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It has been shown that synovial fibroblasts (SF) play a key role in the initiation of inflammation and joint destruction, leading to arthritis progression. Fibroblasts may express major histocompatibility complex class II region (MHCII) molecules, and thus, they could be able to process and present antigens to immunocompetent cells. Here we examine whether different types of fibroblasts (synovial, dermal, and thymic murine fibroblasts, destructive LS48 fibroblasts, and noninvasive NIH/3T3 fibroblasts) may be involved in the initiation of rheumatoid arthritis (RA) pathogenesis and can process and present type II collagen (COL2)—an autoantigen associated with RA. Using a panel of MHCII/Aq-restricted T-cell hybridoma lines that specifically recognize an immunodominant COL2 epitope (COL2259–273), we found that NIH/3T3 fibroblasts activate several T-cell clones that recognize the posttranslationally glycosylated or hydroxylated COL2259–273 epitope. The HCQ.3 hybridoma, which is specific for the glycosylated immunodominant COL2 epitope 259–273 (Gal264), showed the strongest response. Interestingly, NIH/3T3 cells, but not destructive LS48 fibroblasts, synovial, dermal, or thymic fibroblasts, were able to stimulate the HCQ.3 hybridoma and other COL2-specific T-cell hybridomas. Our experiments revealed that NIH/3T3 fibroblasts are able to activate COL2-specific T-cell hybridomas even in the absence of COL2 or a posttranslationally modified COL2 peptide. The mechanism of this unusual activation is contact-dependent and involves the T-cell receptor (TCR) complex.

Radiation-attenuated intracellular parasites are promising immunization strategies. The irradiated parasites are able to invade host cells but fail to fully replicate, which allows for the generation of an efficient immune response. Available radiation technologies such as gamma rays require complex shielding constructions and are difficult to be integrated into pharmaceutical production processes. In this study, we evaluated for the first time low-energy electron irradiation (LEEI) as a method to generate replication-deficient Toxoplasma gondii and Cryptosporidium parvum. Similar to other radiation technologies, LEEI mainly damages nucleic acids; however, it is applicable in standard laboratories. By using a novel, continuous, and microfluidic-based LEEI process, tachyzoites of T. gondii and oocysts of C. parvum were irradiated and subsequently analyzed in vitro. The LEEI-treated parasites invaded host cells but were arrested in intracellular replication. Antibody-based analysis of surface proteins revealed no significant structural damage due to LEEI. Similarly, excystation rates of sporozoites from irradiated C. parvum oocysts were similar to those from untreated controls. Upon immunization of mice, LEEI-attenuated T. gondii tachyzoites induced high levels of antibodies and protected the animals from acute infection. These results suggest that LEEI is a useful technology for the generation of attenuated Apicomplexan parasites and has potential for the development of anti-parasitic vaccines.

The interaction between the mitochondria and the endoplasmic reticulum (ER) is essential for hepatocyte function. An increase in ER–mitochondria contacts (ERMCs) is associated with various metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and its progressive form non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. However, the role of ERMCs in the progression of NAFL to NASH is still unclear. We assessed whether ERMCs could correlate with NAFLD severity. We used a proximity ligation assay to measure the abundance of ERMCs in liver biopsies from patients with biopsy-proven NAFLD (n = 48) and correlated the results with histological and metabolic syndrome (MetS) features. NAFLD patients were included according to inclusion and exclusion criteria, and then assigned to NAFL (n = 9) and NASH (n = 39) groups. ERMCs density could discriminate NASH from NAFL (sensitivity 61.5%, specificity 100%). ERMCs abundance correlated with hepatocellular ballooning. Moreover, the density of ERMCs increased with an increase in the number of MetS features. In conclusion, ERMCs increased from NAFL to NASH, in parallel with the number of MetS features, supporting a role for this interaction in the pathophysiology of NASH.

