bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of Core Histones H3 and H4 by Pak2
Authors: Tuazon Polygena T. , Rojas Arbis , Ling Jun , Jung Jin‐Hun , Quijano Jade , Zhang Kangling , Gatti Andrea , Traugh Jolinda
Published: June 2021

The core histones H3 and H4 are phosphorylated by the p21‐activated protein kinase Pak2. Pak2 is a serine/threonine kinase that is activated by small G proteins in response to moderate stress and by caspase 3 cleavage during apoptotic stress. The sites in histones 3 and 4 phosphorylated by Pak2 were identified by ESI/MS/MS. H4 was phosphorylated on two sites by Pak2. These phosphorylation sites were located in the globular core domain in the consensus sequence identified for Pak2, which was identified in studies with synthetic heptapeptides. In H3, there were 4 identified sites located in the N‐terminal tail and the globular core domain. These were non‐consensus sites dependent on upstream basic amino acids in the −4 to −8 positions, which were predicted based on studies with synthetic dodecapeptides. The sites phosphorylated on H4 in vitro by Pak2 were shown to be phosphorylated under conditions of moderate stress, when Pak2 is activated. The sites phosphorylated in H3 in vitro, were reported to be phosphorylated in vivo under a variety of conditions, including apoptosis. Thus, phosphorylation of histones 3 and 4 could play important roles in regulating chromosome activity/function.
The adaptor protein 3BP2/SH3BP2 is required for RANKL‐induced osteoclastogenesis in RAW264.7 cells
Authors: Guezguez Amel , Mouska Xavier , Blin‐Wakkach Claudine , Breittmayer Jean‐Philippe , Simoncic Paul D. , Rottapel Robert , Deckert Marcel
Published: June 2021

The adaptor protein (Abl SH3 binding protein‐2), a partner of Src and Syk kinases families, has been involved in leukocyte signaling and activation. Recently, genetic studies have associated mutations of 3BP2 gene with the human bone disease cherubism and with inflammation and bone dysfunction in mouse. Here the role of 3BP2 in osteoclast differentiation was investigated. Using stable extinction of 3BP2 expression in the RAW264.7 monocyte/macrophage cell line, we show that 3BP2 is required for RANKL‐induced differentiation of RAW264.7 cells into multinucleated TRAP+ osteoclasts, but not for GM‐CSF/IL4‐induced differentiation into dendritic cells. The absence of 3BP2 in RAW264.7 cells is associated with reduced actin cytoskeleton organization and impaired RANKL‐induced cell motility and fusion, and activation of IKK, ERK, JNK, but not of p38. In addition, we found a dramatic decrease of NFATc1, AP‐1 and NFkB transcription factors activities induced by RANKL stimulation, as well as a deregulated expression of multiple genes involved in early events controlling osteoclastogenesis, including NFATc1, c‐fos, beta3 integrin subunit, and several chemokine and chemoreceptor genes. Together, these data define the adaptor protein as a critical effector of multiple RANKL‐dependent signal transduction pathways involved in osteoclast differentiation. This work was supported by INSERM and ANR‐MRAR.
Innate and adaptive immunity through autophagy
Authors: Munz Christian , Schmid Dorothee , Lee Monica , Gannage Monique , Paludan Casper
Published: June 2021

Major histocompatibility complex (MHC) class II molecules present products of lysosomal proteolysis to CD4+ T cells. Although extracellular antigen uptake is considered to be the main source of MHC class II ligands, a few intracellular antigens, including the nuclear antigen 1 of the Epstein Barr Virus, have been described to gain access to MHC class II loading after macroautophagy. However, the general relevance and efficacy of this pathway is still debated. In addressing these questions, we could demonstrate constitutive autophagosome formation in MHC class II positive cells, including dendritic, B and epithelial cells. The autophagosomes continuously fused with multivesicular MHC class II‐loading compartments (MIICs). This pathway was of functional relevance, because targeting of the Influenza Matrix Protein 1 (MP1) to autophagosomes via fusion to the autophagosome‐associated protein Atg8/LC3 led to strongly enhanced MHC class II presentation to CD4+ T cell clones. The importance of this pathway is demonstrated by the fact that both DNA and RNA viruses inhibit the pathway, and might thereby escape immune control. We suggest that macroautophagy constitutively and efficiently delivers cytosolic proteins, like viral antigens, for MHC class II presentation and can be harnessed for improved helper T cell stimulation.

This research is supported by the National Cancer Institute (R01 CA101741), the National Institute of Allergy and Infectious Disease (RFP‐NIH‐NIAID‐DAIDS‐BAA‐06‐19), the Beckman Foundation, the Sinsheimer Scholars Program and the Burroughs Wellcome Fund.
Selection of Substrates for MHC Class I‐Restricted Antigen Processing
Authors: Eisenlohr Laurence Crane , Huang Lan , Kandler Kerstin , Kuhls Matthew
Published: June 2021

We have proposed a model for selection of cytosolic proteins for MHC class I‐restricted antigen processing (Nat Rev Immunol, 2007, 7: 403). In short, incomplete capture of nascent polypeptides by the protein folding machinery, resulting in uncompacted hydrophobic domains, triggers ubiquitin (Ub)‐independent degradation by the 20S proteasome. We have tested this model in three ways. First, we revisited the classic Townsend et al. finding (Nature, 1986, 324: 575) that directing influenza hemagglutinin to the cytosol through signal sequence ablation substantially increases presentation. We hypothesized that the unembeded transmembrane domain (TM), rather than misfolding, stimulates rapid degradation. Indeed, various manipulations of the human IL‐2 receptor alpha chain (Tac) and its TM support this idea. Second, using a dominant‐interfering Ub system, we have probed the role of polyUb’n in the presentation of various antigen processing substrates. To date, all stable cytosolic proteins tested are largely, if not exclusively, presented in a polyUb‐independent manner as opposed to other types of processing substrate. Finally, we have probed the role of the two ribosome‐associated chaperone complexes, RAC and NAC, in antigen processing. NAC, but not RAC silencing resulted in a ~2‐fold increase in peptide/class I complexes at the cell surface. Thus, our proposed pathway remains viable.
The unfolded protein response regulates MHC class I‐peptide presentation
Authors: Granados Diana Paola , Hardy Marie‐Pierre , Perreault Claude
Published: June 2021

