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During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α. The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62Δ69-251 mice, global p62−/− and Ucp1-Cre p62flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding., Beta-adrenergic stimulation of brown adipose tissue leads to thermogenesis via the activating transcription factor 2 (ATF2) mediated expression of the thermogenic genes Ucp1 and Pgc-1α. Here, the authors show that the scaffold protein p62 regulates brown adipose tissue function through modifying ATF2 genomic binding and subsequent Ucp1 and Pgc-1α induction.

One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT–qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% ( n = 85) of positive samples by RT–qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.

Reliable detection of unwanted organisms in experimental animal colonies is an essential requirement for biomedical research. Since most rodents are housed in Individually Ventilated Cage (IVC) systems which prevent aerogenic transmission of pathogens, soiled-bedding sentinels are commonly used for routine health monitoring. In this study, we investigated whether testing of Exhaust Air Particle (EAP) samples by real-time PCR (EAP PCR) might be a valid alternative to SBS for the detection of H. hepaticus infections. EAP PCR detected H. hepaticus infections at a minimal prevalence of 1/63 cages within one week. EAP PCR provides a sensitive, simple, and reliable alternative to SBS for H. hepaticus identification in laboratory mouse colonies.

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation is an opportunity for nearly all patients lacking an HLA matched stem cell donor. However, graft rejection and graft-versus-host disease (GvHD) as well as infectious complications still result in high treatment-related mortality. Here, we used the dog as a preclinical model for the study of tolerance induction with the aim to optimize and to improve a clinical protocol of haploidentical stem cell transplantation. For this purpose CD6-depleted peripheral blood stem cells (PBSCs) were transfused 6d after transplantation of unmodified bone marrow from dog leukocyte antigen (DLA)-haploidentical littermate donors in order to induce immune tolerance. Besides hematopoietic stem cells CD6-depleted PBSC contain, NK cells and a minority of suppressive CD8-positive cells that may suppress activated T lymphocytes. Recipients were conditioned with, cyclophosphamide and antithymocyte globulin (ATG) preceded by a transfusion of donor buffy coat and either 1, 2 or 3 × 3.3 Gy total body irradiation (TBI). Postgrafting immunosuppression was limited to 30 d of cyclosporine and methotrexate. The additional administration of CD6-depleted PBSCs after unmodified marrow could not prevent GvHD, but it may improve engraftment and chimerism after conditioning with 2 × 3.3 Gy TBI. Reasons for incomplete suppression and possible improvements for clinical applications are discussed.

Objective Donor lymphocyte transfusions (DLT) are effective in the treatment of leukemia after allogeneic stem cell transplantation. Graft-vs-host-disease (GVHD) is the major risk factor of DLT. In dog leukocyte antigen (DLA)−identical littermate dogs, DLT given within 3 weeks after transplantation of marrow depleted of T cells using absorbed antithymocyte globulin produced fatal GVHD, whereas at 2 months or later after transplantation, DLT were tolerated without GVHD. Here, we studied tolerance to DLT in DLA-haploidentical recipients of CD6-depleted bone marrow. Materials and methods Bone marrow recipients were DLA-heterozygous and DLA-haploidentical to their DLA-homozygous donors. Marrow transplantation was performed on day 0, one day after total body irradiation with 10Gy. CD6 depletion was achieved by treatment of the marrow with CD6 antibody and rabbit complement. Natural killer cell activity of CD6-depleted cells was studied against canine thyroid adenocarcinoma cells. DLT were given at various time points after transplantation. Results Seven DLA-heterozygous dogs given undepleted marrow died of GVHD within 28 days. In contrast, three dogs given CD6-depleted marrow had sustained engraftment without occurrence of GVHD. DLT given on either day 3, 7, or 14 produced fatal GVHD. DLT on day 20, however, produced fatal GVHD in only two of four dogs. Mixed chimerism was converted into complete chimerism in all cases. Contrary to T-cell depletion with antithymocyte globulin, CD6 depletion spares canine natural killer cells. Conclusions We conclude that T-cell depletion with CD6 antibody and complement induces graft-vs-host tolerance without jeopardizing engraftment. DLT on days 3, 7, and 14 after transplantation produced GVHD, but it failed to abrogate tolerance in two of four dogs transfused on day 20.

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.

