bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid induced TNF Receptor Ligand (GITRL) transgenic mice develop T and B lymphocyte hyperplasia and expansion of CD4+ FOXP3+ cells
Authors: Whitters Matt J , Miyashiro Joy , Stedman Nancy , Benoit Stephen , Ryan Mark , Small Clayton , Kuang Wen , Collins Mary , Douhan John , Dunussi‐Joannopoulos Kyri
Published: June 2021

The glucocorticoid‐induced TNF‐related receptor (GITR) and its natural ligand (GITRL) belong to the TNF receptor and TNF superfamilies (TNFRSF18 and TNFSF18). The expression profile of both GITR and GITRL suggests that they play primary roles in the immune response. To better understand the role of GITR/GITRL interactions in the mouse immune system, we generated transgenic GITRL mice under the control of a MHC class II promoter. Elevated expression of GITRL was observed on macrophages, dendritic cells and B cells. GITRL transgenic mice had mild microcytic anemia and substantially enlarged spleens. Histologically, these animals exhibited multi‐organ lymphocytic infiltrates which were particularly profound in the spleen, lung, liver, kidneys, and salivary glands. Immunophenotyping of these animals demonstrated higher percentages and numbers of T and B cells in spleens and other lymphoid organs. Germinal center formation was rare in the lymphoid organs. The percentage of total splenic CD4+ T cells in transgenic mice was elevated while the total CD8+ T cell percentage was diminished compared to WT littermates. Interestingly, the percentage of CD4+ FOXP3+ cells in the spleen was greatly increased but very few of these cells were positive for GITR surface expression. In conclusion, enhanced expression of GITRL on MHC class II positive mouse cells leads to expansion of both T and B cell compartments and lymphoid organ enlargement. Further evaluation of T cell subsets and the consequences of enhanced GITR/GITRL interactions in these animals is ongoing.
The formylpeptide receptor FPR exploits the function of the epidermal growth factor receptor to promote the progression of glioblastoma
Authors: Huang Jian , Zhou Y , Bian X , Le YY , Gong W , Chen K , Wang JM
Published: June 2021

In the course of studying the role of chemoattractant receptors in tumor growth and metastasis, we discovered that highly malignant human glioblastoma and anaplastic astrocytoma specimens were stained positively for the formylpeptide receptor FPR, which is normally expressed in myeloid cells and accounts for their chemotaxis and activation induced by bacterial peptides. Screening of human glioma cell lines revealed that FPR was expressed only in glioma cells with a more highly malignant phenotype. FPR expressed in glioblastoma cell lines mediates cell chemotaxis, proliferation and production of VEGF, in response to agonistic ligands released by necrotic tumor cells. Furthermore, FPR in glioblastoma cells transactivates the receptor for epidermal growth factor (EGFR) by a signal transduction pathway that increases the phosphorylation of a selected tyrosine residue in EGFR. This transactivation of EGFR by FPR accounts for part of the function of FPR in tumor cells. Depletion of FPR or EGFR in tumor cells by small interference (si) RNA each reduces the capacity of the tumor cells to form actively growing tumors in nude mice, while depletion of both receptors completely abolishes the tumorigenicity of glioblastoma cells. Thus, FPR aberrantly expressed in human glioblastoma cells by responding to agonistic ligands produced in the tumor microenvironment cooperates with EGFR to promote rapid tumor progression. These results suggest important molecular targets for the design of novel anti‐glioblastoma therapeutics.
Protective Roles of the Fractalkine/CX3CL1‐CX3CR1 Axis against Alkali‐induced Corneal Neovascularization thorough Enhanced Anti‐angiogenic Factor Expression
Authors: Lu Peirong , Mukaida Naofumi , Zhang Xueguang
Published: June 2021

Macrophage infiltration is observed during the course of corneal neovascularization. We examined corneal neovascularization process after alkali injury in mice deficient in fractalkine receptor/CX3CR1, which is expressed by macrophages. The mRNA expression of CX3CR1 and its ligand CX3CL1/fractalkine was augmented together in the corneas after alkali injury. Compared with WT mice, CX3CR1‐deficient (KO) mice exhibited enhanced corneal neovascularization 2 weeks after injury, as evidenced by enlarged CD31‐positive areas. Concomitantly, the infiltration of F4/80‐positive macrophages but not Gr‐1‐positive neutrophils was significantly attenuated in KO mice comparing with WT mice. The intraocular mRNA expression of angiogenic factor, vascular endothelial factor, was enhanced to similar extents in WT and KO after the injury. In contrast, the mRNA expression of anti‐angiogenic factors, a disintegrin and metalloprotease with thrombospondin (ADAMTS)‐1 and thrombospondin (TSP)‐1 was enhanced in wild‐type mice but the enhancement was attenuated in KO mice. A double immunofluorescence analysis demonstrated that F4/80‐positive cells also expressed CX3CR1 and ADAMTS‐1, TSP‐1 and ADAMTS‐1 were detected in CX3CR1‐positive cells. CX3CL1 enhanced the ADAMTS‐1 and TSP‐1 expression by peritoneal macrophages. Moreover, topical application of CX3CL1 inhibited corneal neovascularization at 2 weeks, together with enhanced intraocular ADAMTS‐1 and TSP‐1 expression. Thus, these observations indicate that intraocular infiltrating CX3CR1‐positive macrophages can dampen alkali‐induced corneal neovascularization by producing anti‐angiogenic factors, such as ADAMTS‐1 and TSPs.
The Role of Complement and VEGF on SDF‐1 Mediated Migration of CD34+ Stem Cells
Authors: Pokropinski Sharon , Smith Stephen , Campro Lory , Rausa Francisco , Amrani David , Johnson Richard
Published: June 2021