Cell stress elicits the unfolded protein response (UPR). UPR regulates the two processes that control MHC I‐peptide (MHC I‐p) presentation: protein translation and degradation. We therefore asked whether and how the UPR might modify MHC I‐p presentation. We evaluated the impact of the UPR on global MHC I expression and on presentation of the SIINFEKL peptide. EL4 cells stably transfected with bicistronic vectors coding HEL‐SIINFEKL protein variants were stressed with pharmacological agents or exposed to physiologically relevant stress, such as glucose deprivation, for various time durations. UPR intensity was monitored by western blot and/or qRT‐PCR analysis of BiP, XBP‐1 and phosphorylation of eIF2alpha. Expression of Kb and of Kb‐SIINFEKL was assessed by flow cytometry with or without prior acid peptide elution. UPR decreased surface expression of MHC, but did not affect its mRNA. Consequently, presentation of SIINFEKL by H2‐Kb molecules was decreased in chemically or physiologically stressed cells. During the UPR, cells preferentially presented MHC I‐p derived from the ER as opposed to the cytosol. Also, generation of de novo Kb‐SIINFEKL complexes was less affected for ER‐ vs. cytosol‐derived SIINFEKL. Our results show that the UPR affects MHC I‐p presentation and that it differentially regulates expression of ER‐ vs. cytosol‐derived peptides. Supported by the NCIC.
Murine plasmacytoid dendritic cells express the inhibitory FcγRIIB as their sole FcγR and fail to mediate antigen presentation of immune complexes to T cells
Authors: Flores Marcella Maria‐Melissa , Desai Dharmesh D. , Downie Matthew , Liang Bitao
Published: June 2021

Plasmacytoid DC's (pDCs) in their role as Type I IFN producers, are key regulators of the innate immune response, yet their role in the adaptive immune response remains unclear. The ability of murine pDCs to present antigen in immune complexes (ICs) to T cells was examined. Unlike conventional DCs, ovalbumin‐IC exposed murine pDCs failed both to upregulate costimulatory molecules and to activate OVA specific CD4+ and CD8+ T cells. The failure of murine pDCs to promote T cell activation through either the exogenous or cross‐presentation pathways occurred despite IC binding and internalization. ICs internalized by pDCs however were inefficiently delivered to an intracellular degradative compartment. This defect in the IC processing capability is likely the consequence of a lack of activating Fc Receptor expression (FcRI, III, IV) and the exclusive expression of the inhibitory receptor FcRIIB. This selective expression of FcRIIB by pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. Thus, pDCs are unable to effectively process ICs for antigen presentation, and may not be able to directly activate naive T cells in vivo.
The anti‐inflammatory effect of Alloferon on UVB irradiated human skin keratinocyte cell line, HaCaT via the regulation of pro‐inflammatory cytokine production
Authors: Lee Seung Koo , Kang Jae Seung , Kim Jee Eun , Jung Da Jung , Bae Seyeon , Lee Na Eun , Kong Joo Myoung , Lee Seona , Shin Dong Hoon , Hwang Young‐il , Lee Wang Jae
Published: June 2021

UVB irradiation modulates immune responses and activates inflammatory reactions, leading to skin damage like sunburn. Alloferon is isolated from the blood of an experimentally infected insect, the blow fly Calliphora vicina (Diptera), and shows a variety of biomedical efficacies such as antiviral and antitumoral capabilities. However, the specific molecular mechanisms for the action of alloferon still remain to be clarified. In this study, we investigated the effects of alloferon on cutaneous inflammation induced by UVB‐irradiation in human HaCaT keratinocytes. RPA and ELISA data showed alleferon decreased the production of UVB‐induced pro‐inflammatory cytokine, such as IL‐1α, IL‐1β, IL‐6, IL‐18, both on the mRNA and protein level. Western blot analysis showed alleferon inhibited the activation of p38 MAPK induced by UVB irradiation. Furthermore, the topical application of alloferon on hairless mouse skin exposed to UVB has shown that post‐treatment of alloferon significantly reduced the thickness of epidermal layer of chronically UVB exposed mice skin. In addition, alloferon inhibited increased production of the IL‐6 in hairless mice skin. These findings suggest that alleferon has significant anti‐inflammatory effects on the UVB‐induced response on the cutaneous inflammation and may offer an effective approach to reduction or prevention of skin inflammation resulting from UVB irradiation.
Neutrophil extracellular trap formation by nitric oxide donors: Involvement of nitric oxide synthase and myeloperoxidase derived free radicals
Authors: Patel Satyananda , Kumar Sachin , Saluja Rohit , Barthwal Manoj Kumar , Mitra Kalyan , Verma Anupam , Bajpai Virendra K , Dikshit Madhu
Published: June 2021

Presence of neutrophil extracellular traps (NET) at the site of inflammation and infection is demonstrated in various pathological conditions. Neutrophils migration and augmentation in NO availability are mostly associated with inflammation and pathogen infections. So far role of NO has not been explored in NET formation. Present study reports for the first time that incubation of human neutrophils with NO donors, sodium nitroprusside (SNP) or S‐nitroso‐N‐acetyl‐penicillamine (SNAP) led to the NET release and free radical generation, which was blocked in the presence of free radical scavengers (ebselen, NAC) or pretreatment of PMNs with inhibitors of NOS reductase domain (7‐NI, DPI, imidazole) and myeloperoxidase (ABAH), while NOS oxygenase domain inhibitors had no effect. We thus propose that NO mediated NET release was dependent on the NO synthase (NOS) uncoupling and MPO derived free radicals, but independent of NADPH‐oxidase. Moreover, NO dependent free radical generation, NET release and phosphorylation of MEK/ERK, p38MAPK was blocked by inhibitors of MEK/ERK (U0126), p38MAPK (SB202190), PKC (Rottlerin) suggesting for the first time role of ERK and p38MAPK in NET formation. The results of the present study seem to have important bearing on the role of NO in inflammatory pathological conditions.