The interaction between the mitochondria and the endoplasmic reticulum (ER) is essential for hepatocyte function. An increase in ER-mitochondria contacts (ERMCs) is associated with various metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and its progressive form non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. However, the role of ERMCs in the progression of NAFL to NASH is still unclear. We assessed whether ERMCs could correlate with NAFLD severity. We used a proximity ligation assay to measure the abundance of ERMCs in liver biopsies from patients with biopsy-proven NAFLD (

Mesenchymal stromal/stem cells (MSCs) have great capacity for immune regulation. MSCs provide protective paracrine effects, which are partially exerted by extracellular vesicles (EVs). It has been reported that MSCs-derived EVs (MSC-EVs) contain soluble factors, such as cytokines, chemokines, growth factors and even microRNAs, which confer them similar anti-inflammatory and regenerative effects to MSCs. Moreover, MSCs modulate microglia activation through a dual mechanism of action that relies both on cell contact and secreted factors. Microglia cells are the central nervous system immune cells and the main mediators of the inflammation leading to neurodegenerative disorders. Here, we investigated whether MSC-EVs affect the activation of microglia cells by β-amyloid aggregates. We show that the presence of MSC-EVs can prevent the upregulation of pro-inflammatory mediators such as tumor necrosis factor (TNF)-α and nitric oxide (NO). Both are up-regulated in neurodegenerative diseases representing chronic inflammation, as in Alzheimer’s disease. We demonstrate that MSC-EVs are internalized by the microglia cells. Further, our study supports the use of MSC-EVs as a promising therapeutic tool to treat neuroinflammatory diseases.Significance StatementIt has been reported that mesenchymal stromal/stem cells and MSC-derived small extracellular vesicles have therapeutic effects in the treatment of various degenerative and inflammatory diseases. Extracellular vesicles are loaded with proteins, lipids and RNA and act as intercellular communication mediators. Here we show that extracellular vesicles can be taken up by murine microglial cells. In addition, they partially reduce the activation of microglial cells against β-amyloid aggregates. This inhibition of microglia activation may present an effective strategy for the control/therapy of neurodegenerative diseases such as Alzheimer’s disease. Graphic Abstract

The oxytocinergic system has been assumed to contribute to food intake, possibly via interactions with dopamine. However, so far, it is unknown whether oxytocin influences the underlying motivational behaviour towards food. In the present study, we used a food-based approach-avoidance task (AAT) in a randomised, placebo-controlled, double-blind, cross-over design to compare intranasal oxytocin with a placebo. In the AAT, participants pushed or pulled a joystick when images of foods with a high or low craving rating were presented, where differences in response times typically reflect approach and avoidance motivational biases towards positively and negatively valence stimuli, respectively. Thirty-three healthy male participants (age = 25.12 ± 3.51 years; body mass index = 24.25 ± 2.48 kg/m2 ) completed the two-session study, one with placebo and the other with oxytocin. We used mixed-effects models to investigate effects of treatment (oxytocin, placebo), response type (approach, avoid) and stimulus (high, low craving). The results showed that both approach and avoid responses tended to be faster for foods higher in craving compared to foods lower in craving. Most importantly, we did not observe any significant effects of oxytocin compared to placebo in motivational behaviour towards food. Our study demonstrates a general response bias towards foods with different craving values, which could have implications for future studies investigating food-related behaviour. We discuss possible explanations for the null effects of oxytocin and suggest further investigation of the relationship between oxytocin, dopamine and food-reward processing.