Exposure to high concentrations of Manganese (Mn) is known to potentially induce an accumulation in the brain, leading to a Parkinson related disease, called manganism. Versatile mechanisms of Mn-induced brain injury are discussed, with inactivation of mitochondrial defense against oxidative stress being a major one. So far, studies indicate that the main Mn-species entering the brain are low molecular mass (LMM) compounds such as Mn-citrate. Applying a single low dose MnCl2 injection in rats, we observed alterations in Mn-species pattern within the brain by analysis of aqueous brain extracts by size-exclusion chromatography--inductively coupled plasma mass spectrometry (SEC-ICP-MS). Additionally, electrospray ionization--ion cyclotron resonance-Fourier transform-mass spectrometry (ESI-ICR/FT-MS) measurement of methanolic brain extracts revealed a comprehensive analysis of changes in brain metabolisms after the single MnCl2 injection. Major alterations were observed for amino acid, fatty acid, glutathione, glucose and purine/pyrimidine metabolism. The power of this metabolomic approach is the broad and detailed overview of affected brain metabolisms. We also correlated results from the metallomic investigations (Mn concentrations and Mn-species in brain) with the findings from metabolomics. This strategy might help to unravel the role of different Mn-species during Mn-induced alterations in brain metabolism.

Occupational and environmental exposure to increased concentrations of manganese (Mn) can lead to an accumulation of this element in the brain. The consequence is an irreversible damage of dopaminergic neurons leading to a disease called manganism with a clinical presentation similar to the one observed in Parkinson's disease. Human as well as animal studies indicate that Mn is mainly bound to low molecular mass (LMM) compounds such as Mn-citrate when crossing neural barriers. The shift toward LMM compounds might already take place in serum due to elevated Mn concentrations in the body. In this study, we investigated Mn-species pattern in serum in two different animal models by size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). A subchronic feeding of rats with elevated levels of Mn led to an increase in LMM compounds, mainly Mn-citrate and Mn bound to amino acids. In addition, a single i.v. injection of Mn showed an increase in Mn-transferrin and Mn bound to amino acids 1 h after injection, while species values were more or less rebalanced 4 days after the injection. Results from Mn-speciation were correlated to the brain metabolome determined by means of electrospray ionization ion cyclotron resonance Fourier transform mass spectrometry (ESI-ICR/FT-MS). The powerful combination of Mn-speciation in serum with metabolomics of the brain underlined the need for Mn-speciation in exposure scenarios instead of the determination of whole Mn concentrations in blood. The progress of Mn-induced neuronal injury might therefore be assessed on the basis of known serum Mn-species.

Manganese (Mn) is an essential trace element contributingto various physiological processes like brain development,diverse metabolisms and proper function of antioxidativeenzymes like Acetylcholinesterase (AchE) (Erikson et al.,2005). On the downside, overexposure to Mn can lead to thedegeneration of dopaminergic neurons inducing a Parkinson-like complaint called manganism. Extensive studies havebeen carried out to decipher transport routes into thebrain and to detect cellular mechanisms of oxidative injuryin the affected brain areas (Crossgrove et al., 2003; Yokel,2009 ;Yokel and Crossgrove, 2004 Aschner et al., 2007).Nevertheless, the correlation between the active Mn-species and the occurring conceivable causes of oxidativeinjury has not been drawn so far. The aim of this study istherefore, to elucidate the predominant Mn-species in theexposed brain and link it to levels of glutamate or GABA,AchE activity, Fe(II)/(III) status and levels of other traceelements like Cu, Fe or Zn (Figure 1).The applied model was a feeding trial of rats, where Mnlevels were elevated (500 mg/kg) in a non-toxic manner inthe test group and which lasted for 53 days.After removal of the brain, it was deep frozen androughly homogenized. Levels of Mn, Cu, Fe and Zn weredetermined in the whole brain by ICP-OES or –MS. Mn levelswere significantly higher in Mn-exposed (test) rats comparedto normal exposed (control) rats, which was interestinglyalso true for Cu levels. Furthermore an aqueous extractionof the brain served as matrix for determination of AchEactivity as well as glutamate and GABA levels by commercialavailable kits. Test rats showed significantly elevated AchEactivity as well as increased glutamate levels, indicatingoxidative stress in the affected brain tissue (Erikson andAschner, 2003; Melo et al., 2003). The same brain extractswere applied for determination of the Fe(II)/(III) status by