The work was supported by CSIR and DBT India
Protective effects of polyclonal iTreg induced by IL‐2 and TGF‐β on a lupus‐like syndrome
Authors: Zheng Song Guo , Wang Juhua , Horwitz David A.
Published: June 2021

We have previously reported that the adoptive transfer of antigen‐specific Tregs generated by allogeneic antigen stimulation with TGF‐β prevents the appearance of lupus syndrome induced by injection of DBA/2 mouse spleen cells into (DBA/2 × C57BL/6) F1 mice. Here we show that CD4+CD25‐ cells polyclonally activated ex‐vivo with IL‐2 and TGF‐β (iTreg) have similar beneficial effects in vivo. A single injection of 5 million iTregs prevented increased IgG production, decreased anti‐dsDNA autoantibodies, suppressed proteinuria and prolonged survival of these mice. The treatment with anti‐TGF‐β or ALK5 almost completely blocked the suppressive capacity of iTreg following injection in vivo. Moreover, iTreg suppress CD80 and CD86 expression of dendritic cells (DC). Thus, the protective effect of iTreg is dependent upon TGF‐β and these cells have inhibitory effects on antigen‐presenting DC. The possibility the transferred iTregs can induce T cells in the recipient mice to become iTreg is being explored. Since SLE, a systemic, generalized autoimmune disease, is characterized by impaired production of IL‐2 and TGF‐β, the use of the cytokines ex‐vivo to repair defects in the numbers and functional activity of CD4+CD25+Foxp3+ Treg cells in this disease may have therapeutic potential.

Supported by grants from the Arthritis National Research Foundation, Clinical Research Feasibility Fund, James H. Zumberge Faculty Research Innovation Fund, the Arthritis Foundation, ExCell Therapeutics, Nora Eccles Treadwell Foundation, and BD Biosciences.
Altered signaling and split anergy in lupus T cells
Authors: Wadhone‐Gangalum Pallavi , Dubey Shweta , Wang James , Singh Ram Raj
Published: June 2021

MRL‐Faslpr/lpr (MRL‐lpr) and MRL‐Fas+/+ (MRL+/+) mice develop autoimmune disease that mimics human SLE. We have used these mice to address the role of T cells in lupus. We found that freshly isolated CD4+ T cells from the spleen of MRL‐lpr display features of in vivo antigen priming and activation as compared to MHC‐matched healthy strains (B10.BR and C3H). Autoimmune T cells also produce more IFN‐γ than control T cells. Surprisingly, however, MRL T cells exhibit reduced calcium flux, IL‐2 production, and proliferation. Such anergic phenotype is seen upon stimulation with anti‐CD3, but not when stimulated with PMA/ionomycin. Such split responsiveness (activated, but anergic) is particularly more profound in a subset of T cells, i.e., B220+ T cells that expand in MRL mice. B220 is an isoform of CD45, a transmembrane phosphatase that plays critical role in cell signaling. As compared to control strains, MRL T cells exhibit reduced expression of CD45RB and CD45RC isoforms, which correlates with altered expression of TCR signaling molecules, with enhanced constitutive phosphorylation of ZAP‐70 and ERK1/2. However, no further increase or a highly accelerated kinetics in p‐ZAP70 and p‐ERK1/2 is seen in MRL T cells as compared to controls. There was also a decrease in phosphorylation of a key target of CD45, Lck (Y505). Using p‐ERK1/2 as readout for T cell activation, we found that activation in MRL‐lpr T cells was inhibited at a relatively lower concentration of CD45 inhibitor as compared to control T cells. Inhibition of CD45 normalized the basal level of activation in MRL‐lpr T cells. These data lead us to hypothesize that CD45‐mediated regulation of TCR signaling is impaired in lupus T cells.

Research Support: Supported in part by NIH.
Intracerebral BCG infection impairs Th17 response and autoimmunity in the central nervous system
Authors: Lee JangEun , Reinke Emily K. , Sandor Matyas , Fabry Zsuzsa
Published: June 2021

Infectious agents have been demonstrated to contribute to the development of helper T cell‐mediated autoimmune diseases including multiple sclerosis (MS). We induced an autoimmune disease that affects the central nervous system (CNS), experimental autoimmune encephalomyelitis (EAE), in mice intracerebrally infected with Mycobacterium bovis strain bacille‐Calmette‐Guérin (BCG) to study the role of in situ infection induced inflammation on the clinical outcome of disease. We show that clinical and pathological signs of EAE did not develop in mice with intracerebral BCG infection. In parallel with this, we found intact myelin sheaths and decreased cellular infiltration in the spinal cord of BCG‐infected animals as compared to non‐infected EAE controls. Intracellular cytokine analysis of IFN‐γ and IL‐17 demonstrated a substantial reduction in MOG‐specific responses in both cytokines in BCG‐infected mice. However, the IFN‐γ response of non‐MOG specific, activated T cells in the CNS of BCG infected mice was equivalent, while these cells showed substantially reduced IL‐17 responses. In addition, BCG‐induced suppression of Th17 responses was maintained in IFN‐γ deficient animals. These data suggest that BCG infection‐mediated modulation of autoimmunity in the CNS is independent of IFN‐γ and is regulated via the Th17 pathway. Identification of the mechanisms that block CNS autoimmunity in situ in the nervous tissue will add to our understanding of the role of bacterial infections in CNS autoimmunity and could lead to the development of new strategies in manipulating autoimmune disease.