CD34+ stem cells migrate from the bone marrow niche to sites of injury in response to various chemokines. We have investigated the influence of VEGF and complement peptides C3a and C5a, on the stromal derived factor‐1(SDF‐1)‐dependent migration of freshly isolated (from G‐CSF mobilized mononuclear cell preparations) and cord‐blood derived CD34+ stem cells. Freshly isolated CD34+ cells migrated in a dose‐dependent manner in response to SDF‐1, but were unable to migrate to the complement peptides C3a or C5a alone. However, both complement split products augmented the migration of CD34+ cells in response to low concentrations of SDF‐1. This augmented migration behavior was abrogated when CD34+ cells were incubated with anti‐C3a or anti‐C5a antibodies. Flow cytometry demonstrated that CD34+ stem cell express both the C3a and C5a receptors, and anti‐receptor antibodies also mitigated this synergy. Freshly isolated CD34+ cells responded initially to VEGF, but this response diminished after 1–2 days In contrast, with cultured cord blood‐derived CD34+ cells, we observed enhanced migration to SDF‐1 in the presence of VEGF compared to SDF‐1 alone. In conclusion, the migration of CD34+ stem cells depends on their source and ex vivo treatment, and may be enhanced to ischemic tissues (where SDF‐1 is produced) under the influence of other inflammatory and angiogenic signals.
MARCKS protein regulation of human neutrophil migration in vitro
Authors: Jones Samuel L. , Eckert Rachael E. , Adler Kenneth B.
Published: June 2021

Protein kinase C is required for neutrophil migration as is the regulatory molecule calmodulin. The actin binding protein Myristoylated Alanine‐Rich C‐kinase Substrate (MARCKS), which is phosphorylated by PKC and binds calmodulin, is a potential point of convergence of these two regulatory mechanisms. Stimulation of neutrophils with the chemoattractant peptide fMLF results in rapid MARCKS phosphorylation and dissociation from the cell membrane. The hypothesis that MARCKS has a role in the regulation of neutrophil migration was tested using a myristoylated peptide corresponding to the first 24 amino acids of the MARCKS aminoterminus (MANS peptide). Treatment of isolated human neutrophils with MANS (50 μM) significantly inhibited their migration and beta 2 integrin dependent adhesion in response to fMLF, IL8, or LTB4. A missense peptide (RNS peptide) did not affect migration or adhesion. MANS significantly reduced F‐actin content in neutrophils 30s after fMLF stimulation, although the peptide did not alter the ability of cells to organize the actin cytoskeleton or spread. MANS did not alter fMLF stimulated increase in surface expression of beta 2 integrins. Our data suggest that MARCKS, via its myristoylated aminoterminus, is a key regulator of neutrophil migration and adhesion. Supported by NIH R37 HL36982 and the NC State Center for Comparative Medicine and Translational Research.
Calcineurin Deficiency Decreases Inflammatory Lesions in TGFbeta1‐deficient Mice
Authors: Bommireddy Ramireddy , Bueno Orlando F. , Martin Jennifer , Ormsby Ilona , Chen Dora , Molkentin Jeffery D. , Boivin Gregory P. , Babcock George F. , Doetschman Thomas
Published: June 2021

Tgfb1 knockout mice develop severe autoimmune inflammatory lesions and die within 3 wks after birth. Since the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1−/− thymocytes, and since calcineurin Aβ‐deficient (Cnab−/−)T cells are hyporesponsive to stimulation, we have generated Tgfb1−/− Cnab−/− mice (double knockout mice) to address whether CnAβ deficiency prevents T‐cell activation and inflammation in Tgfb1−/− mice. Here we show that in Tgfb1−/− Cnab−/− mice inflammation is reduced relative to that in Tgfb1−/− mice. Both CD4+ and CD8+ T cells in double knockout (DKO) mice are activated. To our surprise, we have found that T cells undergo spontaneous activation in Cnab KO mice. Further analysis of Cnab KO mice revealed that Foxp3 expression is down regulated and total Treg cell numbers are reduced suggesting that CnAβ could regulate T‐cell activation through generation of Treg cells. These data also suggest a dual role for CnAβ in T‐cell activation and regulation.

Grant support: This study was supported by NIH AI067903 and CA84291 to TD, and by a grant from Shriners of North America to GFB.
Characterization of the immune response in a new murine model of autoimmune arthritis
Authors: McMahon Eileen Joan , Adipue Iris , Wilcox Joel
Published: June 2021

We have discovered a novel mouse strain that spontaneously develops arthritis, and we are currently assessing its viability as a new model of immune‐mediated arthritis. The purpose of this project was to characterize the immune response in this new strain. Cells of the spleen, lymph nodes, and joints were isolated from AR and non‐AR littermates and analyzed for immune cell proliferation and activation using flow cytometry. Serum was also collected and assayed for immunoglobulin levels and pro‐inflammatory cytokines. AR mice had significantly more immune cells in their joints. The vast majority were neutrophils, although T cells and B cells were also present. Surprisingly, AR mice tended to have lower numbers of total cells isolated from their spleen and lymph nodes, possibly indicating that the immune cells were largely localizing to the joints, the major site of inflammation. The CD4+and CD8+ T cells that were in these organs did show elevated signs of activation in the AR mice (i.e. increased CD25 and CD69 and decreased CD45RB). Mean serum levels of total Ig and IgG1 but not IgG2b were elevated in AR mice. Finally, interleukin‐6 was elevated in all the AR mice in the early stages of disease and tumor necrosis factor‐alpha was elevated in half of the AR mice. Overall, our data indicate that there is more robust inflammatory response occurring in the AR mice.
IL‐22 directly induces keratinocyte hyperplasia, up‐regulates anti‐microbial peptide and pro‐inflammatory cytokine gene expression in mouse skin
Authors: Napierata Lee , Liang Spencer , Mason Larry , Cai Jie , Collins Mary , Nickerson‐Nutter Cheryl , Fouser Lynette , Young Debbie , Ma Hak‐Ling Margery
Published: June 2021

IL‐22 is a member of the IL‐10 family of cytokines that signals through the class II cytokine receptor heterodimer IL‐22R/IL‐10R2. It is secreted by activated CD4+ T cells and NK cells and has been shown to regulate keratinocyte hyperplasia and differentiation in vitro. Since the IL‐22 receptor is expressed on a variety of epithelial tissues, the IL‐22/IL‐22R pathway is believed to have effects on epithelial cells. The fact that IL‐23 induced ear swelling is significantly decreased in IL‐22 deficient mice when compared with the control mice further highlights the role of IL‐22 in dermal inflammation and acanthosis.

