Apicomplexan parasites are attenuated by low-energy electron irradiation in an automated microfluidic system and protect against infection with Toxoplasma gondii.

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.

NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

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.

Attenuation of graft-versus-host-disease in NOD scid IL-2Rγ(-/-) (NSG) mice by ex vivo modulation of human CD4(+) T cells.

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.

Bone marrow cell transplantation time-dependently abolishes efficacy of granulocyte colony-stimulating factor after stroke in hypertensive rats.

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.

Allometric dose retranslation unveiled substantial immunological side effects of granulocyte colony-stimulating factor after stroke.

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.

Mucosal immunization with a low-energy electron inactivated respiratory syncytial virus vaccine protects mice without Th2 immune bias.

The respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections associated with numerous hospitalizations. Recently, intramuscular (i.m.) vaccines against RSV have been approved for elderly and pregnant women. Noninvasive mucosal vaccination, e.g., by inhalation, offers an alternative against respiratory pathogens like RSV. Effective mucosal vaccines induce local immune responses, potentially resulting in the efficient and fast elimination of respiratory viruses after natural infection. To investigate this immune response to an RSV challenge, low-energy electron inactivated RSV (LEEI-RSV) was formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) or 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DD-LEEI-RSV) for vaccination of mice intranasally. As controls, LEEI-RSV and formalin-inactivated-RSV (FI-RSV) were used via i.m. vaccination. The RSV-specific immunogenicity of the different vaccines and their protective efficacy were analyzed. RSV-specific IgA antibodies and a statistically significant reduction in viral load upon challenge were detected in mucosal DD-LEEI-RSV-vaccinated animals. Alhydrogel-adjuvanted LEEI-RSV i.m. showed a Th2-bias with enhanced IgE, eosinophils, and lung histopathology comparable to FI-RSV. These effects were absent when applying the mucosal vaccines highlighting the potential of DD-LEEI-RSV as an RSV vaccine candidate and the improved performance of this mucosal vaccine candidate.

Evaluation of a Developed MRI-Guided Focused Ultrasound System in 7 T Small Animal MRI and Proof-of-Concept in a Prostate Cancer Xenograft Model to Improve Radiation Therapy.

Focused ultrasound (FUS) can be used to physiologically change or destroy tissue in a non-invasive way. A few commercial systems have clinical approval for the thermal ablation of solid tumors for the treatment of neurological diseases and palliative pain management of bone metastases. However, the thermal effects of FUS are known to lead to various biological effects, such as inhibition of repair of DNA damage, reduction in tumor hypoxia, and induction of apoptosis. Here, we studied radiosensitization as a combination therapy of FUS and RT in a xenograft mouse model using newly developed MRI-compatible FUS equipment. Xenograft tumor-bearing mice were produced by subcutaneous injection of the human prostate cancer cell line PC-3. Animals were treated with FUS in 7 T MRI at 4.8 W/cm2 to reach ~45 °C and held for 30 min. The temperature was controlled via fiber optics and proton resonance frequency shift (PRF) MR thermometry in parallel. In the combination group, animals were treated with FUS followed by X-ray at a single dose of 10 Gy. The effects of FUS and RT were assessed via hematoxylin-eosin (H&E) staining. Tumor proliferation was detected by the immunohistochemistry of Ki67 and apoptosis was measured by a TUNEL assay. At 40 days follow-up, the impact of RT on cancer cells was significantly improved by FUS as demonstrated by a reduction in cell nucleoli from 189 to 237 compared to RT alone. Inhibition of tumor growth by 4.6 times was observed in vivo in the FUS + RT group (85.3%) in contrast to the tumor volume of 393% in the untreated control. Our results demonstrated the feasibility of combined MRI-guided FUS and RT for the treatment of prostate cancer in a xenograft mouse model and may provide a chance for less invasive cancer therapy through radiosensitization.

Mucosal Application of a Low-Energy Electron Inactivated Respiratory Syncytial Virus Vaccine Shows Protective Efficacy in an Animal Model.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections in the elderly and in children, associated with pediatric hospitalizations. Recently, first vaccines have been approved for people over 60 years of age applied by intramuscular injection. However, a vaccination route via mucosal application holds great potential in the protection against respiratory pathogens like RSV. Mucosal vaccines induce local immune responses, resulting in a fast and efficient elimination of respiratory viruses after natural infection. Therefore, a low-energy electron irradiated RSV (LEEI-RSV) formulated with phosphatidylcholine-liposomes (PC-LEEI-RSV) was tested ex vivo in precision cut lung slices (PCLSs) for adverse effects. The immunogenicity and protective efficacy in vivo were analyzed in an RSV challenge model after intranasal vaccination using a homologous prime-boost immunization regimen. No side effects of PC-LEEI-RSV in PCLS and an efficient antibody induction in vivo could be observed. In contrast to unformulated LEEI-RSV, the mucosal vaccination of mice with PC formulated LEEI-RSV showed a statistically significant reduction in viral load after challenge. These results are a proof-of-principle for the use of LEEI-inactivated viruses formulated with liposomes to be administered intranasally to induce a mucosal immunity that could also be adapted for other respiratory viruses.

Extracellular Vesicles of Mesenchymal Stromal Cells Can be Taken Up by Microglial Cells and Partially Prevent the Stimulation Induced by ß-amyloid.

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 ß-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 ß-amyloid aggregates. This inhibition of microglia activation may present an effective strategy for the control/therapy of neurodegenerative diseases such as Alzheimer's disease.

Helicase primase inhibitors (HPIs) are efficacious for therapy of human herpes simplex virus (HSV) disease in an infection mouse model.

Although the seroprevalence of Herpes simplex virus type 1 (HSV-1) currently amounts to âˆ¼ 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.

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology.

INTRODUCTION: Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice. METHODS: In this study, we compared the use of CD34+ HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR). RESULTS: It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdcscid IL2rγtm1Wjl /SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model. CONCLUSION: From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells.

Unchanged food approach-avoidance behaviour of healthy men after oxytocin administration.

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.

Differential somatostatin, CXCR4 chemokine and endothelin A receptor expression in WHO grade I-IV astrocytic brain tumors.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Age-dependent cellular reactions of the human immune system of humanized NOD scid gamma mice on LPS stimulus.

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.

Degradation of macrolide antibiotics by ozone: a mechanistic case study with clarithromycin.

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.

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 µl makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments. Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes. Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

Long-Term Effects of (-)-Epigallocatechin Gallate (EGCG) on Pristane-Induced Arthritis (PIA) in Female Dark Agouti Rats.

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.

Animal Models of Rheumatoid Arthritis (I): Pristane-Induced Arthritis in the Rat.

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.

Rapid and reliable determination of the halogenating peroxidase activity in blood samples.

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.

Comparison of hematopoietic stem cells derived from fresh and cryopreserved whole cord blood in the generation of humanized mice.

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 IL2Rγ(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.