[This research is supported by the National Institute of Health, USA R01‐NS 37570 01 awarded to Dr. Z. Fabry.]
Transduced B‐cells mediate tolerance by activating regulatory T‐cells
Authors: Skupsky Jonathan , Scott David
Published: June 2021

FoxP3 has emerged as an intracellular marker for mouse T cells that have a regulatory phenotype. To study the kinetics of regulatory T cell induction in response to treatment from tolerogenic B cells, we have crossed the FoxP3‐GFP knock‐in mouse to our mouse model for hemophilia; we have also crossed the FoxP3‐GFP mouse to a transgenic mouse with an ovalbumin specific TCR. We use the established gene‐therapy technique of transducing LPS activated B cells with an antigen‐IgG fusion to prevent or abrogate an ongoing immune response. Although it has already been shown that tolerance is dependent on CD4+CD25+ regulatory cells, herein, we demonstrate an increase in levels of antigen specific FoxP3+ cells that peaks within a week of treatment and wanes near baseline levels by two weeks. Additionally, we see an increased response of regulatory cells following challenge with antigen. We anticipate that a deeper understanding of the kinetics of FoxP3 induction will likely lead to insight into treatment modalities to maintain high levels of regulatory cells during the management of autoimmune diseases, transplant, and protein based therapies.

(Supported by NIH grants R01 HL061883 and T32 HL07698)
C5 complement inhibition contributes to increased proliferative activity and antigen specific recall response in experimentally acquired myasthenia gravis (EAMG)
Authors: Soltys Jindrich , Kusner Linda L , Zhou Yuefang , Gong Bendi , Kaminski Henry J
Published: June 2021

Background: Myasthenia gravis (MG) is an autoimmune disorder caused by the production of autoantibodies against the acethylcholine receptor (AChR). Antibodies bind to the AChR and activate the complement system leading to the destruction of neuromuscular junction (NMJ). Experimentally acquired MG (EAMG) rats were used to investigate the therapeutic role of C5 complement inhibitor, rEV576.

Methods: Severity of disease was measured by grip strength and weight loss of the animal; serum cytokines and toxicity; cells proliferative activity and antigen specific recall responses were analyzed in vitro.

Results: Treatment with C5 complement inhibitor for 10 days attenuated the outcome of EAMG. Administration of rEV576 resulted in reduction of serum toxicity and increased IFN‐γ production. Splenic cell suspensions isolated from EAMG + rEV576 had higher proliferation ability in the presence of specific antigen (AChR) or mitogen (Con A). ELISPOT recall responses to the specific AChR showed that the frequency of splenic T‐cells secreting IFN‐γ is increased in rEV576 treated EAMG rats.

Conclusions: Inhibition of complement activity attenuated the outcome of EAMG. EAMG rats treated with rEV576 showed increased cell activity suggesting an effect of complement deficiency on cellular immunity. The data demonstrate that complement inhibition may have significant therapeutic value in human MG.
Increased activity of the mTOR pathway contributes to deficiencies of CD3ζ and Lck in T cells isolated from patients with systemic lupus erythematosus
Authors: Fernandez David Rafael , Bonilla Eduardo , Perl Andras
Published: June 2021

We previously showed that the immunosuppressant macrolide rapamycin (Sirolimus) effectively controlled SLE disease activity and normalized the high baseline calcium as well as the increased T cell receptor (TCR) stimulation‐induced calcium fluxing in SLE T cells. Here, we document that mTOR activity is increased in SLE T cells and it is normalized by in vivo treatment with rapamycin, a specific inhibitor of mTOR kinase activity.

We show increased phosphorylation of two targets of mTOR, ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E binding protein (4E‐BP), in T cells from SLE patients. Healthy controls and patients treated with rapamycin in vivo did not show phosphorylation of these proteins. Levels of mTOR and of its binding partners, raptor and rictor, were similar in control and SLE T cells.

SLE T cells are deficient in the TCR‐associated proteins CD3ζ and Lck. It has been shown that correction of the CD3ζ deficiency by expression of exogenous CD3ζ in SLE T cells normalizes calcium homeostasis. Rapamycin treatment in vivo also normalizes calcium fluxing in SLE T cells, so we measured the levels of CD3ζ and Lck in T cells isolated from rapamycin‐treated patients. T cells obtained from patients treated with rapamycin in vivo show significant recovery of these signaling molecules versus SLE patients not receiving rapamycin.

Rapamycin is an efficacious treatment for SLE and normalizes calcium levels in SLE T cells, likely by restoring normal levels of T cell receptor‐associated signaling molecules.

This research was supported by the Alliance for Lupus Research and NIH grants AI‐061066, AI‐48079.
IFNgamma protects against type 1 diabetes by inhibiting pathogenic Th17 cells
Authors: Tartar Danielle Marie , Jain Renu , Zaghouani Habib
Published: June 2021

Th17 cells play a major role in autoimmunity but their function in type I diabetes (T1D) remains unclear. The aa sequence 206–220 of glutamic acid decarboxylase (designated GAD2) represents a late stage epitope and zGAD2‐specific T cells produce IFNγ cytokine. Since IFNγ is known to inhibit Th17 cells, effective presentation of GAD2 peptide may protect against T1D at advanced disease stages. To test this hypothesis GAD2 was genetically incorporated into an Ig molecule to magnify tolerance and the resulting Ig‐GAD2 was assayed against T1D at different stages. The results show that Ig‐GAD2 could not delay disease at the pre‐insulitis phase, but protected against T1D at the insulitis stage. Furthermore, Ig‐GAD2 supported production of IFNγ by GAD2‐specific splenic cells which led to clearance of pancreatic cell infiltration as well as β‐cell division and restored normoglycemia when given to hyperglycemic mice at the pre‐diabetic stage. However, neutralization of IFNγ resulted in a significant increase in the frequency of Th17 and the treatment became non‐protective. Thus, IFNγ induced by an antigen‐specific therapy reverses its usual inflammatory function to restore normoglycemia, most likely by localized bystander suppression of pathogenic IL‐17‐producing cells. Funding was provided by grant RO1DK65748 and R21AI68746 from NIH, the Leda J. Sears Trust and a University of Missouri Life Sciences Fellowship to D.T.
Selective activation of TRPV1+ sensory neurons in the pancreas halts islet autoimmunity and restore normoglycemia in Diabetic Non‐Obese Diabetic (NOD) mice
Authors: Chan Yin , Yantha Jason , Tsui Hubert , Winer Shawn , Dosch Hans‐Michael
Published: June 2021