To directly examine the role of IL‐22 in vivo, IL‐22 or saline was injected into the ears of wild type Balb/c mice every other day for two weeks. RNA from the mouse ears was prepared for quantitative RT‐PCR analysis two weeks after the first injection. At the end of the two weeks, IL‐22 induced keratinocyte hyperplasia in the mouse ear, as well as increased defensin β1, S100A8 and S100A9 transcripts. IL‐22 also significantly induced IL‐1α gene expression with a trend towards increased IL‐6 and TNFα transcripts. These results further confirmed that IL‐22 directly induces pro‐inflammatory gene expression and proliferation of keratinocytes that may lead to the recruitment of immune cells and contribute to the development of skin inflammation in vivo.
Migration of Tissue Dendritic cells in Immune‐mediated Inflammatory Diseases: Role of Cooperative Interaction Between Epidermal gd T Cells and Skin Dendritic Cells
Authors: Eriksson Anna Ulrika , Singh Ram Raj
Published: June 2021

The skin‐resident dendritic cells (DC), namely dermal DC (dDC) and Langerhans cells (LC), are believed to sample antigens in the skin and migrate to cutaneous lymph nodes (CLN) to induce immunity or tolerance to these antigens. We report a previously unappreciated defect in the migration of LC prior to the appearance of skin inflammation in genetically autoimmune‐prone MRL‐Faslpr/lpr and MRL‐Fas+/+ mice. In these mice, LC displays a drastic reduction in migration in the steady state as well as upon in vitro or in vivo stimulation. This defect is associated with markedly reduced numbers of γδ dendritic epidermal T cells (DETC) in the epidermis of MRL mice. The role of DETC is not fully investigated but they are believed to be involved in homeostasis of skin, and facilitate wound healing. Both defects are corrected and skin inflammation is reduced by in vivo treatment of these animals with α‐galactosylceramide, a glycolipid that binds CD1d and activates natural killer T (NKT) cells. Surprisingly, DETC numbers and LC migration are also reduced in CD1d‐deficient, but not in NKT cell‐deficient, mice. These data suggest that a glycolipid can induce expansion of skin γδ DETC, which promote the migration of LC from epidermis to skin draining lymph node.
B Cell Receptor Crosstalk: BCR Signaling Via the Alternate and Classical Pathways Induces Expression and Secretion of the Autoimmunity‐Associated Cytokine, Osteopontin
Authors: Guo Benchang , Rothstein Thomas L
Published: June 2021

Classical BCR signaling requires a number of signalosome mediators that are bypassed when BCR signaling follows an alternate pathway produced by prior exposure of B cells to IL‐4. The two pathways, classical and alternate, co‐exist in IL‐4‐treated B cells. Objective: To explore the biological significance of the IL‐4 induced alternate signaling pathway. Methods: Micro array, Real‐time PCR, Western blot, and ELISA were employed. Results: Operation of the IL‐4‐induced alternate pathway changes the nature of the B cell response to BCR engagement so that the cytokine, osteopontin (Opn), is produced. Although Opn expression by B cells has not previously been noted, anti‐Ig‐induced secretion by IL‐4‐treated B cells amounts to levels comparable to those secreted by activated T cells. Unlike T cells, B cell production of Opn does not depend on T‐bet. Conclusions: Because elevated levels of IL‐4 occur in association with severe illnesses, and because Opn is strongly associated with autoimmunity, these results suggest that the IL‐4‐induced alternate BCR signaling pathway may participate in the pathophysiology of autoimmune dyscrasias. This work was supported by Public Health Service grants AI40181 and AI60896 awarded by the National Institutes of Health
NKG2D‐Rea1 recognition between NK cells and Kupffer cells in a novel NK cell‐dependent fulminant hepatitis
Authors: Tian Zhigang , Hou Xin
Published: June 2021

Increasing evidences suggest the contribution of NK cells in the pathogenesis of human hepatitis, but the mechanisms remain to be further investigated. In this study, we demonstrate that poly I:C can induce severe liver injury in D‐galactosamine (D‐GalN) sensitized mice. Further experiments show that the liver injury induced by poly I:C/D‐GalN depends on the cooperation between NK cells and Kupffer cells. poly I:C/GalN induce Kupffer cells to produce TNF‐¦Á and to express the retinoic acid early inducible‐1 (RAE 1) proteins, which then stimulates NK cells to produce IFN‐¦Ã via NKG2D‐RAE 1 interaction. The NK‐derived IFN‐¦Ã and Kupffer cells‐derived TNF‐¦Á induce severe liver injury via synergic effects. These results suggest that NK cells and Kupffer cells have a synergic effect on liver injury and provide the first in vivo evidence that NKG2D/ligand interaction are involved in the cooperation between NK cells and macrophages. Our results also provide an NK cells‐mediated hepatitis model.
Titanium increases CCR4 expression and promotes expression of inflammatory cytokines in human T cells
Authors: Sutanto Michael , Chan Erwin , Mhawi Amir , Filgueira Luis
Published: June 2021

Titanium is increasingly used for implanted bio‐medical devices, including bone fracture treatment and joint replacement therapies. Bio‐corrosion of implants and accumulation of titanium in immune cells, lymph nodes and spleen has been reported. Inflammatory reactions against titanium implants have been reported in up to 15% of cases. However, little is known about the effect of titanium on immune cells. The aim of this study was to investigate the effect of Ti(IV) ions (1 to100μM) on human T‐lymphocytes, freshly isolated from buffy coats or peripheral venous blood of healthy individuals (n=10). No toxic effect was detected in viability assays for the applied titanium concentrations. Using energy filtered transmission electron microscopy, uptake and accumulation of titanium in T‐lymphocytes was documented. Titanium induced cell proliferation (BrdU incorporation assays) in a concentration dependent manner in 40% of the tested individuals. Cytokine measurement in the culture supernatants revealed increased IL‐6 and TNFα production at 12.5μM titanium concentration. Measurement of surface marker expression with flow cytometry showed increased expression of CD3, CD4, CD45 and the chemokine receptor CCR4.