Modified vaccinia virus Ankara (MVA) serves as a versatile platform in vaccine development. This highly attenuated orthopoxvirus, which cannot replicate in mammalian cells, triggers strong innate immune responses, including cell migration. Previously, we have shown that induction of chemokine (C-C motif) ligand 2 (CCL2) by MVA is necessary for the recruitment of monocytes and T cells, but not neutrophils, to the lung. Here, we identified neutrophil-attracting chemokines produced by MVA-infected primary murine lung fibroblasts and murine bone marrow-derived macrophages. We demonstrate that MVA, but not vaccinia virus (VACV) strain WR, induces chemokine expression, which is independent of Toll-like receptor 2 (TLR2) signaling. Additionally, we show that both chemokine (C-C motif) receptor 1 (CCR1) and chemokine (C-X-C motif) receptor 2 (CXCR2) are involved in MVA-induced neutrophil chemotaxis in vitro . Finally, intranasal infection of Ccr1 −/− mice with MVA, as well as application of the CCR1 antagonist J-113863, revealed a role for CCR1 in leukocyte recruitment, including neutrophils, into the lung. IMPORTANCE Rapid attraction of leukocytes to the site of inoculation is unique to MVA in comparison to other VACV strains. The findings here extend current knowledge about the regulation of MVA-induced leukocyte migration, particularly regarding neutrophils, which could potentially be exploited to improve other VACV strains currently in development as oncolytic viruses and viral vectors. Additionally, the data presented here indicate that the inflammatory response may vary depending on the cell type infected by MVA, highlighting the importance of the site of vaccine application. Moreover, the rapid recruitment of neutrophils and other leukocytes can directly contribute to the induction of adaptive immune responses elicited by MVA inoculation. Thus, a better understanding of leukocyte migration upon MVA infection is particularly relevant for further development and use of MVA-based vaccines and vectors.

Occupationally or environmentally caused chronic exposure to Manganese (Mn) can lead to a degeneration of dopaminergic neurons inducing a Parkinson-like complaint called manganism. Deciphering the ongoing neurodegenerative mechanisms in the affected brain is still a major task for understanding the complex modes of action. Therefore, we applied a non-toxic, oral feeding in rats simulating a chronic exposure to Mn. Analysis of brain extracts by electrospray ionization Fourier transform resonance mass spectrometry (ESI-FT-ICR-MS) revealed an increase in markers of oxidative stress like glutathione disulfide (GSSG), prostaglandins, and 15(S)-HETE, a marker of lipid peroxidation. Furthermore, acetylcholinesterase (AchE) activity and glutamate concentrations were elevated in brain samples of Mn-supplemented rats, suggesting oxidative stress in the brain tissue. Application of ion chromatography coupled to inductively coupled plasma-optical emission spectrometry (IC-ICP-OES) further showed a shift of Fe(III) towards Fe(II) in the brain samples enabling for example the action of the Fenton reaction. This is the first time that changes in the Fe-species distribution could be related to Mn-induced neuroinflammation and is therefore enlarging the knowledge of this complex neurodegenerative condition. The combination of our findings provides substantial evidence that Mn-induced neuroinflammation leads to oxidative stress triggered by multifactorial pathophysiological processes.

Female donors produce a strong graft-versus-leukemia (GvL) effect in male patients. Presumably Y-encoded minor histocompatibility antigens (mHA) are recognized by the alloimmune effector lymphocytes. UTY (ubiquitously transcribed tetratricopeptide repeat, Y-linked) is a potential candidate gene for mHA in dogs and men. In the canine model we could show that the minor histocompatibility antigen UTY might be a potential target to further improve the adoptive immunotherapeutic approach. Five HLA-A2 restricted peptides were used for pulsing canine dendritic cells and stimulating canine T cells. T-cells were separated from the blood of female donors using the MACS technique and effector T-cells were generated by incubation of 2x10*6 CD3+ T-cells/ml with autologous mature, peptide-pulsed dendritic cells (DCs) in X-Vivo 15 medium (+ 160 U/ml human IL-2 and 5 ng/ml human IL-7; ratio T:DCs=10:1). T cells were restimulated weekly with peptide-pulsed DCs. After 3 or 4 weeks in culture T-cells were examined for the CD8/CD4 ratio, cytotoxicity assays were assessed after 3–4 re-stimulations in [51Cr]-release assays. The number of peptide-specific T cells was assessed in IFN-g-ELISPOT-assays. UTY-mRNA expression was determined by RT-PCR in several female and male cell types (BM, PBMCs, monocytes, DCs, B-cells). We were able to expand T-cells from 6 female dogs using peptide-pulsed autologous DCs; 3 out of 5 UTY-derived peptides were able to stimulate a specific autologous T-cell response. The number of CD8+ T-cells could be expanded during four weeks of in vitro culture in 3 dogs. In IFN-g-ELISPOT-assays we could demonstrate that the expanded T-cells secrete IFN-g upon recognition of DCs from DLA-identical male littermates. In addition, these T-cells specifically lysed DLA-identical male BM (25-100%), but not autologous or DLA-identical female BM (0–15%) in [51Cr]-release assays (E:T=80:1). Cell types other than BM and DCs (DLA-identical male and female monocytes, PBMCs, B-cells) were not lysed (0–18%). In summary, we could demonstrate that UTY is a naturally processed and presented antigen in the dog. Our data show that HLA-A2 restricted UTY-peptides immunize specific CTLs from female dogs that efficiently recognize DLA-identical male bone marrow and DCs. These findings suggest that UTY may be employed for studies of immunotherapy in the canine model.

