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11 pages, 8 figures.-- PMID: 17998387 [PubMed].-- PMCID: PMC2118532.-- Printed version published Nov 26, 2007. ; Supporting information attached: Information on Western blots, Immunoprecipitation assays, the Rac activation assay, the FACS-based conjugate formation assay, the Actin polymerization assay, and Stimulation with antibody-coated beads and immunofluorescence.-- Also available at: http://jem.rupress.org/cgi/content/full/jem.20070366/DC1 ; Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class I(A) p85/p110 heterodimers, which are activated by Tyr kinases, and the class I(B) p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gα(q/11), lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. ; I. Alcázar holds a predoctoral fellowship from the Association for International Cancer Research, M. Marqués receives a predoctoral fellowship from the Spanish Ministry of Education and Science University Instructor Training Program, and D.F. Barber held a Ramón y Cajal contract from the Spanish Ministry of Education and Science and a Contract-in-Aid from the Fundación Científica de la Asociación Española Contra el Cancer. This work was supported by grants from the European Union (QLRT2001-02171), the Community of Madrid (8.3/0030/2000), the Ramón Areces Foundation (to D.F. Barber and A.C. Carrera), and the Spanish Dirección General de Ciencia y Desarrollo Tecnológico (SAF2004-00815 and SAF2004-05955- C02-01 to D.F. Barber A.C. Carrera, respectively). The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (Consejo Superior de Investigaciones Científicas) and by Pfizer. ; Peer reviewed

Abstract
Background
This paper explores the relationship between chemical intolerance (CI) and mast cell activation syndrome (MCAS). Worldwide observations provide evidence for a two-stage disease process called toxicant-induced loss of tolerance (TILT) as a mechanism for CI. TILT is initiated by a major exposure event or a series of lower-level exposures. Subsequently, affected individuals report that common chemical inhalants, foods, and drugs (i.e., various xenobiotics) trigger multi-system symptoms.

Purpose
To determine whether MCAS provides a plausible biological mechanism for CI/TILT.

Methods
Using the validated Quick Environmental Exposure and Sensitivity Inventory (QEESI), we compared patients diagnosed with MCAS (n = 147) to individuals who reported chemical intolerances (CI/TILT) following various exposures (n = 345) and to healthy controls (n = 76). Using ANOVA, we compared QEESI scores across groups. Clinical scores for the MCAS patient group were used to predict CI status using logistic regression.

Results
More than half (59%) of the MCAS group met criteria for CI. A logistic regression model illustrates that as the likelihood of patients having MCAS increased, their likelihood of having CI/TILT similarly increased, to a near-perfect correspondence at the high ends of the QEESI and clinical MCAS scores. Symptom and intolerance patterns were nearly identical for the CI and MCAS groups.

Discussion
We present data suggesting that xenobiotic activation of mast cells may underlie CI/TILT. The strikingly similar symptom and intolerance patterns for MCAS and TILT suggest that xenobiotics disrupt mast cells, leading to either or both of these challenging conditions. Faced with patients suffering from complex illness affecting multiple organ systems and fluctuating inflammatory, allergic, and dystrophic symptoms, clinicians can now ask themselves two questions: (1) Could MCAS be at the root of these problems? (2) Could environmental exposures be driving MC activation and mediator release? Increasing our understanding of the connection between TILT and MCs has the potential to expose a new link between environmental exposures and illness, offering new opportunities for improving individual and public health.

Conclusion
The close correspondence between QEESI scores and symptom patterns for MCAS and TILT patients supports xenobiotic-driven mast cell activation and mediator release (i.e., MCAS) as a plausible unifying biological mechanism for CI/TILT, with profound implications for medicine, public health, and regulatory toxicology.

