoracic wall and skin were visualized by fluorescence imaging. Pelvic metastases were found after tail vein injection and at other metastatic sites after footpad injection. Flat‐panel volumetric computed tomography scan data allowed highly specific analysis of contrast between tumour and surrounding tissue. Correlation between fluorescence and flat‐panel volumetric computed tomography scan imaging data was observed. Single‐cell imaging visualized tumour cells in the vessels and demonstrated the arrest of tumour cells at vessel junctions followed by extravasation of the tumour cells. Conclusion: We established a model for visualization of experimental metastatic invasion and describe relevant tools for imaging childhood rhabdomyosarcoma metastases at the macroscopic and cellular levels. Imaging of cell trafficking visualized the behaviour of tumour cells and development of metastases by accumulation and extravasation of rhabdomyosarcoma cells.

Background. At early stages of kidney development, the neural cell adhesion molecule (NCAM) is highly expressed on cells of the metanephrogenic mesenchyme. During maturation of the fetal kidney, NCAM gradually disappears. So far, it has been widely accepted that NCAM in the adult kidney is only expressed by nerves, and not by other cell types. Methods. NCAM expression was analysed in human adult healthy and diseased kidneys by immunohistochemistry and western blot analysis. NCAMþ renal interstitial cells were further characterized by double immunofluorescent staining using antibodies against neurofilaments, a smooth muscle actin, vimentin, a5b1 integrin, CD68, CD11c, HLA-DR and the potential progenitor cell markers CD34, CD117, CD133, CD24, nestin and cadherin-11. Results. In adult human kidneys, NCAM expression is restricted to rare interstitial cells with dendritic morphology, which are neurofilament-negative and predominantly localized on the corticomedullary junction. They are also negative for fibroblast cell markers, but co-express the haematopoietic stem cell markers CD34 and CD133. The number of NCAMþ interstitial cells increased in the initial phases of interstitial fibrosis. Western blot analysis of renal tissues with incipient interstitial fibrosis tissues showed the expression of the 140 kDa NCAM isoform. Conclusions. These data indicate that a rare subpopulation of NCAMþ interstitial cells could represent renal progenitors, and that NCAMþ interstitial cells can participate in the initial phase of interstitial fibrosis.

Background/Aims Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm. Procedure The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice. Results After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 × 10 6 ) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system. Conclusion This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine™ 2000 and FuGENE 6™; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61–86%) than the SeVV-GFP (4–24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7–43%) compared to Ad-GFP (4–16%). The Lipofectamine™ 2000 revealed a higher transfection efficiency than the FuGENE 6™ for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

T cells develop in the thymus in a highly specialized cellular and extracellular microenvironment. The basement membrane molecule, laminin-5 (LN-5), is predominantly found in the medulla of the human thymic lobules. Using high-resolution light microscopy, we show here that LN-5 is localized in a bi-membranous conduit-like structure, together with other typical basement membrane components including collagen type IV, nidogen and perlecan. Other interstitial matrix components, such as fibrillin-1 or -2, tenascin-C or fibrillar collagen types, were also associated with these structures. Three-dimensional (3D) confocal microscopy suggested a tubular structure, whereas immunoelectron and transmission electron microscopy showed that the core of these tubes contained fibrillar collagens enwrapped by the LN-5-containing membrane. These medullary conduits are surrounded by thymic epithelial cells, which in vitro were found to bind LN-5, but also fibrillin and tenascin-C. Dendritic cells were also detected in close vicinity to the conduits. Both of these stromal cell types express major histocompatibility complex (MHC) class II molecules capable of antigen presentation. The conduits are connected to blood vessels but, with an average diameter of 2 μm, they are too small to transport cells. However, evidence is provided that smaller molecules such as a 10 kDa dextran, but not large molecules (>500 kDa), can be transported in the conduits. These results clearly demonstrate that a conduit system, which is also known from secondary lymphatic organs such as lymph nodes and spleen, is present in the medulla of the human thymus, and that it might serve to transport small blood-borne molecules or chemokines to defined locations within the medulla.

