bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations of the β‐subunit of human NAD‐specific isocitrate dehydrogenase in patients with Retinitis Pigmentosa
Authors: Dange Mayura , Hartong Dyonne T. , McGee Terri L. , Berson Eliot L , Dryja Thaddeus P. , Colman Roberta F.
Published: June 2021

Mitochondrial NAD‐ and NADP‐isocitrate dehydrogenases (IDH) are crucial in the Krebs cycle. NAD‐IDH is composed of 3 subunits in the ratio 2α:1β:1γ and is allosterically regulated by ADP, by lowering the Km‐isocitrate. Two individuals with autosomal recessive Retinitis Pigmentosa (RP), but no other major symptoms, are homozygous for mutations in the β‐subunit of NAD‐IDH: the missense βL98P, and a 1‐basepair deletion in codon βI163 (ATT to –TT) resulting in a frameshift and premature termination of the β‐subunit. The activities of NAD‐ and NADP‐IDH were evaluated in extracts of lymphoblasts from unaffected individuals, heterozygote carriers of the mutations, and two RP patients. The NADP‐IDH activity was normal in all samples. In contrast, both homozygotes exhibited a Km‐NAD 260–300 times normal, while the Km‐NAD of the heterozygote samples was 4‐8 times that of normal. When ADP is added, Km‐isocitrate decreases only 3‐fold in the RP patient with the I163delA mutation, unlike normals in which ADP decreases Km‐isocitrate 9‐fold. This allosteric effect is completely lost in the homozygote βL98P mutant. The results show that these beta mutations primarily affect the enzyme's NAD affinity and ADP response. NADP‐IDH is the dominant mitochondrial IDH expressed in most tissues, while NAD‐IDH predominates in the eye. This differential expression may explain why the phenotype of NAD‐IDH mutations is confined to the eye.
Polyunsaturated Fatty Acids and Oxidative Stress in a Model of Hepatic Steatosis, the (fa/fa) Zucker Rat, In Vivo Effect of Chlorogenic Acid
Authors: de Sotillo Delcy Rodriguez , Hadley Mary
Published: June 2021

Altererations in lipid metabolism gives rise to hepatic steatosis, due to increased lipogenesis affecting polyunsaturated fatty acids (PUFA). Excessive formation of reactive oxygen species is associated with the onset and progression of steatosis. Previously, we reported that chlorogenic acid (CGA), a phenolic antioxidant, decreased plasma and liver triacylglycerols in Zucker (fa/fa) rats, a model of insulin resistance and steatosis. This study aimed to investigate the in vivo effect of CGA on lipid peroxidation in Zucker (fa/fa) rats. Rats were implanted with jugular vein catheters and CGA was infused (5 mg/Kg body weight/day) for 3 weeks. Analysis were carried out when rats reached 22 weeks old of age. Malondialdehyde (MDA), a biomarker of oxidative stress, was determined in plasma, urine, liver, and kidney. Fatty acid methyl esters were analyzed in liver and adipose tissues. Chlorogenic acid treatment decreased MDA in plasma by 8%; in urinary by 35%, and in liver by 18.4 % compared to control rats. No differences in MDA concentration due to CGA treatment were found in the kidney (p>0.05). Chlorogenic acid decreased the concentration of plasma non‐esterified fatty acids and in the liver increased cocentrations of 18:2 (n‐6), 18:3 (n‐3), and 20:4 (n‐6) were found when compared to control (p<0.05). Chlorogenic acid decreased lipid peroxidation and the ratio of n‐6:n‐3 fatty acids in an animal model of hepatic steatosis.
The uptake and metabolism of lyso‐phospholipids by Saccharomyces cerevisiae
Authors: Riekhof Wayne Russell , Wu James , Jones Jennifer L , Gijon Miguel A , Zarini Simona , Murphy Robert C , Voelker Dennis R
Published: June 2021

We have characterized the uptake and metabolism of lyso‐PtdEtn and lyso‐PtdCho by yeast. The reactions involved in the utilization of these compounds are collectively termed the exogenous lysolipid metabolism (ELM) pathway, and mutants defective in both the uptake and acylation steps of the ELM pathway have been isolated. The P‐type ATPases Dnf1p and Dnf2p, in consort with their β‐subunit Lem3p, catalyze the transport reaction. An enzyme designated Ale1p (acyltransferase for lyso‐PtdEtn), a member of the membrane bound O‐acyltransferase (MBOAT) family, serves as the acyltransferase component of this pathway for both lyso‐PtdEtn and lyso‐PtdCho. Ale1p is the major lyso‐phospholipid acyltransferase in yeast, and shows a preference for mono‐unsaturated acyl‐CoA species, and a broad lyso‐lipid substrate specificity that includes lyso‐PtdOH, lyso‐PtdGro, lyso‐PtdIns, and lyso‐PtdSer. The ale1Δ mutant is devoid of lyso‐PtdEtn and lyso‐PtdCho acyltransferase activities, making this strain an ideal host for expression and analysis of putative acyltransferases from other species. We now report the expression and enzymatic characterization of previously uncharacterized human MBOAT family members in an ale1Δ mutant yeast strain. Supported by NIH grants GM32453 (to D.R.V), GM076798 (to W.R.R.), and HL25785 (to R.C.M.), and by American Cancer Society grant PF‐06‐288‐01‐CSM (to W.R.R.)
RGS4 attenuates parasympathetic signaling in the sinoatrial node: implications for heart rate regulation
Authors: Cifelli Carlo , Zhang Hangjun , Rose Robert Alan , Backx Peter , Heximer Scott
Published: June 2021