Allergic skin diseases caused by low molecular weight chemical compounds are a major health concern among the general population, especially in certain occupational groups. To limit exposure to such compounds, it is important that thorough risk assessments are performed before products reach the consumer market. Traditionally, such risk assessments have been based on animal tests, but due to several factors, including legislations and ethical concerns, the use of animal testing has become increasingly unjustifiable. In this context, one focus in predictive toxicology has been the development of alternative methods to replace animal experiments. Although several alternative methods have been validated for regulatory decision making, they lack certain endpoints for complete risk assessments, and molecular mechanisms underlying skin sensitization are still not entirely understood. The work described in this thesis aims at increasing our knowledge about the mechanisms in dendritic cell activation in response to skin sensitizers and to further develop a dendritic cell-based in vitro assay.In Paper I, we investigated the microRNA regulation in response to stimulation of a DC model with structurally similar rubber sensitizers. The changes triggered by the rubber sensitizers in both mRNA and microRNA expression suggest a chemical-specific regulation, despite the structural similarity of the rubber sensitizers. In Paper II, the skin sensitizing properties of herbicidal formulations are investigated. Here, we predicted the herbicide glyphosate as a non-sensitizer, while the co-formulant polyethylated tallow amine and two glyphosate-based formulations were predicted as sensitizers. Additionally, we also investigated the proteomic alterations in response to these chemicals and formulations, and identified cellular responses associated with the differentially expressed proteins. In the last two papers, Paper III and Paper IV, we present additional applications for the in vitro assay setup used in this thesis. In Paper III, we identified a biomarker signature for the prediction of skin sensitizer potency, demonstrating a balanced accuracy of 78% targeting three potency classes, i.e., 1A (strong sensitizers), 1B (weak sensitizers) and no cat (non-sensitizers). In Paper IV, we applied a statistical method, the conformal prediction framework, to investigate the predictive boundaries of the in vitro assay, and concluded, based on 70 chemicals, that the assay can be applied to a large chemical space.In conclusion, the work presented here can contribute to a better understanding of the mechanisms underlying the immunological response to skin sensitizing chemicals, which together with state-of-the-art predictive assays could be used for improved risk assessment of chemical compounds and to develop tools for prevention and treatment of allergic skin diseases.

32 p.-5 fig.-1 graph.abst ; Brucella canis is a small intracellular Gram-negative bacterium that frequently leads to chronic infections highly resistant to antibiotic therapy in dogs. Also, it causes mild human brucellosis compared to other zoonotic Brucella spp. Herein we characterize the cellular immune response elicited by B. canis by analysing human and canine CD4+ T cells after stimulation with autologous monocyte-derived dendritic cells (MoDCs). Human and canine B. canis-primed MoDCs stimulated autologous CD4+ T cells; however, a Th1 response was triggered by human MoDCs, whereas canine MoDCs induced Th1/Th17 responses, with increased CD4+ T cells producing IFN-γ and IL-17A simultaneously. Each pattern of cellular response may contribute to host susceptibility, helping to understand the differences in B. canis virulence between these two hosts. In addition, other aspects of canine immunology are unveiled by highlighting the participation of IL-17A-producing canine MoDCs and CD4+ T cells producing IFN-γ and IL-17A. ; This work was supported by the Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) from the Chilean Government, grant FONDECYT 1140904, grant FONDECYT 1181780, and MP PhD fellowship CONICYT 21130626. ; Peer reviewed

Abstract
Dysregulation of cholesterol metabolism underlies neurodegenerative disease and is increasingly implicated in neuroinflammatory diseases, such as multiple sclerosis (MS). Cytochrome P450 family 7 subfamily B member 1 (CYP7B1) is a key enzyme in alternative cholesterol metabolism. A recessive mutation in the gene CYP7B1 is known to cause a neurodegenerative disease, hereditary spastic paraplegia type 5 and oxysterol accumulation. However, the role of CYP7B1 in neuroinflammation has been little revealed. In this study, we induced experimental autoimmune encephalomyelitis (EAE), as a murine model of MS, using CYP7B1 homozygous knockout (KO) mice. We found that CYP7B1 deficiency can significantly attenuate EAE severity. CYP7B1 deficiency is sufficient to reduce leukocyte infiltration into the central nervous system, suppress proliferation of pathogenic CD4+ T cells, and decrease myeloid cell activation during EAE. Additionally, live-animal imaging targeting translocator protein expression, an outer mitochondrial membrane protein biomarker of neuroinflammation, showed that CYP7B1 deficiency results in suppressed neuroinflammation. Using human monocyte-derived microglia-like cellular disease model and primary microglia of CYP7B1 KO mice, we also found that activation of microglia of CYP7B1 deficiency was impaired. These cumulative results suggest that CYP7B1 can regulate neuroinflammation, thus providing potential new targets for therapeutic intervention.

The human cytomegalovirus (HCMV) US12 family consists of ten sequentially arranged genes (US12-21) with poorly characterized function. We now identify novel natural killer (NK) cell evasion functions for four members: US12, US14, US18 and US20. Using a systematic multiplexed proteomics approach to quantify ~1300 cell surface and ~7200 whole cell proteins, we demonstrate that the US12 family selectively targets plasma membrane proteins and plays key roles in regulating NK ligands, adhesion molecules and cytokine receptors. US18 and US20 work in concert to suppress cell surface expression of the critical NKp30 ligand B7-H6 thus inhibiting NK cell activation. The US12 family is therefore identified as a major new hub of immune regulation. ; This work was supported by funds from the Wellcome Trust WT090323MA and MRC G1000236. MR/L018373/1. This work was also supported by a Wellcome Trust Principal Research Fellowship (WT101835) to PJL and a Wellcome Trust Senior Fellowship (108070/Z/15/Z) to MPW and a strategic award to Cambridge Institute for Medical Research from the Wellcome Trust (100140). JAP was supported by NIH/NIDDK grant K01 DK098285. BV was supported by projects MEYS – NPS I – LO1413 and Czech Science Foundation P206/12/G151. ER was supported by MH CZ-DRO (MMCI, 00209805). CC and JT were supported by grant G0901682 from the MRC and funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 695551), with partial funding from the National Institute of Health Cambridge Biomedical Research Centre.