Although the seroprevalence of Herpes simplex virus type 1 (HSV-1) currently amounts to ∼ 67% worldwide, the annual incidence of a severe disease progression, particularly herpes encephalitis, is approximately 2–4 cases per 1,000,000 infections. Nucleoside analogues, such as acyclovir (ACV), valacyclovir (VACV) or famciclovir, are still the therapeutic treatment of choice for HSV infections. However, nucleoside drugs have limited efficacy against severe HSV disease and for treatment of nucleoside-resistant viral strains, alternative therapies such as helicase-primase inhibitors (HPIs) which are highly potent by inhibiting viral replication are under development. In preclinical studies we analyzed the antiviral efficacy of drug candidates of a novel compound class of HPIs for the treatment of HSV to identify the most active eutomer structure in an intranasal infection mouse lethal challenge model. HSV-1 infected BALB/c mice treated with vehicle control developed fatal disease according to humane endpoints after 5–7 days. In contrast, the animals dosed orally once daily with the HPI compounds at 10 or 4 mg/kg/day showed a significantly increased survival (70% and 100% for 10 mg/kg/day; 90% and 100% for 4 mg/kg/day, respectively) compared to the vehicle treatment (0–10%), when therapy was initiated 6 h post HSV-1 inoculation. We observed a significantly improved outcome in clinical parameters and survival over 21 days in the group receiving novel HPI candidates using even the lowest dose of 4 mg/kg/day. With VACV treatment of 75 mg/kg daily survival was also significantly increased (80%–90% for 75 mg/kg/day) but to lesser extent. Initial IM-250 therapy at 10 mg/kg/day could be delayed up to 72 h resulting in significantly increased survival compared to the vehicle control. Furthermore, we detected significantly fewer viral genome copies in the lungs and brains of HPI treated animals compared to vehicle (440-fold reduction for 4 mg/kg/day IM-250 in the brain) or VACV controls by quantitative PCR. In conclusion the preclinical studies of the novel HPI compounds showed superior efficacy in comparison to the current standard HSV treatment represented by VACV with respect to the survival according humane endpoints, the clinical score and virus load in lungs and brains. Thus, candidates of this new drug class are promising antivirals of HSV infections and further translation into clinical trials is warranted.

Despite sepsis being a life-threatening disease, targeted drugs that improve the therapy of affected patients are still lacking. Infants and adults differ in the maturity level of their immune system and this results in distinct reactions to Gram-negative bacteria. To study reactions of human immune cells in vivo, we used NOD scid gamma mice transplanted with human CD34+ stem cells to engraft a functional human immune system. Human cells undergo differentiation and maturation in these mice after transplantation and, accordingly, animals were divided into two groups: 8–13 wk and 15–22 wk after transplantation. Endotoxemia was induced by injecting LPS. Six h later, mice were euthanized. In both groups, LPS stimulation induced a decrease of CD14+ monocytes in peripheral blood, an up-regulation of activation markers on different cell subsets such as myeloid dendritic cells, and a release of the human cytokines TNF-α, IL-6 and IL-10. However, significant differences were detected with regard to the amounts of released cytokines, and 8–13-wk-old mice produced more IL-6, while PTX3 was mainly released by 15–22-wk-old animals. Thus, here we provide a potential model for preclinical research of sepsis in infants and adults.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-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 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 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(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) 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 pre-incubation 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. © 2016 International Society for Advancement of Cytometry.

Glioblastomas represent the most common primary malignant tumor of the nervous system and the most frequent type of astrocytic tumors. Despite improved therapeutic options, prognosis has remained exceptionally poor over the last two decades. Therefore, new treatment approaches are urgently needed. An overexpression of somatostatin (SST) as well as chemokine CXCR4 and endothelin A (ETA) receptors has been shown for many types of cancer. Respective expression data for astrocytic brain tumors, however, are scarce and contradictory. SST subtype, CXCR4 and ETA expression was comparatively evaluated in a total of 57 grade I–IV astrocytic tumor samples by immunohistochemistry using well-characterized monoclonal antibodies. Overall, receptor expression on the tumor cells was only very low. SST5 was the most prominently expressed receptor, followed by SST3, ETA, SST2 and CXCR4. In contrast, tumor capillaries displayed strong SST2, SST3, SST5, CXCR4 and ETA expression. Presence of SST5, CXCR4 and ETA on tumor cells and of SST3, CXCR4 and ETA on microvessels gradually increased from grade II to grade IV tumors. Ki-67 values correlated significantly with CXCR4 expression on tumor cells and with vascular SST3, CXCR4 or ETA positivity. SST5 or CXCR4 positivity of tumor cells and vascular SST3 or CXCR4 expression negatively correlated with patient outcome. Though having some prognostic value, SST, CXCR4 or ETA expression on astrocytic tumor cells is clearly of no therapeutic relevance. Indirect targeting of these highly vascularized tumors via SST3, SST5, CXCR4 or ETA on the microvessels, in contrast, may represent a promising additional therapeutic strategy.