Background/Purpose Enhanced green fluorescent protein (eGFP) is widely used as a marker in different biologic systems. After vector transfection, eGFP is expressed by eukaryotic cells and can be visualized using fluorescent microscopy. The aim of this study was to establish an eGFP-transfected human hepatoblastoma (HB) cell line as tool for further basic research studies. Methods The HB cell line HUH6 was transfected with the pEGFP-N1 vector by liposomal transfection. Enhanced green fluorescent protein–positive cells were sorted out by fluorescence-activated cell sort and selected using G418 resistance. Expression of eGFP-messenger RNA was assessed by single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Original and transfected cells were compared biologically and cytomorphologically. Results Vector transfection produced up to 15% eGFP-positive cells. After fluorescence-activated cell sort and G418 selection, a pure cell line was established with 100% eGFP-positive cells. Reverse transcriptase polymerase chain reaction revealed constant expression of eGFP-messenger RNA in fluorescending cells. Analysis of cell characteristics revealed no differences between transfected and original cells. Conclusions For the first time, the authors established an eGFP-transfected HB cell line. This cell line can serve as a promising tool for further studies investigating HB in vitro and in vivo. Our model might also be a basis for similar work on other pediatric solid tumors.

Costimulatory signals play a key role in regulating T cell activation and are believed to have decisive influence in the inciting and perpetuating cellular effector mechanisms in autoimmune diseases such as multiple sclerosis (MS). Inducible costimulator protein (ICOS), a recently identified member of the CD28-family, presumably affects the differentiation of Th1/Th2 cells after primary activation and modulates the immune response of effector/memory T cells. This study examines the expression and functional role of ICOS costimulation in healthy donors and patients with MS. After nonspecific or antigen-specific stimulation, ICOS is preferentially expressed on CD4 Th2-T cells. ICOS-costimulation affects the production of Th1 and Th2 cytokines both in the absence and presence of B7/CD28 costimulation, thus suggesting that ICOS costimulation can modulate cytokine secretion also in a CD28-independent manner. Levels of constitutive and inducible ICOS expression on human T cell subsets from peripheral blood were quantified in healthy donors and patients with MS. Constitutive expression of ICOS on T cells varies between 0.1% and 42.3%. There were no significant differences between both groups in the baseline expression or inducibility of ICOS on CD4 or CD8 T cells. ICOS expression could be demonstrated on CSF T lymphocytes in patients with acute MS relapses but was not elevated compared with peripheral blood. In essence we show that ICOS is upregulated on human T cells after stimulation and can modulate both Th1 and Th2 cytokine production in the absence and presence of B7-costimulation. In MS patients we demonstrate the functionality of the ICOS costimulatory pathway. Potential implications of ICOSL/ICOS interactions for MS immunopathogenesis are discussed.

Here we describe the in vitro generation of a novel adherent cell fraction derived from highly enriched, mobilized CD133+ peripheral blood cells after their culture with Flt3/Flk2 ligand and interleukin-6 for 3 to 5 weeks. These cells lack markers of hematopoietic stem cells, endothelial cells, mesenchymal cells, dendritic cells, and stromal fibroblasts. However, all adherent cells expressed the adhesion molecules VE-cadherin, CD54, and CD44. They were also positive for CD164 and CD172a (signal regulatory protein-α) and for a stem cell antigen defined by the recently described antibody W7C5. Adherent cells can either spontaneously or upon stimulation with stem cell factor give rise to a transplantable, nonadherent CD133+CD34−stem cell subset. These cells do not generate in vitro hematopoietic colonies. However, their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice induced substantially higher long-term multilineage engraftment compared with that of freshly isolated CD34+ cells, suggesting that these cells are highly enriched in SCID-repopulating cells. In addition to cells of the myeloid lineage, nonadherent CD34− cells were able to give rise to human cells with B-, T-, and natural killer–cell phenotype. Hence, these cells possess a distinct in vivo differentiation potential compared with that of CD34+ stem cells and may therefore provide an alternative to CD34+ progenitor cells for transplantation.

Cadherins are a family of cell adhesion molecules that mainly mediate homotypic homophilic interactions, but for E-cadherin, heterophilic interactions with the integrin alpha(E)(CD103)beta(7) have also been reported. In the human thymus, where thymocytes develop in close contact with thymic stromal cells, E-cadherin expression was detected on thymic epithelial cells. By immunofluorescence staining, the strongest expression of E-cadherin was observed on medullary thymic epithelial cells. These cells also express cytosolic catenins, which are necessary to form functional cadherin-catenin complexes. Regardless of their developmental stage, human thymocytes do not express E-cadherin, indicating that homophilic interactions cannot occur. Flow cytometric analysis revealed that the E-cadherin ligand CD103 is expressed on subpopulations of the early CD4(-) CD8(-) double-negative and of the more mature CD8(+) single-positive thymocytes. Using an in vitro cell adhesion assay, double-negative and CD8(+) single-positive thymocytes adhered strongly to isolated thymic epithelial cells. These adhesive interactions could be inhibited by antibodies against E-cadherin or CD103. CD8(+) thymocytes showed a proliferative response when incubated with thymic epithelial cells. This mitogenic effect was inhibited by antibodies against CD103, which strongly indicates a direct involvement of the adhesive ligand pair CD103-E-cadherin in human thymocyte cell proliferation.