This study indicates that titanium affects human T‐lymphocytes by enhancing their inflammatory properties.
Defective TLR‐2‐Dependent Induction of IL‐6 by Macrophages in Aged Humans
Authors: Nguyen Joseph Tri , Gollapudi Sastry , Agrawal Sudhanshu , Gupta Sudhir
Published: June 2021

Aging represents a state of paradoxical chronic inflammation and immune deficiency. However, a role of monocytes in inflammation associated with aging has not been studied in detail. Recent evidence suggests that monocytes are heterogeneous with regards to phenotype and functions. In this study, we have compared the phenotypically heterogeneous monocytes for their innate immune functions in elderly subjects. Whole peripheral blood mononuclear cells were stimulated with Pam‐3Cys (TLR‐2 agonist) and LPS (TLR‐4 agonist). Monocytes were surface stained with anti‐CD14 and anti‐CD16 antibodies, and isotype controls, then permeabilized and stained for intracellular cytokines IL‐6 and TNFalpha. Production of cytokines and TLR‐2 and TLR‐4 expression were analyzed in four subsets of monocytes (CD14+CD16+; CD14++CD16+; CD14+CD16−; CD14++CD16−) by multicolor flow cytometry. The proportion of all four subsets of monocytes as well as expression of TLR‐2 and TLR‐4 were comparable in young and aged subjects. LPS‐induced IL‐6 and TNFa producing monocytes were also comparable. However, Pam‐3cys‐induced IL‐6 production by all four monocyte subsets was significantly decreased in aged humans. This would suggest a defect in signaling downstream of TLR‐2 in monocyte subset in aged humans, which is currently being investigated.
Comparison of the neuroinflammatory pathways induced by TLR7, TLR8 and TLR9 agonists in the neonatal brain
Authors: Butchi Niranjan Babu , Pourciau Susan , Du Min , Morgan Timothy , Peterson Karin
Published: June 2021

Neuroinflammation in the central nervous system is a common finding in a variety of neurological diseases induced by viruses, bacteria and injury as well as those of unclear etiologies. The TLR9 family members are strong candidates for regulating neuroinflammatory responses as ligands for these receptors are expressed by a number of pathogens that can infect the CNS and induce neuronal damage. To examine the potential role of TLR7, 8 and 9 in glia activation, neuroinflammation and neuronal damage, a single inoculum of Imiquimod, 3M002 or CpG DNA respectively were injected intracerebrally into new born mice. TLR9 stimulation induced a stronger neuroinflammatory response as compared to either TLR7 or TLR8 stimulation, despite the use of much lower molar dose of TLR9 agonist. In addition, we analyzed the influence of one TLR stimulation on the expression of other TLRs in neonatal brain in vivo and in astrocyte and mixed glia cultures ex vivo. Our preliminary results indicate that TLR7 stimulation alters the expression of TLR8 and 9, but not TLR7. Stimulation of TLR9 alters expression of TLR8. As these receptors have been shown to interact with each other's signaling, the alteration of TLR expression may have auto‐regulatory functions. We are currently analyzing the interaction between these receptors in neuroinflammation utilizing TLR7 and TLR9 deficient mice. This research is supported by NCRR # 1P20RR20159‐01.
Aging impairs plasmacytoid dendritic cell function in response to TLR9 activation
Authors: Stout‐Delgado Heather , Tesar Bethany M , Goldstein Daniel R
Published: June 2021

Plasmacytoid dendritic cells are innate sensors that produce IFNα in response to type A unmethylated CpG sequences or viral infections. However, the impact of aging on plasmacytoid dendritic cell function is unknown. Determining how aging alters the function of these cells may explain why older people are more susceptible to viral infections. Hence, we examined the effect of aging on plasmacytoid dendritic cell function in response to TLR9 stimulation with either type A CpG or herpes simplex virus type 2. We found that aged murine plasmacytoid dendritic cells produced lower levels of IFNα but preserved IL‐12p40 production after TLR9 activation as compared to young plasmacytoid dendritic cells. Transfer of young plasmacytoid dendritic cells into aged hosts recovered IFNα production and led to control of viremia during herpes simplex viral infection. We found that aged plasmacytoid dendritic cells manifested decreased gene expression of several signal adaptors, including interferon regulatory factor 7, which are critical for IFNα production during Toll‐like receptor activation. In sum, aging impairs the type 1 IFN‐signaling pathway in plasmacytoid dendritic cells, which may explain the increased susceptibility of older people to certain viral infections.

This work was supported in part by NIH grants AG028082 and AG026772 to DRG. HWSD is supported by NIH grant T32AG019134. WEW is supported by NIH grant 5T32HL007778.
Dendritic cells as a producer cell of beta‐defensins
Authors: Kawasaki Takashi , Kobayashi Makiko , Herndon David N , Suzuki Fujio
Published: June 2021

Beta‐defensins (BDs) and dendritic cells (DC) have been described as major effectors on host antimicrobial innate immunities. In the present study, the ability of DC to produce BDs was explored using DC from mice with different severities of burn injuries.