Background and Purpose— We aimed to determine a possible synergistic effect of granulocyte colony-stimulating factor (G-CSF) and bone marrow–derived mononuclear cells (BM MNC) after stroke in spontaneously hypertensive rats. Methods— Male spontaneously hypertensive rats were subjected to middle cerebral artery occlusion and randomly assigned to daily injection of 50 μg/kg G-CSF for 5 days starting 1 hour after stroke (groups 1, 2, and 3) with additional intravenous transplantation of 1.5×10E7 BM MNC per kilogram at 6 hours (group 2) or 48 hours (group 3) after stroke, or control treatment (group 4). Circulating leukocyte counts and functional deficits, infarct volume, and brain edema were repeatedly assessed in the first week and first month. Results— G-CSF treatment led to a significant neutrophilia, to a reversal of postischemic depression of circulating leukocytes, and to a significantly improved functional recovery without affecting the infarct volume or brain edema. BM MNC cotransplantation was neutral after 6 hours, but reversed the functional effect of G-CSF after 48 hours. Short-term investigation of combined G-CSF and BM MNC treatment at 48 hours indicated splenic accumulation of granulocytes and transplanted cells, accompanied by a significant rise of granulocytes in the circulation and the ischemic brain. Conclusions— G-CSF improved functional recovery in spontaneously hypertensive rats, but this effect was abolished by cotransplantation of BM MNC after 48 hours. In the spleen, transplanted cells may hinder the clearance of granulocytes that were massively increased by G-CSF. Increased circulation and infiltration of granulocytes into the ischemic brain may be detrimental for stroke outcome.

Animal models are a useful tool to study pathomechanisms and therapeutic targets in biomedical research. In the last decade the investigation of different humanized mouse models became important in research. The existence of human immune cells in immunodeficient mice allows the bridging between rodent model and human immune system. The method of choice to characterize these humanized mice is flow cytometry. Here, we explain facts about human immune cells in mice and show how to detect them.

NOD.Cg-Prkdc(scid) IL-2rg(tm1Wjl) /SzJ (NSG) mice are a valuable tool for studying Graft-versus-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 × 10(7) PBMCs without anti-human CD4 pre-treatment induced a severe GvHD (0% survival). In animals receiving 2 × 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(+) /CD8(+) cytotoxic T cells and CD3(+) /CD4(+) 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 pre-incubation 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. © 2016 International Society for Advancement of Cytometry.

Rheumatoid arthritis (RA)--a widespread chronic inflammatory disease in industrialized countries--is characterized by a persistent and progressive joint destruction. The chronic pro-inflammatory state results from a mutual activation of the innate and the adaptive immune system, while the exact pathogenesis mechanism is still under discussion. New data suggest a role of the innate immune system and especially polymorphonuclear granulocytes (PMNs, neutrophils) not only during onset and the destructive phase of RA but also at the chronification of the disease. Thereby the enzymatic activity of myeloperoxidase (MPO), a peroxidase strongly abundant in neutrophils, may be important: While its peroxidase activity is known to contribute to cartilage destruction at later stages of RA the almost MPO-specific oxidant hypochlorous acid (HOCl) is also discussed for certain anti-inflammatory effects. In this study we used pristane-induced arthritis (PIA) in Dark Agouti rats as a model for the chronic course of RA in man. We were able to shown that a specific detection of the HOCl-producing MPO activity provides a sensitive new marker to evaluate the actual systemic inflammatory status which is only partially detectable by the evaluation of clinical symptoms (joint swelling and redness measurements). Moreover, we evaluated the long-term pharmacological effect of the well-known anti-inflammatory flavonoid epigallocatechin gallate (EGCG). Thereby only upon early and continuous oral application of this polyphenol the arthritic symptoms were considerably diminished both in the acute and in the chronic phase of the disease. The obtained results were comparable to the treatment control (application of methotrexate, MTX). As revealed by stopped-flow kinetic measurements, EGCG may regenerate the HOCl-production of MPO which is known to be impaired at chronic inflammatory diseases like RA. It can be speculated that this MPO activity-promoting effect of EGCG may contribute to the pharmacological mode of action of this polyphenol.