The α-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, α-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of α-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of α-defensins. The in vitro and in vivo findings suggest that α-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.

The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures., Secondary ion mass spectrometry is effective for imaging cellular turnover, but it cannot identify subcellular structures such as organelles. Here the authors show a method for correlating this technique with high-resolution fluorescence microscopy, enabling the measurement of turnover in cellular compartments.

Human gammadelta T lymphocytes play an important role in nonadaptive reactions to infection and early tumor defense. This is the first report that freshly isolated, native gammadelta T cells of some healthy donors can kill human neuroblastoma cells to varying degrees. Their killing ability was increased and maintained during expansion and cultivation with interleukin-2 (IL-2; 400 IU/mL) for as long as 30 days (100% specific lysis at an effector-to-target cell (E:T) ratio of 20:1). gammadelta T lymphocytes without this spontaneous killing ability gained a specific cytolytic activity of 81% +/- 10.4% SD after stimulation with IL-2 for 24 hours. gammadelta cells were isolated from peripheral blood by positive enrichment (using a magnetic cell sorting system; purity, 95.2% +/- 3.2% SD, n = 21). High natural cytotoxic activity against human neuroblastoma cell lines (>50% specific lysis at an E:T ratio of 20:1) was exhibited by one of 11 donors, whereas two of 11 showed medium cytotoxicity (30% to 50% specific lysis). Eight of 11 donors showed very slight or no lytic activity against human neuroblastoma cells (

Activation of the nuclear transcription factor κB (NF-κB) regulates the expression of inflammatory genes crucially involved in the pathogenesis of inflammatory diseases. NF-κB governs the expression of adhesion molecules that play a pivotal role in leukocyte–endothelium interactions. We uncovered the crucial role of NF-κB activation within endothelial cells in models of immune-mediated diseases using a “sneaking ligand construct” (SLC) selectively inhibiting NF-κB in the activated endothelium. The recombinant SLC1 consists of three modules: (i) an E-selectin targeting domain, (ii) a Pseudomonas exotoxin A translocation domain, and (iii) a NF-κB Essential Modifier-binding effector domain interfering with NF-κB activation. The E-selectin–specific SLC1 inhibited NF-κB by interfering with endothelial IκB kinase 2 activity in vitro and in vivo. In murine experimental peritonitis, the application of SLC1 drastically reduced the extravasation of inflammatory cells. Furthermore, SLC1 treatment significantly ameliorated the disease course in murine models of rheumatoid arthritis. Our data establish that endothelial NF-κB activation is critically involved in the pathogenesis of arthritis and can be selectively inhibited in a cell type- and activation stage-dependent manner by the SLC approach. Moreover, our strategy is applicable to delineating other pathogenic signaling pathways in a cell type-specific manner and enables selective targeting of distinct cell populations to improve effectiveness and risk–benefit ratios of therapeutic interventions.

Clinical outcome of patients suffering from head neck squamous cell carcinomas is still poor due to recurrent disease and surgical limitations. There is still a demand for multimodality approaches and new therapeutic options. Hypericin is a promising phototoxic drug which was investigated for its effects on head neck squamous cell carcinoma cells in vitro. FaDu cells incubated with or without hypericin were illuminated (450-700 nm, 50,000 lx) for different time periods. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide- and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay were used to score metabolic and apoptotic activity. Even after the shortest illumination FaDu cells incubated with hypericin showed massive reduction of metabolism and excessive apoptosis. This was present even with the lowest hypericin concentration. Cells without hypericin or without illumination were not affected. These photosensitizing effects of hypericin could be suitable for clinical application and could lead to the development of an intraoperative photodynamic therapy of head neck squamous cell carcinomas.

Previous studies indicate that the release of proteases, including the gelatinase matrix metalloproteinase (MMP)-9, from mature granulocytes plays a crucial role in cytokine-induced hematopoietic stem and progenitor cell (HSPC) mobilization. However, studies with MMP-9-deficient mice revealed that HSPC mobilization was normal in these animals, suggesting that additional proteases must be active at clinically relevant cytokine concentrations. In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. During granulocyte colony-stimulating factor-induced HSPC mobilization, highly elevated serum concentrations of MMP-8 were observed on days 4 to 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34(+) cells. Elevated serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking.