Traumatic brain injury is accompanied by glial cell activation around the site of the injury. In this study, we investigated the role of toll-like receptor 2 (TLR2) in glial cell activation using a stab-wound injury (SWI) model with TLR2 knock-out mice. Penetration of a normal mouse brain with a 26-G needle using a stereotaxic instrument resulted in an 18- and 4-fold upregulation of GFAP and CD11b mRNA, respectively, along the needle track in the injury area. However, in the TLR2 knock-out mice, the induced expression of these genes was reduced by 70% and 40%, respectively. Likewise, there was a reduction in the area of activated glial cells detected by immunohistochemistry and the glial cells had a less-activated morphology in the TLR2 knock-out mice. In addition, the expression of the heme oxygenase-1 (HO-1) gene, a glia-expressing wound-responsive gene, was reduced after SWI in TLR2 knock-out mice. Taken together, these data argue that TLR2 contributes to the glial cell activation and HO-1 gene expression associated with traumatic brain injury. ; This work was supported by the Neurobiology Research Program at the Korea Ministry of Science and Technology (M10412000014-07N1200-01410), the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R01-2007-000-10243-0), and by a Korea Research Foundation grant (KRF-2005-070-C00096).

Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. ; This work was supported by grants from Fondo de Investigación Sanitaria [PI11/00298] [PS09/00181]; from the Spanish Ministry of Economy and Competitivity (MINECO) [SAF2012-32293], and from the Regional Government of Madrid [grant number S2010/BMD-2326 INMUNOTHERCAN]; EG-C was a recipient from a fellowship from Fundación La Caixa and received the Immunotools IT-special-Award 2014; SL-C was a recipient from a fellowship from the Spanish Ministry of Education, partially supported by CSIC [grant 201020E086 (to MV-G)] and received the Immunotools IT-BOX-139 award 2012; GR-C was a recipient of a JAE-predoc fellowship from CSIC; RC-C was supported by a BBSRC studentship; MR was funded by a postdoctoral fellowship from the Spanish Cancer Foundation (AECC). ; Peer reviewed ; Peer Reviewed

Transplantation of mesenchymal stem cells (MSCs) into injured or diseased tissuefor the in situ delivery of a wide variety of MSC-secreted therapeutic proteinsis an emerging approach for the modulation of the clinical course of several diseases and traumata. From an emergency point-of-view, allogeneic MSCs have numerous advantages over patient-specific autologous MSCs since off-the-shelf cell preparations could be readily available for instant therapeutic intervention following acute injury. Although we confirmed the in vitro immunomodulatory capacity of allogeneic MSCs on antigen-presenting cells with standard coculture experiments, allogeneic MSC grafts were irrevocably rejected by the host's immune system upon either intramuscular or intracerebral transplantation. In an attempt to modulate MSC allograft rejection in vivo, we transduced MSCs with an interleukin-13 (IL13)-expressing lentiviral vector. Our data clearly indicate that prolonged survival of IL13-expressing allogeneic MSC grafts in muscle tissue coincided with the induction of an alternatively activated macrophage phenotype in vivo and a reduced number of alloantigen-reactive IFN- and/or IL2-producing CD8(+) T cells compared to nonmodified allografts. Similarly, intracerebral IL13-expressing MSC allografts also exhibited prolonged survival and induction of an alternatively activated macrophage phenotype, although a peripheral T cell component was absent. In summary, this study demonstrates that both innate and adaptive immune responses are effectively modulated in vivo by locally secreted IL13, ultimately resulting in prolonged MSC allograft survival in both muscle and brain tissue. ; We thank A. Liston and S. Humblet-Baron (VIB Translational Immunology Lab, KU Leuven, Belgium) for sharing their optimized staining protocols for intracellular cytokine analysis by flow cytometry. This work was supported by research grants G.0130.11 and G.0136.11 (granted to A.V.d.L., Z.N.B. and P.P.), G.0131.11 (granted to Z.N.B. and P.P.), G.0834.11 (granted to S.H. and P.P.) and G.0851.11 (granted to C.V. and Z.N.B.) of the Research Foundation Flanders (FWO Vlaanderen, Belgium) and in part by a Methusalem research grant from the Flemish government (granted to H.G.). C.J.H. and E.Lu. hold a PhD studentship from the FWO Vlaanderen. C.G. and D.L.B. hold a PhD studentship from the Flemish Institute for Science and Technology (IWT Vlaanderen).