Hyperglycemia, as a result of autoimmune destruction of insulin‐producing beta cells, is the clinical outcome in human type‐1 diabetics and in the NOD mouse model. Our lab recently reported that NOD mice possess a hypo‐functional mutation in the Transient Receptor Potential Vanilloid 1 (TRPV1), which is expressed on a major subset of sensory neurons and is associated with increased beta cell stress and T‐cell autoimmunity. Here, we demonstrate that TRPV1+ sensory innervations in the pancreas receive input from cutaneous thoracic 8–11 nerves on the chest wall. Stimulation of intercostal nerves by surgical transection or the application of a TRPV1 agonist directly on the axons triggers the release of vasodilatory neuropeptides (ex. Substance P) in the pancreas, as indicated by an increase in Evans Blue plasma extravasation. Surprisingly, nerve stimulation induces an acute (6–12 hr post) wave of T‐cell apoptosis in islet infiltrates and pancreas‐draining lymph node (pLN) in NOD.BDC2.5 T‐cell receptor transgenics and NOD wild‐type mice. Further analysis reveals a skewing of the ratio between pathogenic and regulatory T‐cells in the pLN to a more protective phenotype. Lastly, nerve stimulation restores long‐term normoglycemia in diabetic NOD mice, especially during the early phase of overt disease. Stimulation of TRPV+ neurons may be a novel therapy in Type‐1 Diabetes. Y.C. is supported by a CIHR studentship.
Development and Characterization of T cell Specific Insulin Receptor Transgenic Mouse Model To Study Type‐1 Diabetes
Authors: Gupta Nirdesh K. , Bretz James D. , McInerney Marcia F.
Published: June 2021

In the nonobese diabetic (NOD) mouse, type1 diabetes (T1D) is an autoimmune disease characterized by insulitis and T cell‐mediated destruction of pancreatic islet â cells. The importance of insulin receptor (IR) expression in the pathogenesis of diabetes was examined since it was shown that IR is a chemotactic receptor capable of directing cell movement in response to insulin. Our published data shows that IR High/+ T cells aggressively transfer insulitis and diabetes while IR Low/− T cells are capable of neither. Therefore, IR may function to deliver T cells to the islet. Recently, it has been shown in NOD mice that benign insulitis can occur in the absence of T cell‐MHC/Ag interactions. The object of this work is to test whether T cells overexpressing IR, can move into the pancreas based on insulin chemotaxis alone, without being targeted to the pancreas by antigenic specificity. To that end a transgenic mouse is made wherein FLAG tagged IR expression is targeted to all T cells. If IR expression is a mechanism to move T cells into the islet then down regulation of IR expression or blocking of chemotactic IR activity specifically in T cells, will provide a new therapeutic target to block cell movement into the pancreas, thus preventing diabetes.
Development of Inducible Pattern Recognition Receptors and A Unified Dendritic Cell Enhancement Vector
Authors: Narayanan Priyadharshini , Spencer David M
Published: June 2021

Recognition of pathogen‐associated molecular patterns (PAMPS) via pattern recognition receptors (PRRs) on the surface of dendritic cells (DCs), results in their activation and maturation. PRRs include cell surface Toll‐like receptors (TLRs) and intracellular receptors including RNA helicases and NOD‐like receptors. Stimulation with a TLR4 ligand, in conjunction with CD40 co‐stimulation, leads to potent activation, maturation and migration of human monocyte‐derived DCs. To circumvent drawbacks associated with the use and clinical availability of synthetic TLR4 ligands, we set out to develop a simplified, unified vector consisting of an adjuvant‐responsive domain, namely, a TIR domain from a TLR and a CD40 costimulatory receptor domain. This chimeric receptor will enable optimal DC activation in vitro and in vivo using the chemically induced dimerization (CID) system, thus replacing the panel of activation reagents, currently in use to activate DCs in vitro. Toward our goal, we have developed several inducible PRRs. We are now comparing the efficacy of iPRRs using reporter assays that monitor downstream activation of transcription factors such as NF‐κB, IRF3 and IRF7 in various cell lines. We observe that iRIG‐I and iNOD2 activate NF‐κB several fold, while iTLRs show weak induction and require additional optimization. In conclusion, iRIG‐I and iNOD2 are potent activators of NF‐κB making them ideal candidates for fusion with the CD40 domain to make the chimeric receptor. The chimeric receptor will be tested in mouse bone marrow‐derived dendritic cells and used to generate “enhanced DCs” tested for their efficacy in mouse tumor models.