In the pathogenesis of rheumatoid arthritis (RA), synovial fibroblasts (SF) play a key role as they secrete distinct patterns of cytokines and express variable levels of costimulatory and adhesion molecules. The murine fibroblast cell line LS48 has been shown to be invasive in the cartilage destruction models in vivo and in vitro. The purpose of this study was to examine in detail the LS48 phenotype, to obtain a better understanding of the SF-mediated cartilage destruction in RA. The destructive fibroblasts line LS48 and the nondestructive 3T3 cells were cultured and characterized with slide-based and flow cytometry, using antibodies against several adhesion molecules, immunological acting molecules, and marker proteins. The invasive LS48 fibroblasts are characterized by significantly higher expression of adhesion molecules such as CD47 (IAP), CD51 (integrin αV), CD61 (GPIIIa), and CD147 (EMMPRIN), and immunological acting molecules such as CD40 (Bp50), CD55 (DAF), and TLR-2. The results from the slide-based and flow cytometry analyses were exactly the same, except for the selected CD147 and TLR-2. This study demonstrated that the destructive fibroblast cell line LS48 has the characteristics of RA SFs. The high expression of specific costimulatory and adhesion molecules underlines the aberrant phenotype of these cells when compared with noninvasive fibroblasts. Furthermore, slide-based and flow cytometry complement each other in fibroblast phenotyping. Overall, this study shows that LS48 is an excellent tool to gain a deeper understanding of SF in RA. © 2008 International Society for Analytical Cytology

The determination of acidic pharmaceutical and endocrine disrupting compounds at low ng/L levels in surface and wastewater requires highly selective and sensitive analytical procedures. Therefore, the water samples under study were prepared by means of solid phase extraction (SPE) for analyte enrichment and clean up. Prior to GC-NCI-MS, the polar analytes were derivatized using pentafluorobenzyl bromide. The performance of this analytical method has been revealed in fortification experiments by limits of detection ranging from 0.01 to 0.2 ng/L for, e. g., ibuprofen and 17α-ethinylestradiol, respectively. SPE may be the most rate determining step of the analytical procedure. Therefore, the performance of SPE disks and cartridges was additionally compared. Method evaluation demonstrated that even complex sample matrices, such as model wastewater or even synthetic humic acids, did not interfere with the quantification and identification of target analytes. The performance of the analytical method developed for water monitoring was further emphasized by the investigation of surface water of the River Saale and effluents of the wastewater treatment plant (WWTP) at Halle, Saxony-Anhalt, Germany. In those samples, acidic pharmaceuticals and corresponding metabolites occurred at 0.1 ng/L for clofibric acid up to 498 ng/L for bezafibrate. Technical nonylphenol and bisphenol A were found in every water sample. Meanwhile, 17α-ethinylestradiol was determined only in one WWTP effluent sample at 1 ng/L.

Neutrophilic granulocytes, the most abundant immune cell type in the blood, are essential for the initiation as well as for the regulation and termination of acute inflammatory reactions. Thereby the enzyme myeloperoxidase (MPO) formed by these cells could be a key player. Thus a newly developed method for the quick purification of neutrophilic granulocytes from blood samples and a subsequent activity staining for MPO is presented which may contribute to a better understanding of the (patho-)physiological role of this enzyme.