The sentinel and immune functions of microglia require rapid and appropriate reactions to infection and damage. Their Toll-like receptors (TLRs) sense both as threats. However, whether activated microglia mount uniform responses or whether subsets conduct selective tasks is unknown. We demonstrate that murine microglia reorganize their responses to TLR activations postnatally and that this process comes with a maturation of TLR4-organized functions. Although induction of MHCI for antigen presentation remains as a pan-populational feature, synthesis of TNFα becomes restricted to a subset, even within adult central nervous system regions. Response heterogeneity is evident ex vivo, in situ, and in vivo, but is not limited to TNFα production or to TLR-triggered functions. Also, clearance activities for myelin under physiological and pathophysiological conditions, IFNγ-enforced upregulation of MHCII, or challenged inductions of other proinflammatory factors reveal dissimilar microglial contributions. Notably, response heterogeneity is also confirmed in human brain tissue. Our findings suggest that microglia divide by constitutive and inducible capacities. Privileged production of inflammatory mediators assigns a master control to subsets. Sequestration of clearance of endogenous material versus antigen presentation in exclusive compartments can separate potentially interfering functions. Finally, subsets rather than a uniform population of microglia may assemble the reactive phenotypes in responses during infection, injury, and rebuilding, warranting consideration in experimental manipulation and therapeutic strategies.

Endothelial progenitor cells (EPCs) protect the kidney from acute ischemic injury. The aim of this study was to analyze whether pretreatment of murine “early outgrowth” EPCs (eEPCs) with the hormone melatonin increases the cells' renoprotective effects in the setting of murine acute ischemic renal failure. Male (8–12 wk old) C57Bl/6N mice were subjected to unilateral ischemia-reperfusion injury postuninephrectomy (40 min). Postischemic animals were injected with either 0.5×106 untreated syngeneic murine eEPCs or with cells, pretreated with melatonin for 1 h. Injections were performed shortly after reperfusion of the kidney. While animals injected with untreated cells developed acute renal failure, eEPC pretreatment with melatonin dramatically improved renoprotective actions of the cells. These effects were completely reversed after cell pretreatment with melatonin and the MT-1/-2 antagonist luzindole. In vitro analysis revealed that melatonin reduced the amount of tumor growth factor-β-induced eEPC apoptosis/necrosis. Secretion of vascular endothelial growth factor by the cells was markedly stimulated by the hormone. In addition, migratory activity of eEPCs was enhanced by melatonin and supernatant from melatonin-treated eEPCs stimulated migration of cultured mature endothelial cells. In summary, melatonin was identified as a new agonist of eEPCs in acute ischemic kidney injury.

Purpose Elderly people often develop visceral obesity accompanied by osteoporosis. Visceral adipocytes secrete a number of adipokines and cytokines which augment the development of arteriosclerosis and type 2 diabetes. Bone marrow fat cells also secrete these pro-inflammatory cytokines which stimulate osteoclast and inhibit osteoblast activity. Ovariectomized (ovx) rats also develop general and bone marrow obesity and osteoporosis both of which can be partially prevented by estradiol (E2) and the special extract of Cimicifuga racemosa (CR) BNO 1055. Whether this extract or the thereof isolated triterpene-saponins or polar substances can also prevent bone marrow obesity and thereby the development of osteoporosis was compared with the effects of estradiol (E2). Methods Rats were ovx and fed with food containing either CR BNO 1055 or its triterpene-saponin or polar constituents or with E2 for 4 weeks. Histomorphometry and STRUT analyses were applied to histological preparations to determine the amount of trabecles, hematopoietic and fat tissue in the bone marrow. Results Ovx rats lost significant amounts of trabecular BMD, surface and nodes while the number of free trabecular ends and fat load in the marrow increased. This was totally prevented by E2 and partially by CR BNO 1055 and the triterpene-saponin but not by the polar fraction. High serum osteocalcin and CrossLaps levels were reduced by E2 and the S-fraction. Conclusions It is well established that E2 prevents osteoporosis. It is also known that CR BNO 1055 does not contain estrogenic substances. CR BNO 1055 and the triterpene-saponin-fraction reduced the development of osteoporosis most likely by a reduction of the bone marrow fat load and possibly by reducing the secretion of pro-inflammatory cytokines. Hence, the triterpene-saponin-fraction may serve as a basis for a new osteoporosis preventing preparation also in human patients.