FT‐burned mice (BALB/c mice with 25% TBSA flame burn, 3rd degree) and PT‐burned mice (BALB/c mice with 25% TBSA scald burn, 1 to 2nd degree) were prepared as described previously. DC were isolated from spleens of mice 3 days after burn injuries, and 1×106 cells/ml of them were cultured with LPS or SAC. Culture fluids harvested 24 hrs after cultivation were assayed for BD1 and BD3 (ELISA) and a bactericidal activity (colony‐counting, P. aeruginosa). Also, DC were tested for BD mRNAs by RT‐PCR.

90% of the bacterial growth was inhibited by the culture fluids of splenic DC from PT‐burned mice, while only 30% growth inhibition was shown by the culture fluids of splenic DC from FT‐burned mice. As compared with DC from normal mice, DC from PT‐burned mice produced increased amounts of BD1 and BD3 in their culture fluids, while decreased amounts of BDs were produced by DC from FT‐burned mice. The results were reproduced in 2 different assays for BDs (ELISA and RT‐PCR).

These results indicate that 1) DC from spleens of mice have an ability to produce BDs, and 2) the production of BDs by DC is influenced strongly by thermally injured stress. Since PT‐burned mice are resistant and FT‐burned mice are susceptible to P. aeruginosa infection, BDs produced by DC may play an important role on the host's antibacterial resistance. (Supported by SHC NA #8610.)
IgG1 and IgG2a/c induce IL‐10 from different FcγR on macrophages as well as DCs in Leishmania mexicana infection
Authors: Buxbaum Laurence U. , Thomas Bolaji N.
Published: June 2021

Chronic L. mexicana disease in C57BL/6 mice requires both FcRγ and IL‐10. The initial response to infection includes anti‐leishmanial IgG1 followed by a chronic phase mixed IgG1/IgG2a/c response. FcγRIII KO and IL‐10 KO mice resolve lesions with a stronger IFN‐γ response, with parasite control by 12 weeks. We also found that bone marrow‐derived macrophages (BMMΦ) from FcγRIII KO and FcRγ KO mice have the same profound defect in IL‐10 production induced by IgG1‐opsonized amastigotes. Previous reports have shown that FcγRI and IV have higher affinity binding of IgG2a/c than other FcγR. We now show that in BMMΦ, IgG1‐opsonized parasites induce IL‐10 primarily via FcγRIII, while IL‐10 induction by IgG2a/c‐opsonized parasites is mostly via FcγRI and to a lesser extent, FcγRIII. Although FcγRIV was present on BMMΦ, blockade of this receptor did not have an effect on IL‐10 induction. Similar results were obtained for BMDCs indicating both macrophages and DCs are equally capable of secreting IL‐10 in response to parasite immune complexes. Furthermore, in vivo we found that although healing FcγRIII KO mice have no alteration in IL‐10 from T cells in the draining LN, there was a 7‐fold decrease in IL‐10 from lesions, where macrophages are abundant and T cells are rare. This supports a role for both IgG1 and IgG2a/c, and macrophages and possibly DCs in generating the suppressive IL‐10 response in the lesion during infection.

Funded by VA Merit.
PD‐1 deficiency protects mice from the lethality of sepsis by balancing efficient pathogen clearance and inflammatory cytokine production
Authors: Huang Xin , Venet Fabienne , Wang Yvonne L , Lepape Alain , Swan Ryan , Chen Yaping , Chung Chun‐shiang , Monneret Guillaume , Ayala Alfred
Published: June 2021

Recent studies suggest that the tolergenic receptor, PD‐1, is upregulated on leukocytes of the severely injured, however, its’ contribution to septic morbidity/mortality is unknown. Here we report that PD‐1−/− mice showed profound resistance to the lethal effects of septic challenge in mice (cecal ligation and puncture [CLP]). Surprisingly, this was associated with a marked decline in bacterial burden in blood and peritoneal lavage of the PD‐1−/− as compared to the WT CLP mice. Moreover, both systemic blood and local peritoneal inflammatory cytokine levels were markedly attenuated in the PD‐1−/− vs the CLP WT mice. To the extent that this is a macrophage specific aspect of PD‐1's effects we found: 1st, peritoneal macrophage expressed significantly higher levels of PD‐1 during sepsis; 2nd, when peritoneal macrophage had been depleted (using clodronate liposomes) from PD‐1−/− mice, the animals' bactericidal capacity was lowered, the cytokines levels were elevated and, their protection from septic lethality diminished; and 3rd, both blood monocytes PD‐1 levels from septic shock patients and mice are markedly increased. In conclusion, we not only show that PD‐1 plays a role in septic morbidity/mortality but PD‐1 mediates these deleterious effects, in part, through its’ actions on macrophages. (NIH GM46354)
Murine CD1d‐restricted gamma‐delta T cells respond to cardiolipin
Authors: Dieudé Mélanie , Zajonc Dirk , Behar Samuel M. , Levine Jerrold S , Rauch Joyce
Published: June 2021

CD1d recognizes both microbial and self‐antigens, but the nature of the endogenous lipids bound by CD1d remains poorly elucidated. Cardiolipin (CL) is a major phospholipid component of bacteria that have been shown to trigger strong CD1d‐restricted responses. CL is also present in mammalian cells, but normally hidden from the immune system because of its location in the mitochondrial inner membrane. Based on growing evidence that CD1‐restricted T cells recognize phospholipids, we hypothesized that T cells in the normal immune repertoire respond to CL. Splenocytes, or liver‐derived lymphocytes, from C57BL/6 mice were incubated with bovine heart CL or control phospholipids. Proliferation occurred exclusively with CL, in a dose‐dependent manner, and was greatest in liver‐derived cells. CL‐dependent T cell stimulation was abrogated by monoclonal anti‐CD1 antibody, but was unaffected by antibody against MHC I or II. Furthermore, CL‐dependent T cell stimulation was undetectable in cells isolated from CD1d‐deficient mice. Using flow cytometry, CL‐responsive cells were shown to express gamma‐delta T cell receptor (TCR) and to produce interferon‐gamma. These findings demonstrate that a subset of CD1d‐restricted gamma‐delta T cells responsive to CL exists in the liver of healthy mice, and suggest that these cells could play a role in T cell responses to bacteria and, potentially, to self‐CL.