Joint destruction in rheumatoid arthritis (RA) starts typically at sites of mechanically stressed inserts of the synovial membrane near the cartilage/bone border. In the therapy of RA, tumour necrosis factor (TNF) antagonists have rapidly emerged as a valuable class of anti-rheumatic agents that reduce joint destruction. The aim of this study was to investigate and profile genes involved in the interaction between articular movement and anti-TNF therapy in an in vitro model. Murine LS48 cells, an established substitute for invasive RA synovial fibroblasts, were cultured, stretched and/or treated with anti-TNF-alpha antibody for 24 h. RNA was isolated and gene transcript levels were determined using U74Av2 Affymetrix GeneChips to identify transcriptional events. Positive findings were verified by polymerase chain reaction (PCR). We identified 170 differentially regulated genes, including 44 of particular interest. Gene expression fell into different functional groups that can be explained by RA pathogenesis and experimental conditions. For 21 genes of the 44 of particular interest, regulation could be confirmed by real-time PCR. Remarkably, we found structural as well as functional genes differently regulated between stretched cells, anti-TNF-treated cells, and stretched cells treated with anti-TNF antibody. Additionally, we also found a large number of genes that are apparently not related to the experimental conditions. Mechanical exertion modulates gene expression and subsequently cellular response to anti-TNF therapy. Results in exerted cells correspond to current knowledge regarding RA pathogenesis and underline the relevance of our experimental approach. Finally, the central function of the interleukin-18 system in joint destruction could be confirmed by our findings.

The key pathologic mechanism in rheumatoid arthritis (RA) is the destruction of cartilage by fibroblasts. In a severe combined immunodeficient (SCID) mouse model, this process can be modulated by gene transfer using in- vasive LS48 fibroblasts. This study aims to investigate the effect of interleukins (IL) -11 and -12 on cartilage destruction when transferred into LS48, and of IL-15 when transfected into non-invasive 3T3 cells; to compare three transduc- tion systems (a lentiviral vector system, a retroviral vector system, and a par- ticle-mediated gene transfer); and to establish an in vitro cartilage destruction system based on LS48 cells. Transduced fibroblasts were injected into SCID mice knee joints, and disease progression assessed microscopically. Distinctive morphologic pattern revealed invasion of fibroblasts into the articular carti- lage by transfected, as well as non-transfected, LS48 cells. IL-12 and IL-15 did not alter swelling or cartilage destruction. Animals treated with IL-11-trans- fected cells showed reduced cartilage damage but no changes in swelling. Effi- cacy of gene transfer to establish transfected fibroblasts was shown to be > 85% for lentiviral transfer, compared to < 10% for retroviral transfer and gene gun. Furthermore, cells were co-incubated with porcine cartilage. Transduction of IL-11 led to a reduction of apoptosis in chondrocytes. These findings suggest that cartilage destruction by invasive fibroblasts can be modulated by gene transfer. Lentiviral vector systems offer the most effective approach for gene transduction. In vitro fibroblast/cartilage co-cultures present a convenient sys- tem for the assessment of novel therapeutic strategies towards reduction of articular destruction.

Objective: To investigate the mode of action of methotrexate (MTX) in different types of models for rheumatoid arthritis (RA) and multiple sclerosis (MS). Methods: Models for RA and MS were selected known to have different pathogenesis—that is, fibroblast induced arthritis in SCID mice, collagen induced arthritis (CIA), anticollagen II antibody induced arthritis (CAIA), and experimental autoimmune encephalomyelitis (EAE) in (Balb/cxB10.Q)F1 and B10.Q mice, and Pristane induced arthritis in DA rats (PIA). The MTX treatment was started 1 day after the onset of disease and continued for 14 days to compare effects on the different models. Results: All models known to be critically dependent on T cell activation (CIA, PIA, and EAE) were effectively down regulated by titrated doses of MTX. In contrast, no effects were seen on fibroblast induced arthritis or CAIA. No effects were seen on the levels of anticollagen II antibodies in the CIA experiment. Conclusion: The data show that MTX has strong ameliorative effect on both classical models of RA, like CIA and PIA, but also on a model for MS, EAE. It also suggests that MTX operates only in diseases which are preceded by, and dependent on, T cell activation. A comparison of CAIA and CIA suggested that MTX operates independently of arthritogenic antibodies. These results demonstrate that different animal models reflect the complexity of the corresponding human diseases and suggest that several models should be used for effective screening of new therapeutic agents.