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis., Author Summary Infection with mycobacteria results in a host response which results in the formation of granulomas, highly organized structures characterized by the presence of macrophages, which are considered to rely solely on invariant immune receptors. On the other hand, the presence of variable immune receptors is required for granuloma formation but this process is not solely dependent on T cells. Furthermore, TNF is required for the maintenance of the mycobacterial granuloma structure in humans. We now find evidence for subpopulations of human and mouse macrophages that express variable αβ T cell receptors (TCRαβ). Engagement of the macrophage-TCRαβ triggers CCL2 release and phagocytosis of baits directed to this receptor is enhanced. TCRαβ bearing macrophages accumulate in human tuberculosis granulomas and anti-TNF treatment of macrophages results in downregulation of the TCRαβ, which is associated with caspase 3 cleavage and suppression of TCRξ. Anti-TNF treatment reduces mycobacteria induced cluster formation of TCRαβ positive macrophages, which is in line with reduced granuloma formation in rag1–/–(T cell rag1+/+) and TNF–/–(T cell TNF+/+) chimeric mice. Consequently, both chimeras show reduced CCL2 staining after mycobacterial infection. In summary, we have identified a recombinatorial immunoreceptor in monocytes/macrophages and demonstrate its implication in mycobacterial infection.

This review focuses on technical advances in fluorescence microscopy techniques including laser scanning techniques, fluorescence-resonance energy transfer (FRET) microscopy, fluorescence lifetime imaging (FLIM), stimulated emission depletion (STED)-based super-resolution microscopy, scanning confocal endomicroscopes, thin-sheet laser imaging microscopy (TSLIM), and tomographic techniques such as early photon tomography (EPT) as well as on clinical laser-based endoscopic and microscopic techniques. We will also discuss the new developments in the field of fluorescent dyes and fluorescent genetic reporters that enable new possibilities in high-resolution and molecular imaging both in in vitro and in vivo. Small animal and tissue imaging benefit from the development of new fluorescent proteins, dyes, and sensing constructs that operate in the far red and near-infrared spectrum.

In recent years, slide-based cytometry has become a key technology for polychromatic cytometric investigations, and many efforts have been made to increase the number of measurable fluorochromes for multiparametric analysis. Sequential photobleaching of fluorochromes next to very photostable dyes is one approach for this technology. As the ALEXA dyes are known to be photostable as compared to the conventional fluorochromes FITC, PE (Riggs et al., Am J Pathol 1958;34:1081-1097), and APC, a differentiation within a fluorochrome pair is possible. Here, we have analyzed the newly available NorthernLights secondary antibodies for use in slide-based cytometry and microscopy. Currently, these fluorochrome-conjugates are now available with three distinct excitation- and emission maxima (NL493, NL557, NL637). Their spectral properties are similar to the frequently used fluorochromes FITC, PE, and APC and can, therefore, be used with most common excitation sources of cytometers or microscopes. As the NorthernLights are bright, resistant to photobleaching, stable in alcohols and xylene and of affordable price, these dyes are promising candidates for use with most laser- and HBO/XBA-based fluorescence microscopy-like techniques.

Background: Traditionally, specific immune recognition is thought to be restricted to lymphoid cells. Recent evidence of T cell receptor (TCR) expression in neutrophil and eosinophil granulocytes, however, indicates that variable immunoreceptors are not restricted to lymphocytes. In this study, we tested whether monocytes/macrophages, the second major population of professional phagocytes next to neutrophils, express a variable immunoreceptor. Methods and Findings: We demonstrate that subpopulations of monocytes, monocyte-derived macrophages and tissue macrophages express a TCRab immunoreceptor and essential components of the TCR signaling complex. Consistent with V(D)J rearrangement of the TCRb locus, we find individual-specific expression of TCR repertoires in human monocytes/macrophages. In vitro, Th1/Th2 cytokines (IFNg, IL-4) and forced import and export of cholesterol elicit expression of differential TCR Vab-repertoires in monocyte derived macrophages from healthy individuals. This indicates that normal macrophages have the capacity to respond to various stimuli in a flexible manner. Engagement of the macrophage-TCR stimulates secretion of the major monocyte chemoattractant MCP-1. In vivo, analysis of freshly obtained artery specimens from patients (n = 10) with severe carotid atherosclerosis consistently revealed the excessive presence of TCRab+ macrophages that express highly restricted Vb repertoires including Vb16 and Vb22. Similar results were obtained for advanced coronary artery disease. Experimental atherosclerosis in mouse carotids induces accumulation of TCRab bearing macrophages in the vascular wall and TCR deficient rag-/- mice have a reduced inflammatory response in their media relative to wildtype controls. Conclusions: We have identified an as yet undiscovered myeloid machinery for variable immune recognition in macrophages which is implicated in a major inflammatory disease as paradigmatically shown for atherosclerosis. These results may further our understanding of macrophage-dependent clinical diseases.