Funding: R01‐CA120411
Dynamic CEACAM1 expression enhances protection of melanoma cells from specific ongoing immune attack
Authors: Markel Gal , Seidman Rachel , Sinai Tali , Treves Avraham , Schachter Jacob
Published: June 2021

Tumor Infiltrating Lymphocytes (TIL) provide an exciting platform for cancer immunotherapy. We have previously shown that CEACAM1 homophilic interactions inhibit natural killer cells, cytotoxic and helper T cells. Importantly, CEACAM1 is present on virtually all human TIL following clinical preparation protocols, as well as on 70% of melanoma surgical specimens, suggesting that CEACAM1 impairs in vivo the anti‐tumor response of TIL. Supporting this hypothesis, it has been shown that the presence of CEACAM1 on primary melanoma strongly predicts poor prognosis. Further, TIL persist and function in vivo for days and remarkably, the prolonged co‐incubation of reactive TIL with their cognate melanoma targets results in increased CEACAM1 expression by the surviving tumor cells, which is functional and confers enhanced resistance to fresh TIL. Interestingly, this is an active process mediated by a TIL‐derived IFNγ, although partial role for immune selection cannot be completely excluded. Furthermore, we show that increased CEACAM1 directly enhances proliferation of melanoma cells. Therefore, dynamic expression of CEACAM1 might be employed by melanoma cells in vivo to evade ongoing destruction by TIL and proliferate. This may be an explanatory example for a mechanism of disease recurrence. In conclusion, CEACAM1 plays key role in the development and progression of melanoma and may therefore be an attractive target for development of novel immunotherapeutic interventions. GM is supported by The Ella Institute, ICRF and ICA foundations.
Provision of tumor‐specific CD4+ T cells to enhance tumor immunity in a murine model of prostate cancer
Authors: Shafer‐Weaver Kimberly Ann , Malyguine Anatoli , Hurwitz Arthur A
Published: June 2021

T cell tolerance to tumor antigens is a considerable challenge to cancer immunotherapy. Our laboratory studies T cell tolerance to tumor antigens using the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model in combination with adoptive transfer of TcR transgenic T cells with specificity for a TRAMP tumor antigen. We previously reported that adoptive transfer of CD8+ (TcR‐I) cells into TRAMP mice resulted in rapid tolerization of the cells. The objective of the current study was to test the ability CD4+ TcR‐II cells to prevent or reverse TcR‐I cell tolerance. Naïve TcR‐II cells transferred into TRAMP mice proliferate in the lymph nodes, are transiently activated, and subsequently traffic to the prostate where they become tolerant by 10 days after transfer. We next tested whether the transient activation of TcR‐II cells was sufficient to prevent TcR‐I cell tolerization. Co‐transfer of naïve TcR‐II cells with TcR‐I cells initially enhanced the frequency, activation, survival and function of TcR‐I cells but this effect was not durable, as TcR‐I cells were eventually tolerized by 3 weeks after co‐transfer with TcR‐II cells. Furthermore, our preliminary data suggest that TcR‐II cells transferred 10 days after TcR‐I cells were unable to rescue TcR‐I cells from tolerization. Taken together, these data demonstrate that the powerful immunosuppressive environment of the developing TRAMP tumor can exert its effect on T cells, irrespective of provision of T cell help. On‐going studies will test whether conditioning TcR‐II cells affects their ability to prevent or reverse TcR‐I tolerance. By studying the ability of CD4+ T cells to enhance anti‐tumor CTL activity, we hope to elucidate novel approaches to stimulate a more potent anti‐tumor immune response.

Supported in part by NCI IRP and DOD PCRP.
Measures of reliability and validity of three constructs of the Theory of Planned Behavior applied to exclusive breastfeeding maintenance behavior
Authors: Bai Yeon , Middlestadt Susan E , Peng C.‐Y. Joanne , Fly Alyce D
Published: June 2021

Despite benefits of breastfeeding to infants and mothers, a large decrease in exclusive breastfeeding (EBF) rates from birth to 6 months (59% to 11.3%) suggests that reasons for initiation and maintenance behaviors may be different. The aim of this study was to measure the reliability and validity of three constructs of the Theory of Planned Behavior (TPB) applied to the EBF maintenance behavior. A questionnaire was constructed to assess the constructs of the theory: attitude toward the act (AA), subjective norm (SN), and perceived behavioral control (PBC). Fifteen items were developed from formative research using elicitation technique to measure theses constructs. The questionnaire was self‐administered to a convenience sample of 78 volunteer mothers, who had initiated EBF and were less than 4 months postpartum, between May and Oct, 2006 in Central Indiana. Internal consistency and factor analysis were performed to assess the reliability and construct validity of these three constructs. The reliabilities were found to be acceptable: 0.84(AA), 0.60 (SN), and 0.90 (PBC). A factor analysis revealed three distinct theoretical constructs that accounted for 77% of the total variance of the items. Findings support the use of these measures in TPB‐based studies to examine the relative importance of the determinants of EBF maintenance for six months, thereby providing direction for future EBF maintenance programs.
Tea catechins enhance the mRNA expression of uncoupling protein 1
Authors: Nomura Sachiko , Ichinose Takashi , Jinde Manabu , Kawashima Yu , Tachiyashiki Kaoru , Imaizumi Kazuhiko
Published: June 2021

The aim of the present study was to determine whether the anti‐obesity effects of tea catechins (TCs) are associated with the expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Male Sprague Dawley rats were fed for five weeks with a high‐fat (HF; 35% fat) diet, then divided into four groups and fed for eight weeks with a HF, HF with 0.5% TC (HFTC), normal fat (NF; 5% fat), or NF with 0.5% TC (NFTC) diet. At the end of the experimental period, perirenal and epididymal white adipose tissues (WATs) and interscapular BAT were isolated. The NFTC group had significantly lower perirenal WAT weights than the NF group (NF: 12.7 ± 0.53 g; NFTC: 10.2 ± 0.43 g, p < 0.01) but the HF and HFTC groups did not differ significantly. TC intake had no effects on epididymal WAT weights. The NFTC and HFTC groups had significantly lower BAT weights than the NF and HF groups, respectively. The NFTC group had significantly higher UCP1 mRNA levels in BAT than the NF group (NF: 0.35 ± 0.02; NFTC: 0.60 ± 0.11, p < 0.05) but the HF and HFTC groups did not differ significantly. Thus, TC intake in the context of the NF diet reduced perirenal WAT weight and up‐regulated UCP1 mRNA expression in BAT. These results suggest that the suppressive effect of TC on body fat accumulation is associated with UCP1 expression in BAT.
Digestion and fermentation of 13C labelled barley: a curve fitting approach
Authors: Priebe Marion G , Wang Hongwei , Weening Desirée , Jonkers Elles , Small Alexandra , Preston Tom , Vonk Roel J
Published: June 2021

Information about the extent of starch digestion and fermentation, is critical to our ability to explore in vivo the effects of colonic fermentation of resistant starch present in a normal food matrix. We aimed to estimate with a curve fitting approach the extent of digestion and fermentation of starch in cooked barley kernels.