Supported by CIHR (JR).
Mast cells and interleukin‐6 are required for the optimal accumulation of dendritic cell subpopulations in the lymph node in response to bacterial peptidoglycan
Authors: Dawicki Wojciech , Marshall Jean S.
Published: June 2021

Acquired immune responses are shaped by the nature and activation status of dendritic cell (DC) subpopulations in the lymph node. To better understand the process of DC accumulation in the lymph node during infection we examined the importance of several proinflammatory cytokines and mast cells, which can be a potent source of factors that promote accumulation of DC in the lymph node.

The number of plasmacytoid DC (pDC), CD8+ DC and CD11b+ DC in the lymph nodes that drained the site of intradermal injection of S. aureus peptidoglycan (PGN) or saline was determined at 18h post injection. To examine the importance of tumor necrosis factor (TNF), interleukin (IL)1α/β and IL‐6 in PGN induced DC recruitment, we assayed responses in mice deficient in these mediators. TNF and IL‐1 receptor were dispensable for the accumulation of pDC, CD8+ DC and CD11b+ DC in response to PGN. IL‐6 deficient mice had significantly reduced accumulation of CD11b+ DC. Examination of responses to PGN in mast cell deficient mice and mice locally reconstituted with mast cells revealed that mast cells are essential for the optimal recruitment of CD8+ DC and pDC, but not the IL‐6 dependent CD11b+ DC subset.

These data suggest that PGN activated mast cells can drive the accumulation of DC subpopulations in the lymph node and thus could play a role in modulating the ensuing acquired immune responses. This work is funded by the Canadian Institutes of Health Research.
Nutrition and education intervention in asthmatic Head Start children in the District of Columbia
Authors: Lockwood Susan D , Harland Barbara F
Published: June 2021

The purpose of this intervention is to improve the nutrition and asthma management of low‐income, asthmatic children in the Washington, DC Head Start Program. The goals are to reduce missed school days, hospital emergency room visits, and improve their nutrition. Emergency departments in the District of Columbia experience approximately 7000 pediatric, asthma‐related visits per year. Studies have concluded that the prevalence of overweight is significantly higher in children with moderate to severe asthma. Overweight asthmatic children experience greater asthma symptoms. Asthma severity and parental health beliefs contribute to the lower activity level of these children. Two, three‐hour educational interventions for parents/caregivers relative to asthma management include nutritional and activity components. Measurement of body mass index identifies the degree of overweight, and a 3‐day dietary intake, food allergies, and a record of activity level provide data for the development of an individualized “Asthma Action Plan”. Telephone follow‐up monitors compliance. (Research Support: Research Infrastructure in Minority Institutions Project, 2006–2008)
A comparative study on the nutritional status of the families with working housewives and with non‐working ones
Authors: Lee Joung‐won
Published: June 2021

In order to investigate if the employment of housewives may affect the nutritional status of their family members, an analysis was made for the data of 2001 Korean NHANES. Housewives aged 20 or over were divided into two groups of the employed(43.8%) and the unemployed(56.2%), and household income levels were divided into 4 groups of low, middle, high, and high above according to the minimum cost of living. Nutrients intakes were assessed by using RDA for Koreans revised in 2000. Employed housewives(E) showed similar levels to those of unemployed housewives(UE) in RDA% of energy and most nutrients. However whole family members excluding housewives of E, than those of UE, took less protein, calcium, iron, vitamin A, thiamin, riboflavin, niacin, and vitamin C in RDA% and took more sodium. Such differences were very strong in the age of 7 to 12 and 13 to 19, and also in the middle income households. Red blood cell count were lower and blood HDL‐cholesterol and BMI were higher in the families of E than in those of UE. Interestingly, both systolic and diastolic BP were higher in the family members aged 30 to 49 of E than in those of UE. In conclusion, employment of housewives could have negative influences on the nutritional status of their family members, especially of their school and adolescent children and of the middle income class.
Effect of intermittent fasting on markers of oxidative stress and serum biochemical markers of cellular damage in healthy subjects
Authors: Ibrahim Wissam H. , Habib Hosam M. , Jarrar Amjad H. , Albaz Samer A.
Published: June 2021

During the lunar month of Ramadan Muslims abstain from eating, drinking, and smoking from dawn to sunset. This makes Ramadan a unique model for studying the effects of intermittent fasting in humans. The aim of this study was to determine the effect of Ramadan fasting on markers of oxidative stress and serum biochemical markers of cellular damage in healthy subjects. Fourteen healthy volunteers (9 men and 5 women aged 25 – 58) who fasted during Ramadan participated in the study. Blood sampling was conducted 2 days before Ramadan, the 14th and 28th days of Ramadan. The following were measured: 1) Serum: malondialdehyde, AST, ALT, creatine kinase, ALP, LDH, BUN, total proteins, uric acid, albumin, glucose, triglycerides, and total cholesterol, 2) Plasma: protein‐bound carbonyls, alpha‐tocopherol, gamma‐tocopherol, retinol, vitamin C, and carotenoids, 3) Erythrocytes: MDA, glutathione, glutathione peroxidase and catalase. Erythrocyte malondialdehyde, serum glucose and triglycerides, and plasma total carotenoids were significantly lower (P < 0.05) at the 28th day of Ramadan compared to before Ramadan. The rest of the variables were not significantly altered by Ramadan fasting. The results obtained indicate that Ramadan fasting confers protection against lipid peroxidative damage in erythrocytes as noted by significant decreases in malondialdehyde and does not adversely affect cellular damage.
Increased dietary protein attenuates C‐reactive protein and creatine kinase responses to exercise‐induced energy deficit
Authors: Blakely Shirley R. , Blumberg Jeffrey , O'Neill Karen Rene , Smith Tracey J. , Pikosky Matthew A. , Young Andrew J.
Published: June 2021