In rheumatoid arthritis (RA), fibroblasts have been shown to be crucial for disease progression as well as joint destruction. In the model of human/murine SCID arthritis, synovial explants as well as fibroblasts from human rheumatoid synovial membrane induce destructive arthritis in immunodeficient mice. Hereby, the underlying cartilage destruction is accomplished by murine fibroblasts. Therefore, murine destructive fibroblasts represent a promising tool to investigate destruction of articular cartilage and bone. In this context, a novel destructive murine fibroblast line (LS48) was examined for morphological, ultrastructural, immunological and functional cellular parameters. These cells were injected into knees of SCID mice. Subsequently, the animals were monitored for joint swelling and serological parameters of arthritis by radiological methods. Finally, cartilage destruction was assessed morphologically. Cultured LS48 cells exhibit characteristic features that resemble those of activated synovial fibroblasts in human RA. Expression levels of inducible nitric oxide synthase, interleukin-6, tumour necrosis factor-alpha and matrix metalloproteinases were comparable to those detected in invasive human fibroblasts. The instillation of 5 x 10(5) LS48 cells into the knee joints of SCID mice initiated a rapid progressive process, that caused cartilage destruction within 10 days, and morphological examinations revealed that articular cartilage was infiltrated by the fibroblasts injected previously. In summary, the intra-articular application of LS48 cells represents a rapid and highly reproducible model to investigate the initiation and progression of cartilage destruction in connection with RA therapy and represents an easy-to-handle animal model.

Lithiation of 6-methyl-2-(trimethylsilylamino)pyridine (APyTMSH) occurs smoothly in tetrahydrofuran (thf) affording [Li(APyTMS)(thf)]2 (1). Treatment of anhydrous lanthanoid chlorides (LnCl3, Ln=Gd, Er) with 1.5 equivalents of (1) yields the solvent-free homoleptic tris–amido complexes [Ln(APyTMS)3], (Ln=Gd (2); Ln=Er (3)). Similar treatment of LnCl3 (Ln=Gd, Er) with one equivalents of 1 putatively generates the heteroleptic species [Ln(APyTMS)2Cl], (Ln=Gd (4); Ln=Er (5)) in situ, however, these compounds undergo redistribution in hexane to yield homoleptic 2 and 3 and the anhydrous lanthanoid halides (Ln=Gd, (6), Ln=Er (7)) and were therefore not fully characterised. These lanthanoid reagents are extremely moisture sensitive as examplified by the low yield isolation of [APyH2·H]2[ErCl5(thf)] during one prepartion of 3. The structures of compounds 1, 2, 3 and 8 were characterised by X-ray crystallographic methods. The X-ray structure of 1 is a centrosymmetric dimer similar to its diethyl ether analogue. Compounds 2 and 3 are six-coordinate homoleptic mononuclear species and compound 8 comprises the unprecedented [ErCl5(thf)]− anion within an intricate hydrogen-bonded ionic system.

By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases.

Background and Purpose— Granulocyte colony–stimulating factor (GCSF) showed robust neuroprotective and neuroregenerative properties after stroke in rodents but failed to meet study end points in patients. Because immunologic side effects of GCSF may have escaped preclinical testing because of nonallometric dose translation, we hypothesized those as possible reasons. Methods— Stroke was induced in C57BL/6 mice by 45-minute filament middle cerebral artery occlusion. GCSF was administered at 50 and 832.5 μg/kg body weight. Treatment was controlled by vehicle injection, sham surgery, and naive animals. Immune cell counts were assessed in blood, spleen, and brain by multidimensional flow cytometry 1 day after stroke. Results— High-dose GCSF significantly altered myeloid and T-cell subpopulations in blood and spleen and caused a tremendous increase of monocytes/macrophages infiltrating the ischemic brain. Conclusions— Dose-dependent immunomodulation superimposes central nervous system-specific effects of GCSF after stroke. Adaption of dose or treatment time may overcome this drawback.