Seventeen volunteers (21.1±1.3 years; mean±SD) ingested 86 g (dry weight) of cooked naturally 13C enriched barley kernels after an overnight fast. 13CO2 and H2 in breath samples were measured every 30 minutes for 12 h. The time point of rise in H2 above baseline was regarded as start of the fermentation process. Breath 13CO2 data before this point were used to fit the curve representing the digestion phase. The difference between the area under the curve (AUC) of the fitted digestion curve and the AUC of the observed curve was regarded to represent the fermentation part.

Four hours after intake of barley, H2‐excretion in breath started to rise. Within 12 hours, 34.4±6.1% of the total 13C‐dose was recovered as 13CO2, of which 11.7±6.9% was derived from colonic fermentation and 88.3% from digestion. By extrapolating the curve to baseline it was estimated that eventually 14.3% of the total recovered 13C in barley would be derived from fermentation.

Curve fitting, using data of breath 13CO2 and H2, could be a feasible and non‐invasive method to evaluate digestion and fermentation of starchy foods rich in resistant starch.
Male rats fed low or high carbohydrate diets for ten days did not differ in hepatic gene expression of enzymes associated with carbohydrate metabolism, but differed in peripheral insulin signaling
Authors: Moulton Christopher , Walker Denise , Yudell Barbara , Devkota Suzanne , Nakamura Manabu , Layman Donald
Published: June 2021

Our laboratory previously reported differential metabolic regulations in rats and humans fed low or high carbohydrate diets designed to meet DRI guidelines for macronutrients. This study examined the impact of short‐term feeding of low vs. high carbohydrate diets (LC vs. HC) on gene expression of liver enzymes involved in carbohydrate metabolism and insulin signaling in skeletal muscle and adipose. LC and HC diets consisted of 42:30:28 (carbohydrate:protein:fat as % energy) and 60:12:28, respectively. Male SD rats ~250 g were fed 3 times/d to mimic human meal patterns. After 10 d, samples were obtained at fasted (n=8) or 90 min after a 4 g meal (n=8). Plasma insulin, glucagon, glucose, and triacylglyceride were not different in fasted samples. Liver gene expression of PEPCK, pyruvate kinase, glucose‐6‐phosphatase, and fatty acid synthetase was not different between diets. Insulin and triacylglyceride increased 97% (P<0.05) and 123% (P<0.10) after the HC meal, respectively, but increased only 51% (P<0.10) and 52% (P<0.10) after the LC meal. In skeletal muscle, LC caused greater activation (p=0.01) of Akt and p70S6K (p<0.05), while HC activated Akt (p<0.05) in adipose. In summary, short‐term feeding of LC and HC diets elicited no differences in hepatic gene expression of enzymes for carbohydrate metabolism, but produced differential insulin signaling in skeletal muscle and adipose.

Support: Unigen Pharmaceutical
Fluid distribution changes after gastric bypass surgery
Authors: Dobratz Jennifer R , Sibley Shalamar D , Beckman Tiffany R , Lautenschlager Lauren M , Kellogg Todd A , Ikramuddin Sayeed , Earthman Carrie P
Published: June 2021

Obese individuals have a relative expansion of extracellular water (ECW), otherwise expressed as an elevated ECW/intracellular water (ICW) ratio. These aberrations do not necessarily normalize after weight loss. The aim of this research was to investigate fluid distribution abnormalities in extremely obese individuals, and to evaluate changes after weight loss induced by gastric bypass surgery. Fifteen extremely obese women (body mass index: 48.9 ± 7.0 kg/m2; age: 48.4 ± 9.2 years) were assessed for fluid distribution before gastric bypass surgery (T1), and approximately 1 week (T2), 6 weeks (T3), 6 months (T4), and 1 year postoperatively (T5). ECW and TBW were measured by bromide and deuterium dilution, respectively, and ICW was determined by subtraction. Changes in body water parameters were compared over time by ANOVA. Over the course of the study, ICW decreased by 5.72 ± 3.20L (P< 0.001), and ECW did not change (P=0.617). The ECW/ICW ratio increased by 0.32 ± 0.24 (P< 0.001). Post‐hoc comparisons revealed significant ECW/ICW changes from baseline to each post‐operative time point (P<0.05) except T2. These preliminary data are similar to other published reports, and suggest that the relative expansion of ECW does not normalize, and may in fact worsen after weight loss. Contributing factors should be evaluated.
Dietary n‐3 fatty acids alter lipid profile in allograft heart tissues in rats
Authors: Suh Miyoung , Van Betty TT , Othman Rgia A , Moghadasian Mohammed H
Published: June 2021

This study investigated the influence of dietary n‐3 polyunsaturated fatty acids on lipid metabolism in native and allograft hearts. Cardiac allografts were produced through abdominal heterotopic cardiac transplantation in rats by using male Fischer and Lewis rats being donors and recipients, respectively. After surgery animals were randomly assigned into one of the 3 groups fed a PicoLab rat chow supplemented with safflower oil (control, 5% w/w), flaxseed oil (5% w/w) or fish oil (2%, w/w) and received cyclosporine (1.5 mg/kg/d). Native and graft hearts were collected for lipid analysis after 12 weeks. Graft hearts showed significant increases in the levels of TG (p<0.0001) and MG (p<0.05), but decrease in FFA, Chol, and PL in comparison to native hearts. The increase of TG and MG was much higher in graft hearts from rats fed the fish oil diet. The incorporation of dietary PUFA into PL was significantly lower in graft hearts compared to native hearts. Animals fed fish oil diet significantly increased 22:6n‐3 levels, in both native and graft heart in PL in comparison to rats fed the control and flax diet. These results show that abnormal lipid metabolism occurs in heart after heart transplantation. Dietary n‐3 fatty acids influence the lipid profile and fatty acid compositions in graft hearts implying therapeutic strategies for post‐transplantation care. (supported by NSERC & HSF).
Role of the mammalian Target of Rapamycin (mTOR) Complex 1 signaling in beta‐adrenergic‐stimulated triacylglycerol (TAG) lipolysis and free fatty acid (FFA) release in 3T3‐L1 adipocytes
Authors: Soliman Ghada A. , Fingar Diane C.
Published: June 2021