Background and aim Endothelial progenitor cells (EPCs) have been shown to promote neovascularization under physiologic and pathologic conditions. Statins have been documented to increase the total number of circulating EPCs in long-term treated patients. Lipid apheresis is used to treat patient with refractory hyperlipidemia. The aim of our study was to evaluate whether lipid apheresis is associated with EPC mobilization. Methods Thirteen patients with refractory hyperlipidemia (analysis at the beginning and at the end of a single lipid apheresis treatment) and 10 healthy controls were included into the study. For quantifying total peripheral EPCs, CD133+/Flk-1+ myelo-monocytic blood cells were enumerated by flow cytometry. The proliferative potential of EPCs was evaluated by a "colony-forming unit" assay. In some patients, EPC eNOS expression was evaluated before and after treatment. Results Circulating EPCs and the cells' proliferative activity were lower in hyperlipidemia patients as compared to controls (0.14 +/- 0.07 vs. 0.6 +/- 0.14, P = 0.01, and 13.9 +/- 4.9 vs. 45.6 +/- 8.1, P = 0.0007). Lipid apheresis treatment was not associated with an increase in total EPCs. The cells' proliferative activity was strongly stimulated by lipid apheresis as reflected by an increase in the number of EPC colonies (13.9 +/- 4.9 to 34.1 +/- 7.3, P = 0.035). Analysis of EPC eNOS expression revealed a threefold increase in the cellular expression intensity after lipid apheresis. Conclusions Patients with refractory hyperlipidemia exhibit lower peripheral EPC numbers and a lower proliferative activity of circulating EPCs than healthy controls. A single lipid apheresis treatment significantly stimulates EPC proliferation, it furthermore increases cellular eNOS. In summary, these results show that lipid apheresis mediates beneficial effects on the EPC system as an essential element in the process of vascular repair in the human organism.

The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2-8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94-97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively.

Treatment of childhood rhabdomyosarcoma is limited by recurrent disease and the development of multidrug resistance. Therefore, novel treatment options are desirable. Photodynamic therapy (PDT) using the photodynamic agent hypericin is proposed as an alternative approach for intraoperative visualization and treatment of this disease. The aim of this study was to investigate in vitro effects of hypericin on childhood rhabdomyosarcoma and to evaluate photodynamic therapy as a possible basis for treatment. Rhabdomyosarcoma cells and fibroblasts (control) were incubated with increasing concentrations of hypericin. in vitro uptake and visualization of hypericin was evaluated by fluorescence microscopy and FACS. For photodynamic therapy, cells were exposed to white light for different time periods. Cytopathologic effects were assessed using standard histology. Cancer cells were investigated for cell viability (MTT assay), proliferative activity (Ki-67 assay), and apoptosis (TUNEL test). A 100% uptake of hypericin was found within the population of rhabdomyosarcoma cells. Uptake of hypericin in the fibroblasts was much less than in rhabdomyosarcoma cells. Hypericin without exposure to white light had no effect on tumor cell viability. After irradiation, PDT resulted in a nearly complete inhibition of cell proliferation of rhabdomyosarcoma cells with a corresponding increase in the frequency of apoptosis. In fibroblasts, PDT was less effective compared to tumor cells. Our data suggest hypericin as a novel tool for visualization and photodynamic therapy of childhood rhabdomyosarcoma.

The fluorescence of protophorphyrin IX (PpIX) synthesized after incubation with 5-aminolevulinic acid (5-Ala) is used for the intraoperative visualisation of glioma cells in vivo. Such fluorescence may also be useful for the photodynamic therapy (PTD) of gliomas. A significant difference of fluorescence intensity in tumor cells compared to neurons is required for this application. To explore this, eight human glioma cell lines (LN-18, LN-428, U87MG, U373MG, D247MG, U251MG, LN-308, T98G) were compared with human astrocytes (SV-FHAS) and rat neurons after incubation for different periods of time in vitro with 5-Ala (1 mg/ml). Fluorescence intensity profiles were measured by a digital camera comparing glioma cell lines with control cells. All glioma cell lines could be discriminated from neural cells by their intensity of fluorescence by post-hoc tests for pairwise comparisons using Tukey's honestly significant difference test, at the global significance level of 5%. The glioma cell lines showed significant variation in this possibly limiting clinical use of fluorescence as a guide for resection.