Cordyceps militaris (C. militaris) has various biomedical applications in traditional oriental medicine for different diseases including inflammatory and immune-dysregulated diseases. It is a reservoir of nutritional components such as cordycepin, polysaccharides, and antioxidants. To improve its bioactivity, we fermented C. militaris with a Pediococcus pentosaceus strain isolated from a salted small octopus (SC11). The current study aimed to evaluate whether P. pentosaceus (SC11) fermentation could enhance the anti-allergic potential of C. militaris cultured on germinated Rhynchosia nulubilis (GRC) against a type I hypersensitive reaction in in vitro and in vivo studies. Total antioxidant capacity and cordycepin content were significantly increased in GRC after SC11 fermentation. GRC-SC11 showed significantly enhanced anti-allergic responses by inhibiting immunoglobulin E (IgE)/antigen-induced degranulation in RBL-2H3 cells, compared to GRC. The results demonstrated the significant inhibition of phosphorylated spleen tyrosine kinase (Syk)/ p38/GRB2-associated binding protein 2 (Gab2)/c-jun in IgE/Ag-triggered RBL-2H3 cells. Furthermore, suppressed mRNA levels of interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α) in IgE/Ag-activated RBL-2H3 cells were observed. GRC-SC11 significantly ameliorated IgE-induced allergic reactions by suppressing the ear swelling, vascular permeability, and inflammatory cell infiltration in passive cutaneous anaphylaxis (PCA) BALB/c mice. In conclusion, GRC fermented with P.pentosaceus exerted enhanced anti-allergic effects, and increased the cordycepin content and antioxidants potential compared to GRC. It can be used as bio-functional food in the prevention and management of type I allergic diseases.

Optimizing activation and deactivation of base station transmissions provides an instrument for improving energy efficiency in cellular networks. In this paper, we study the problem of performing cell clustering and setting the activation time of each cluster, with the objective of minimizing the sum energy, subject to a time constraint of serving the users traffic demand. Our optimization framework accounts for inter-cell interference, and, thus, the users achievable rates depend on cluster formation. We provide mathematical formulations and analysis, and prove the problems NP hardness. For problem solution, we first apply an optimization method that successively augments the set of variables under consideration, with the capability of approaching global optimum. Then, we derive a second solution algorithm to deal with the trade-off between optimality and the combinatorial nature of cluster formation. Numerical results demonstrate that our solutions achieve more than 40% energy saving over existing schemes, and that the solutions we obtain are within a few percent of deviation from global optimum. ; Funding Agencies|European Union FP7 Marie Curie Project MESH-WISE [434515]; European Union FP7 Marie Curie Project WiNDOW [318992]; Chinese Scholarship Council; Institute for Infocomm Research (I2R); A*STAR, Singapore, through overseas Ph.D. research internship scheme; Swedish Research Council; European Union FP7 Marie Curie IOF [329313]

Evidence continues to emerge that the social determinants of health play a role in adverse outcomes related to COVID-19, including increased morbidity and mortality, increased risk of long COVID, and vaccine adverse effects. Therefore, a more nuanced understanding of the biochemical and cellular pathways of illnesses commonly associated with adverse social determinants of health is urgently needed. We contend that a commitment to understanding adverse outcomes in historically marginalized communities will increase community-level confidence in public health measures. Here, we synthesize emerging literature on mast cell disease, and the role of mast cells in chronic illness, alongside emerging research on mechanisms of COVID illness and vaccines. We propose that a focus on aberrant and/or hyperactive mast cell behavior associated with chronic underlying health conditions can elucidate adverse COVID-related outcomes and contribute to the pandemic recovery. Standards of care for mast cell activation syndrome (MCAS), as well as clinical reviews, experimental research, and case reports, suggest that effective and cost-efficient remedies are available, including antihistamines, vitamin C, and quercetin, among others. Primary care physicians, specialists, and public health workers should consider new and emerging evidence from the biomedical literature in tackling COVID-19. Specialists and researchers note that MCAS is likely grossly under-diagnosed; therefore, public health agencies and policy makers should urgently attend to community-based experiences of adverse COVID outcomes. It is essential that we extract and examine experiential evidence of marginalized communities from the broader political–ideological discourse.