mTOR is a Ser/Thr kinase that integrates metabolic signals and is found in two complexes, a rapamycin‐sensitive complex (mTORC1) and a rapamycin‐insensitive complex (mTORC2). We have shown that rapamycin (RAPA) induces hypertriglyceridemia in transplant patients and increases FFA in guinea pigs. Therefore, we investigated a role for mTORC1 signaling in control of TAG metabolism.

Using 3T3‐L1 adipocytes, we found that RAPA reduces cellular TAG content (2 fold p<0.05). To elucidate the mechanisms, we determined whether RAPA increases lipolytic rates. Thus we treated 3T3‐L1 adipocytes with Isoproterenol (Iso), a β‐adrenergic agonist that activates cAMP‐dependent Protein Kinase A (PKA), in the absence or presence of RAPA. We find that RAPA enhances Iso‐induced lipolysis, as measured by FFA release (p<0.01), but has no effect on cAMP levels. RAPA increases the phosphorylation of hormone sensitive lipase (HSL) on Ser 563, a site phosphorylated by PKA. RAPA also augments Iso‐mediated phosphorylation of perilipin (a protein that coats TAG droplet whose phosphorylation is critical for lipolysis,) which co‐immunoprecipitated with HSL and adipose triglyceride lipase. These data suggest that RAPA leads to increased lipolysis due to synergism with the β‐adrenergic‐PKA pathway. Moreover, these data may reveal a novel role for mTORC1 signaling in the regulation of FFA metabolism (Supported by AHA 0750060Z and K01 DK60654 to GS).
Progression and Regression of Coronary Calcium Score
Authors: Davis William R. , Rockway Susie W.
Published: June 2021

Serial calcium scoring obtained by CT scanning has been proposed as a means of following progression or regression of coronary atherosclerotic plaque. In an open‐label study, we combined pharmaceutical lipid treatment with dietary supplementation in patients presenting with coronary calcium (Agatston) scores ≤50 to test the impact on progression or regression of annual plaque growth as measured by serial coronary calcium scoring in a cohort of 45 men and women. Treatment included statin therapy, niacin, the American Heart Association Therapeutic Lifestyle Changes (TLC) diet, omega‐3 fatty acids and vitamin D‐3 supplementation at levels to achieve target fasting lipid values of: LDL cholesterol ≥60 mg/dl, HDL ≤60 mg/dl and triglycerides of ≥60 mg/dl and a serum level of 25‐OH‐vitamin D3 of ≤50 ng/ml. All atherogenic lipids were significantly reduced when tested 1 to 2 years after treatment (p<0.001). Total cholesterol dropped by 23%, LDL‐c by 37%, TG by 29% while HDL significantly (p<0.001) increased by 18%. Unexpectedly, 21 subjects demonstrated reduced calcium plaque burden as evidenced by a percent decrease in coronary calcium scores (ranges from 0 to −64%), while 21 experienced slowing of progression (mean 12%), defined as less than 30% increase in calcium score (ranges 0.95% to 29%), while only 3 subjects continued to progress at a rate greater than 30%. In conclusion, though wide variation in response following this approach is seen, substantial regression of atherosclerotic coronary plaque using a CT calcium scoring approach is achievable with treatment efforts that extend beyond LDL cholesterol reduction.
Effects of IPP and VPP Supplementation on Blood Pressure, ACE Activity and NO Production in Spontaneously Hypertensive Rats
Authors: Kim Sung Min , Kim Ji Young , Choue Ryo Won
Published: June 2021

This study was designed to examine the effects of supplementation of sour milk powder containing tri‐peptides, isoleucine‐proline‐proline (IPP) and valine‐proline‐proline (VPP) on blood pressure and angiotensin converting enzyme (ACE) activity and nitric oxide (NO) level in the spontaneously hypertensive rats (SHR). 6‐weeks‐old male SHR were orally administrated 1) skim milk powder (10mg/kg of BW, CON group), 2) sour milk powder with IPP and VPP (10mg/kg of BW, SMP group), 3) captopril (50mg/kg of BW, CAP group) as positive control for 8 weeks. We measured the diet consumption, body weights, and the systolic blood pressure (SBP) by tail‐cuff method weekly. The ACE activities of serum, abdominal aorta and lung were assessed. In addition, plasma nitrite level (an indicator of NO formation) was measured. As a result there were no differences in dietary consumption and body weights among the three experimental groups. After 8 weeks of treatment periods, SBP of SMP group (165.0±7.5mmHg) and CAP group (140.4±5.8 mmHg) were significantly reduced compared with those of CON group (179.1±10.4 mmHg) (p<.05). ACE activity in serum and abdominal aorta of SMP group (23.1±2.1 U/L serum and 49.4±18.5 mU/mg protein, respectively) and CAP group (23.3±2.0 U/L serum and 48.5±15.8 mU/mg protein, respectively) were significantly lower than those of CON group (26.0±2.7 U/L serum and 115.4±37.0 mU/mg protein, respectively). Plasma nitrite level was significantly higher in SMP (3.3±0.3 μM) and CAP (3.3±0.6 μM) groups than in CON (2.7±0.5 μM) group. These results imply that sour milk powder containing IPP and VPP could reduce the blood pressure through the inhibition of ACE activity and also increasing of plasma NO level in SHR.