The alpha-defensins human neutrophil peptides (HNPs)-1, -2, and -3 have been described as cytotoxic peptides with restricted expression in neutrophils and in some lymphocytes. In this study we report that HNPs-1, -2, and -3 are also expressed in renal cell carcinomas (RCCs). Several RCC lines were found to express mRNA as well as the specific peptides of HNP-1, -2, and -3 demonstrated by reverse transcriptase-polymerase chain reaction, mass spectrometric, and flow cytometric analyses. At physiological concentrations HNPs-1, -2, and -3 stimulated cell proliferation of selected RCC lines in vitro but at high concentrations were cytotoxic for all RCC lines tested. As in RCC lines, alpha-defensins were also detected in vivo in malignant epithelial cells of 31 RCC tissues in addition to their expected presence in neutrophils. In most RCC cases randomly, patchy immunostaining of alpha-defensins on epithelial cells surrounding neutrophils was seen, but in six tumors of higher grade malignancy all tumor cells were diffusely stained. Cellular necrosis observed in RCC tissues in association with extensive patches of HNP-1, -2, and -3, seemed to be related to high concentrations of alpha-defensins. The in vitro and in vivo findings suggest that alpha-defensins are frequent peptide constituents of malignant epithelial cells in RCC with a possible direct influence on tumor proliferation.

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different alpha, three beta and three gamma subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin alpha1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the alpha2 chain (LN-2/4) or the alpha5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin alpha4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the alpha3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4- CD8- thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin alpha6beta1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN-5 were integrin alpha6beta4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.

Complete experimental data sets of HLA-ligand motifs and T-cell recognition patterns can be derived from combinatorial peptide libraries. These data provide the exact molecular basis for a fast development of synthetic vaccines, T-cell superagonists and non-peptide antagonists. Patient-specific peptides, peptidomimetics and vaccines of highest reactivity can be derived directly from the data sets via our prediction programme EPIPREDICT. The resulting lead structures may be developed into valuable diagnostics and therapeutic tools for the treatment of viral infections, autoimmune diseases and tumors. As one example, antibody and T cell recognition in the intestinal auto-immune disease, coeliac disease was investigated in more detail concerning the deamidation of gamma-gliadin peptides by tissue transglutaminase 9tTG) leading to autoreactive peptides specific for HLA-DQA1*0501, DQB1*0201.

Synthetic peptide libraries have been shown to represent tools in elucidating molecular interactions in T cell mediated immune response [1,2]. New reliable synthetic concepts and bioassays were developed to describe peptide-MHC interactions in a quantitative way [3]. Two-dimensional databases, describing perfectly the allele-specific peptide binding to many MHC class I and II alleles, have been generated using purified MHC molecules. The databases are used for the design and synthesis of so-called directed peptide and nonpeptide libraries, which are applied to study antigen recognition of T cells from patients with autoimmune diseases. The knowledge about interactions within the trimolecular complex (MHC-peptide-TCR, Figure 1) is used for our new algorithm EPIPREDICT predicting MHC class II self and foreign ligands T cell epitopes.

paper by Fujiki et al. entitled ‘‘Quantification of greenfluorescence protein by in vivo imaging, PCR and flow cyto-metry: comparison of transgenic strains and relevance for fetalcell microchimerism’’ has a very promising approach to imagefetal cell microchimerism (1). The paper compares two typesof transgenic mice expressing green fluorescent protein (GFP)for the future study of fetal microchimerism—the processwhere fetal cells enter the maternal circulation, and in humanscan persist in the maternal blood and tissues for decades. TheGFP transgenic mice chosen for study are the C51B6/6-Tg(ACTB-EGFP) Osb/J (CAG) and the ROSA 26-EGFP. Theauthors conclude that the CAG mice have much brighter cells,but the ROSA 26-EGFP cells more consistently express GFP.The authors compare imaging, PCR, and flow cytometry(FCM) to image cells from these mice and conclude that flowcytometry is the method of choice, since ‘‘. . . due to the exist-ing status of this imaging technology and the purely quantita-tive nature of PCR amplification, FCM provides the best over-all approach for the quantitative and qualitative evaluation ofrare transgenic microchimeric fetal cells.’’The authors, however, are apparently unaware of thepower of fluorescence protein-imaging in vivo. Chishima et al.(2) pioneered this area and showed that single cancer cellsexpressing GFP could be readily imaged in autopsied miceagainst the large background of normal tissue. Subsequently,Yang et al. (3) showed that tumors could be whole-bodyimaged with GFP in live unrestrained mice. Yang et al. (4)then made two more important advances, and showed in skinflaps in live mice that single GFP cells could be imaged inmany organs. Yang et al. (5) then showed that tumor cellsexpressing red fluorescent protein (RFP) in the cytoplasm andGFP in the nucleus, as well as GFP host stromal cells, couldnoninvasively be imaged when grown in the mouse foot pad.Yamamoto et al. (6) and Yamauchi et al. (7,8) showedthat single cells labeled in the nucleus with GFP and in thecytoplasm with RFP could be imaged in mice both noninva-sively as well as in skin flaps. Yamauchi et al. (7,8) imaged thedual color cells in blood vessels as they trafficked in real time.Imaging cell trafficking was greatly enhanced by use of thevariable-magnification Olympus OV100 imaging system.Hayashi et al. (9) used the dual-colored cells and OlympusOV100 to image cells trafficking in lymphatics.Use of a high-powered variable-magnification imagingsystem, such as the OV100, rather than the IVIS 200 photoncounting device used by Fujiki et al. which does not produceimages, enables single fluorescent-protein-labeled cells to bevisualized in real time even during trafficking (10–12). Suchan approach offers many advantages over the IVIS 200 andflow cytometry, since cells can be visualized in the live animalwhich is very important for studying microchimerism (13).In parallel to the rapid development of instrumentation,many efforts have been made to optimize the existing ‘‘classical’’