Background To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. Methods We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. Results Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. Conclusions PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

Macrolide antibiotics are widely used (in the order of 1g per person per year). They pass the body largely unchanged and are also not degraded in wastewater treatment plants. With not too much effort, they may be eliminated from their effluents by ozonation. The macrolide antibiotics have all a dimethylamino group at one of the carbohydrate residues in common. This functional group is the target of the ozone reaction, and clarithromycin has been selected here for a more detailed study. Since only the free amine reacts with ozone, the rate of reaction is pH dependent (at pH 7: k = 4 x 10(4) M(-1) s(-1)). In analogy to the ozonolysis of trimethylamine, the main reaction is a transfer of an O-atom yielding the N-oxide (identified by HPLC/MS-MS). A minor product (10%, based on formaldehyde yields) is demethylated clarithromycin (identified by HPLC/MS-MS). The dimethylamino group is thought to be essential for the binding of the macrolide antibiotics to their target. As a consequence, chemical changes of this functional group, notably the formation of the N-oxide that is no longer a proton acceptor, inactivates these drugs as assayed by the suppression of the growth of Pseudomonas putida. This is most important for wastewater treatment, as mineralization of clarithromycin by ozone would require 100 times as much ozone.

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.

After transplantation with human umbilical cord blood cells NOD-SCID IL2Rγ(null) rodents develop an human immune system which is characterized by the presence of B cells, T cells, monocytes, granulocytes and dendritic cells. The extent of the several subpopulations differs during the time after transplantation. A clear development of the immune system can be seen from immature immune system which contains mainly B cells, a few T cells in peripheral blood, during a transitional state with an additional existence of monocytes, granulocytes and denritic cells in bone marrow and spleen of the mice to a state which reminds of Graft-versus-Host-Disease. This development can be observed during a period of 10 to 20 weeks after transplantation. The B cells are able to produce high amounts of IgM in the early state and few IgG. The antigen presenting cells in the transitional state are able to express activation molecules like HLA-DR and CD86 after the injection of LPS as an immune modifying stimulus. Moreover the cells are able to produce human TNFα, IL-10, IL-1b, IFNγ and high amounts of IL-6 after stimulation with LPS in every state. Release of murine TNFα, IL10, IL-6, MCP and IL-12 was detected as well. The presence of human and murine PTX3 was also measureable after in vivo stimulation with LPS. Depending on the time point after transplantation the human immune cells are able to produce human cytokines and to express several activation markers after immune modifying stimulus.

The purpose of our study was to investigate whether the immune modulator WF10 has an impact on clinical signs of experimentally induced rheumatoid arthritis in DBA/1 mice. Female DBA/1 mice were sensitized with a collagen II emulsion on day 0 and boosted on day 21. The animals were scored from day 17 for clinical signs of collagen induced arthritis (CIA). If the evaluated score exceeded a value of 5, CIA was considered to be established. From the day of CIA onset animals were treated with WF10 or saline in different doses and treatment regimens. Scoring was done for a total of 21 days after CIA onset. Animals which developed CIA within the first 35 days after the immunization were included into the study. Control animals as well as mice treated with WF10 exhibited CIA and showed an increasing score within the observation period, whereas animals treated with the compound showed a reduction of clinical symptoms. 21 days after CIA onset, animals were sacrificed and samples were preserved for further investigations like cytokine measurements, histology and mRNA expression analyses. In conclusion, WF10 had a reducing effect on the severity of CIA symptoms in mice and may therefore present a novel therapeutic approach for patients suffering from rheumatoid arthritis and other inflammatory autoimmune diseases.

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34(+) stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2R gamma(null) (NSG) rodents. Interestingly, the isolation of CD34(+) cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34(+) isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34(+) stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.