We determined if dietary protein (P) modulates responses of C‐reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise‐induced energy deficit (DEF). Thirteen healthy men (22 ± 1 y, VO2peak 60 ± 2 ml·kg−1·min−1) balanced energy expenditure (EE) with energy intake (EI) during baseline (BL) for 4‐d (D1–D4). BL was followed by 7‐d of 1000 kcal·d−1 energy deficit (D5–D11) induced by increased exercise. Throughout D1–D11, kg−1 d−1. Serum CRP and CK were measured on D4, D5, D6 and D11 while resting after an overnight fast, and again post‐exercise. Both groups experienced a time‐dependent interaction for CRP at fasting and post‐exercise. CRP increased for DEF from D4 to D11 (0.18±0.02 to 0.43±0.3 mg/L, respectively). In contrast, although CRP on D4 was higher for DEF‐P vs. DEF due to dietary protein adaptation, levels decreased from D4 to D11 (3.9±2.2 mg/L to 0.26±0.02 mg/L, respectively). CK increased in both groups on D4 to D6 and was higher post‐exercise than at fasting. CK leveled‐off in DEF and decreased in DEF‐P on D11. These results suggest that increased dietary protein attenuates the CRP response and normalizes post‐exercise CK during exercise‐induced energy deficit. Research funded by Medical Research Materiel Command.
Cellular responses to cysteine deprivation are mediated by the amino acid deprivation pathway but not the oxidative stress pathway.
Authors: Sikalidis Angelos K. , Lee Jeong‐In , Hirschberger Lawrence L. , Stipanuk Martha H.
Published: June 2021

Several genes in cysteine (Cys) metabolism are transcriptionally regulated in response to either Cys deprivation or oxidative stress. These include GCLC and GCLM, which encode glutamate:Cys ligase subunits that catalyze the first step of glutathione synthesis, and SLC7A11, which encodes the cystine‐glutamate exchanger responsible for cystine uptake. In Cys‐deprived HepG2/C3A cells, GCLC, GCLM and SLC7A11 were upregulated as assessed by microarray analysis and qRT‐PCR. Expression of many genes with antioxidant response elements was not affected by Cys deprivation while genes with amino acid response elements (AAREs) were consistently upregulated, suggesting that Cys deprivation can induce the eIF2α kinase 4 mediated amino acid deprivation pathway without inducing oxidative stress. Induction of GCLC, GCLM and SLC7A11 was also observed in rats fed 10% (w/w) protein diets, and activation of the AARE‐mediated pathway was evidenced by phosphorylation of eIF2α and upregulation of mRNA and protein levels for genes with AAREs. The pattern of gene expression in growing rats fed diets marginally deficient in sulfur amino acids suggests that the amino acid deprivation response can be activated in response to mild, as well as severe, amino acid deficiencies. SLC7A11 contains an AARE and is likely a direct target, but the details of the regulation of GCLC and GCLM expression in response to Cys deficiency need further study. Funded by NIH Grant PHS DK0664303.
MEK signaling is required for phosphorylation of eIF2a following amino acid limitation of HepG2 human hepatoma cells
Authors: Thiaville Michelle M. , Pan Yuan‐Xiang , Gjymishka Altin , Kilberg Michael S.
Published: June 2021

The mammalian amino acid response (AAR) pathway is up‐regulated by protein or amino acid depletion. This pathway involves detection of uncharged tRNA by the GCN2 kinase, phosphorylation of eIF2α, and enhanced de novo synthesis of the transcription factor ATF4. The present studies demonstrate that inhibition of MEK activation, by treatment of HepG2 human hepatoma cells with PD98059, blocked the increased phosphorylation of eIF2α and ATF4 synthesis triggered by amino acid limitation, showing that AAR signaling requires the MEK‐ERK pathway. Consequently, inhibition of MEK activation blocked transcriptional induction of ATF4 target genes, but the induction was rescued by over‐expression of ATF4 protein. Furthermore, the enhanced ERK phosphorylation following amino acid deprivation required GCN2 kinase activity, but not eIF2α phosphorylation. Inhibition of protein phosphatase 1 action on phospho‐eIF2α, by siRNA knockdown of GADD34, did not block the sensitivity to PD98059 suggesting that the MEK‐ERK pathway functions to enhance GCN2‐dependent eIF2α phosphorylation, rather than to suppress dephosphorylation. These results document a critical interdependence between the MEK‐ERK MAPK signaling pathway and the AAR pathway. Funded by grants from the NIH (DK‐52064, DK‐70647).
Iron supplementation modulates genes involved in oxidoreductase activity, including prostaglandin‐endoperoxide synthase 2 in intestinal tumors
Authors: Swain James , Li Chunbiao , Leahy Patrick
Published: June 2021

To determine the effect of excess dietary iron on gene expression of intestinal tumors in an animal model of human familial adenomatous polyposis, male C57BL/6J ApcMin/+ mice were fed adequate and excess amounts of iron (ferrous sulfate: 45 vs. 450 mg iron/kg AIN‐93M[M] diet). After 10 wk, mice were sacrificed and intestines removed. Tumors and adjacent non‐tumor tissue were excised and RNA isolated for micro‐array analysis (n=3/group; Mouse Genome 430A 2.0 arrays; Affymetrix) and RT‐PCR. Pathway mapping was performed to reveal gene networks (Pathway Studio 4.0, Ariadne Genomics, Inc.; GeneSpring, Agilent, Inc.; Searcher, Lucidyx). Bayesian ANOVA was used for micro‐arrays. Data show that intestinal tumors alter expression of a group of genes involved in oxidoreductase activity when exposed to excess iron. Differentially expressed genes include prostaglandin‐endoperoxide synthase 2, retinol saturase, alcohol dehydrogenase, deiodinase, cytochrome P450 (family 2, 4), and glutathione peroxidase. Findings reveal that iron supplementation modulates oxidoreductase activity of intestinal tumors, possibly in response to the pro‐oxidative, pro‐inflammatory effects of excess intraluminal iron. Supported in part by grants IRG‐91‐022 from ACS (to JS) and P30CA43703 from NIH. Ferrous sulfate provided by Crown Technologies, Inc.
DMT1 is not involved in uptake of copper from the blood plasma by hepatic and mammary epithelial cells
Authors: Moriya Mizue , Ho Yi‐Hsuan , Sauble Eric , Ackland Leigh , Mercer Julian , Linder Maria C.
Published: June 2021