Introduction Sepsis is characterized by systemic microvascular dysfunction. Endothelial progenitor cells (EPCs) are critically involved in maintaining vascular homeostasis under both physiological and pathological conditions. The aim of the present study was to analyze the endothelial progenitor cell system in patients suffering from sepsis with acute renal dysfunction. Methods Patients with newly diagnosed sepsis were recruited from the ICU in a nonrandomized prospective manner. Blood samples were obtained within the first 12 hours after the diagnosis of sepsis. For quantifying endothelial progenitor cells (EPCs), CD133+/Flk-1+ cells were enumerated by cytometric analysis. Analysis of EPC proliferation was performed by a colony-forming units (CFU) assay. Blood concentrations of proangiogenic mediators were measured by ELISA. Acute renal dysfunction was diagnosed according to the Acute Kidney Injury Network (AKIN) criteria. Depending on the overall mean creatinine concentration during the stay at the ICU, patients were either assigned to a 'normal creatinine group' or to a 'high creatinine group'. Survival rates, frequency of dialysis, the simplified acute physiology score (SAPS) II scores, and different laboratory parameters were collected/used for further clinical characterization Results Circulating EPCs were significantly higher in all sepsis patients included in the study as opposed to healthy controls. Patients within the 'high creatinine group' showed an even more pronounced EPC increase. In contrast, EPC proliferation was severely affected in sepsis. Neither total circulating EPCs nor EPC proliferation differed between patients requiring dialysis and patients without renal replacement therapy. Cell numbers and cell proliferation also did not differ between surviving patients and patients with sepsis-related death. Serum levels of vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF-1), and Angiopoietin-2 were higher in sepsis than in healthy controls. Sepsis patients within the 'high creatinine group' showed significantly higher mean serum levels of uric acid. Conclusions Sepsis significantly affects the endothelial progenitor cell system, as reflected by increased EPC numbers, increased concentrations of proangiogenic mediators, and reduced proliferative capacity of the cells. This occurs independently from the frequency of dialysis and from patient survival. Increased serum levels of uric acid are possibly responsible for stronger EPC mobilization in sepsis patients with higher average creatinine levels. peerReviewed

Immunological dogma holds that variable immunoreceptors are restricted to lymphocytes. However, recent studies demonstrate that a subpopuation of human neutrophils expresses the TCRαβ (PNAS 103:14441, 2006) and mouse thymic granulocytes exhibit rearrangement of their TCR loci (Nature 452:764, 2008). Here, we provide evidence that a subpopulation of circulating human monocytes, monocyte-derived macrophages and tissue macrophages constitutively express a TCRαβ-based variable immunoreceptor. TCRαβ + monocyte/macrophage subpopulations were assessed using with laser scanning cytometry and repertoire diversities were determined by CDR3 spectratyping. We find that human macrophages express individual-specific TCR Vαβ repertoires. Th1 or Th2 cytokine dependent in vitro differentiation triggers expression of distinct Vαβ repertoires in macrophages. Moreover, Th1 polarized macrophages show enhanced secretion of the major monocyte chemoattractant MCP-1 in response to costimulation with αCD3/CD28 . The presence of a variable immunoreceptor in macrophages together with our previous identification of the TCR in a subset of neutrophils challenge the traditional dichotomy of the vertebrate immune system into non-specific/myeloid and antigen-specific/lymphoid immunity. They unravel a much closer kinship between both lineages than commonly assumed. Supported by DFG grants KA 1078/4-1, BE 1768/5-1