Graft-versus-host disease (GvHD) is the major obstacle of allogeneic stem cell transplantation. Depletion of T-cells from the graft reduces the risk of GvHD, but results in a higher risk of leukemia relapse. Adoptive immunotherapy with donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after T-cell depleted allogeneic stem cell transplantation. However, GvHD may occur, if DLT is given too early after transplantation. In canine models of DLA-identical and DLA-haploidentical bone marrow transplantation, we compared different methods of T-cell depletion (TCD) and investigated the potential of DLT at different times after transplantation to induce GvHD. T-cell depletion was performed either with absorbed anti-thymocyte globuline (aATG) or with a combination of CD6-antibody and baby rabbit complement. ATG was absorbed with erythrocytes, liver, kidney and spleen for eliminating antibodies against stem cells. CD6-antibody (M-T606) and rabbit complement depleted T-cells effectively without affecting hematopoietic progenitor cells. Unlike aATG, monoclonal CD6-antibody spares natural killer (NK) cells and some CD8-positive cells. Treatment of bone marrow with aATG prevented GvHD in 9 dogs following DLA-identical transplantation. DLT on days 1 and 2 or 21 and 22 induced fatal GvHD in two dogs each. However, it did not induce GvHD when given on days 61 and 62 and later. In DLA-haploidentical bone marrow recipients, non-manipulated marrow produced fatal GvHD in all dogs (n=7), whereas marrow treated with aATG (vol:vol 1:100 and 1:200) produced fatal GvHD in 5 out of 16 dogs only. CD6-depletion prevented GvHD in 3 of 3 DLA-haploidentically transplanted dogs. DLT produced fatal GvHD in one dog each, when given on day 3, 7 or 14 after CD6-depleted haploidentical bone marrow transplantation. However, it produced fatal GvHD in only 2 of 4 dogs transfused on day 20 post grafting. Thus, DLT could be given earlier in DLA-haploidentical animals transplanted with CD6-depleted marrow than in DLA-identical animals transplanted with aATG treated marrow without producing GvHD. These findings support the hypothesis that graft-versus-host tolerance can be induced earlier with grafts not depleted of NK cells. NK cells in the graft may inactivate host dendritic cells necessary for the induction of GvHD. In grafts depleted with aATG, NK cells are depleted as well, because aATG still retains broad specificity despite extensive absorptions. This leaves host DCs unaffected. Transfused donor T-cells encountering this environment will thus be activated which results in severe GvHD. In contrast, monoclonal CD6-antibody spares NK cells, so that donor lymphocytes cannot be activated by host DCs at the time of DLT and thus won’t trigger GvHD. CD6-depletion is the preferred method if adoptive immunotherapy with DLT is planned.

Adoptive immunotherapy with donor lymphocyte infusion (DLI) following bone marrow transplantation may produce a graft-versus-leukemia (GVL) effect after transplantation of T-cell-depleted bone marrow. However, this method is limited by a high risk of GvHD. We studied the occurrence of graft-versus-host disease (GVHD) and chimerism after DLI at various times after CD6-depleted bone marrow transplantation in a canine model of DLA-homozygous donor and DLA-heterozygous littermate recipient. Using a cross reactive mouse-anti-human antibody which recognizes the canine CD6 antigen, a simple and economical depletion method could be established, which makes use of the ability of the CD6 antibody MT-606 to activate the complement cascade. In vitro experiments using MT-606 and rabbit complement showed the effective depletion of CD6-positive cells whereas hematopoietic progenitor cells remained unaffected at the same time. For in vivo studies, DLA heterozygous recipients with one DLA haplotype being identical to the donor were treated with 10 Gy total body irradiation (TBI) and received CD6-depleted bone marrow of their DLA-homozygous littermate donors. In the control group 7 dogs given unmanipulated marrow died of GVHD within 28 days of transplantation. Two dogs showed sustained engraftment without the occurrence of GvHD after the infusion of 1,8 · 108/kg – 2,0· 108/kg CD6-depleted mononuclear marrow cells. The establishment of tolerance to the bone marrow donor was shown by the transplantation of skin from the donor to the host. Transfusion of donor lymphocytes (1.0 · 108 MNCs/kg body weight) on day 3, 7 or 14 induced GvHD in the host. However, with DLI on day 20, only 2 out of 4 dogs developed GvHD, but DLI always converted mixed into complete chimerism. We conclude from this study that the cross reactive CD6 antibody MT606 effectively depletes T cells in the presence of rabbit complement and induces GVH tolerance without jeopardizing engraftment, and the risk of GVHD after DLI is reduced with time after transplantation.