Our long term goal is to understand exactly how Cu is taken up by cells from its blood plasma carriers and how this is regulated in lactation. To investigate the possibility that DMT1 might at least partly be involved, we compared the organ distribution of tracer 64Cu(II) 1h after injection i.p. into non‐lactating and lactating Belgrade rats (with defective DMT1) and their wild type (Fisher) counterparts. No differences were observed. Both kinds of nonlactating rats had ~70% of the administered 64Cu in the liver and kidney. Two‐5 days post‐partum this was reduced to ~45% in both kinds of rats, and the lactating mammary gland took up ~25%. The proportion of radioactivity appearing in the milk (relative to blood plasma) was also unaltered. Confirmatory studies carried out in cultured cell models for hepatocytes and mammary epithelial cells (HepG2 and PMC42, respectively) determined that 1–10 uM Cu uptake from its plasma protein carriers (purified human albumin and alpha‐2‐macroglobulin) was not inhibited by Fe(II)‐ascorbate or Fe(II)‐nitrolotriacetate, except at very high concentrations. HepG2 cell pretreatment with 100 uM Fe for 24h increased Fe uptake but not Cu uptake, and did not alter expression of DMT1 mRNA. We conclude that, although DMT1 may play a role in uptake of ionic Cu by the intestinal epithelium, it plays no such role for cells in internal organs that absorb it from the blood. Supported by PHS Grant No. RO1 HD 46949.
Functional analysis of mammalian selenocysteine synthase
Authors: Xu Xue‐Ming , Carlson Bradley A , Zhang Yan , Berry Marla J. , Gladyshev Vadim N , Hatfield Dolph L
Published: June 2021

Selenocysteine (Sec) is co‐translationally inserted into protein in response to UGA codons as the 21st amino acid in the genetic code. The biosynthetic pathway for Sec has been established in the three domains of life, prokaryotes, archaea and eukaryotes. Mammalian Sec synthase (SecS) uses O‐phosphoseryl‐tRNA as a substrate and accepts monoselenophosphate as the active selenium donor to yield Sec‐tRNA. We have found, in addition, that the Sec biosynthetic factors, SECp43 and SPS1, bind to SecS in vivo and that SECp43 binds to SecS in vitro as well. These bindings did not affect the activity of SecS in vitro. The in vitro reactions which showed that these factors are not essential for the activity of SecS indicate that the in vivo binding of SECp43 and SPS1 to SecS is likely involved in other functions.
Zinc status in male collegiate cross‐country skiers and its relationship to athletic performance
Authors: Walker Sarah , Bobilya Dennis
Published: June 2021

The relationship between dietary zinc, zinc status, and athletic performance was investigated in 11 men on the University of New Hampshire cross‐country ski team through one training year. Subjects completed 3‐day diet records, anthropometric measurements, and blood draws at the end of each of the 4 training phases: Volume phase, May – Aug.; Intensity phase, Sept. – Nov.; Competition phase, Dec. – March; and Recovery phase, March – April. Performance tests were performed after the Volume, Intensity, and Recovery phases. Plasma zinc was within the normal range for all athletes at all time periods, with a mean of 14.4 μmol/L and a range of 11.1 to 17.3 μmol/L. Plasma zinc was positively correlated (r = 0.221) with dietary zinc. Plasma zinc was lower (P < 0.05) after the recovery phase than after each of the other phases, perhaps because the contribution of zinc from breakfast cereals in the recovery phase was half what it was during the other three phases. Plasma zinc was positively correlated (r = 0.463, p = 0.020) with the max number of pull ups in 1 min (a test of endurance) and negatively correlated (r = −0.410, p ≤ 0.05) with max bench press (a test of power). Though plasma zinc was in the normal range for all athletes, their performance in exercises requiring competitive motions (muscle endurance) was positively correlated with their zinc status, while their performance in exercises requiring maximal exertion (muscle power) was not.
Almonds (Amygdalus communis L.) as a possible source of prebiotic functional food
Authors: Mandalari Giuseppina , Tomaino Antonio , Faulks Richard M. , Arcoraci Teresita , Bisignano Giuseppe , Saija Antonella , Wickham Martin S. , Narbad Arjan
Published: June 2021

A prebiotic is “a nondigestible food ingredient which beneficially affects the host by selectively stimulating the growth of one or a limited number of species of bacteria in the colon thereby improving host health”. Here we describe the potential prebiotic properties of almond seeds and skins. Finely (FG) and defatted (DG) ground almonds, raw (RS) and blanched (BS) almond skins were initially digested in a simulated model of human gastric and duodenal digestion. Post‐duodenal digests were tested for potential prebiotic properties using in vitro batch fermentation systems seeded with human colonic bacteria.

A significant increase in bifidobacteria and eubacteria was observed after 24h incubation with FG, whereas no significant change in the bacterial population occurred with addition of DG. Both RS and BS produced an increase in bacteria beneficial to the human colon. A prebiotic index (PI), giving a relative indication of the prebiotic potential, was calculated to be 5.0 with FG and compares well with the value of 6.4 obtained with fructo‐oligosaccharide (FOS), an established prebiotic carbohydrate. The effect on the production of short chain fatty acids, indicators of beneficial bacterial growth, was also determined. These results indicate the presence of bioactive components within almonds that stimulate growth of beneficial intestinal bacteria.

This work was funded by the Almond Board of California.