Parasympathetic signaling in the SAN is mediated by ACh‐dependent stimulation of M2 muscarinic receptors that are coupled to the heterotrimeric G‐protein subunits GiÎ± and GoÎ±ï€ in the SA node. RGS proteins are potent inhibitors of G‐protein signaling. The RGS family members that regulate parasympathetic signaling in pacemaker cells are unknown. We here provide evidence that RGS4 is required for normal inhibition of parasympathetic control of sinus rhythm. Real time RT‐PCR shows that RGS4 is expressed at higher levels in the SAN compared to right atrium. Moreover, the LacZ staining pattern of hearts and right atria from Rgs4tm1Dgen/J knockout/reporter mice indicates robust and selective expression of RGS4 in the SAN. RGS4‐deficient animals showed significantly lower basal heart rates than wild type controls and greater heart rate increases following systemic atropine administration. Lastly, retrograde perfused hearts from RGS4‐deficient mice showed prolonged recovery kinetics following carbachol washout. Taken together these data indicate greater parasympathetic control of heart rate in the knockout strain, and suggest an important role for RGS4 in the normal inhibition of parasympathetic signaling and regulation of sinus rhythm in the mouse heart.
PhLP1 acts as a general mediator of Gβγ and Gβ5 RGS protein dimer assembly
Authors: Howlett Alyson Cerny , Gray Amy J. , Willardson Barry M.
Published: June 2021

Phosducin‐like protein 1 (PhLP1) has been recently shown to be an essential chaperone in the folding and assembly of nascent G protein βγ subunit dimer (Gβγ). However, the role of PhLP1 has only been tested for the most common Gβγ combination Gβ1γ2, yet there are 5 Gβ subunits and 12 Gγ subunits that form dimers in various combinations. In addition, Gβ5 forms important dimers with the R7 family of regulators of G protein signaling (RGS). The current study was designed to answer three questions with regard to the generality of the chaperone role of PhLP1: 1) Does PhLP1 catalyze the assembly of all Gβγ combinations or just a subset? 2) Does PhLP1 influence the specificity of Gβγ dimer formation? and 3) Does PhLP1 contribute to the assembly of Gβ5 RGS complexes? Our data show that PhLP1 is necessary for the assembly of all five Gβ subunits with Gγ2 and of all Gγ subunits that form dimers with Gβ2. Moreover, PhLP1 did not change the preference of Gβ2 for certain Gγs. Finally, PhLP1 was required for Gβ5‐RGS9 assembly, but less so for Gβ5‐RGS7 assembly. These results show that PhLP1 is a general chaperone for dimer formation of all Gβγ combinations and for at least a subset of Gβ5‐RGS complexes.
Control of metamorphic timing by unliganded thyroid hormone receptor through the recruitment of histone deacetylase‐containing corepressor complexes during frog development
Authors: Shi Yun‐Bo
Published: June 2021

Thyroid hormone (T3) is important for vertebrate development and adult organ function. We have been using amphibian metamorphosis as a model for studying the role of T3 receptor (TR) in vertebrate development. We previously proposed a dual function model for TR during development. That is, during premetamorphosis, unliganded TR recruits corepressor complexes to repress gene expression and prevent premature metamorphic transformation, while during metamorphosis, T3‐bound TR recruits coactivators to activate transcription and initiate metamorphosis. Here we have investigated the role of unliganded TR during frog development. We had previously shown that unliganded TR recruits histone deacetylase complexes containing TR‐binding corepressors N‐CoR/SMRT. Thus, we designed a dominant negative form of N‐CoR (dnN‐CoR) containing only the TR‐interacting domain of N‐CoR. We overexpressed dnN‐CoR under the control of a heat shock inducible promoter in tadpoles through transgenesis. RT‐PCR revealed significant derepression of T3‐response genes in transgenic animals. More importantly, transgenic tadpoles developed faster than wild type siblings, with an acceleration of as much as 7 days out of the 30‐day experiment. These data thus demonstrate a role of unliganded TR‐induced gene repression in controlling metamorphic timing by recruiting histone deacetylase‐containing corepressor complexes.
Insulin Induces Intracellular Calcium Transients through Activation of NADPH Oxidase in Myotubes
Authors: Espinosa Berta Alejandra , Hidalgo Cecilia , Jaimovich Enrique
Published: June 2021

In cultured myotubes, insulin triggers intracellular calcium increases measured as fluo‐3 fluorescence. We occasionally see a fast calcium transient (first signal), but around 80s later, myotubes produce a second, oscillatory calcium signal. It is known that insulin signaling involves reactive oxygen species (ROS) production in many cell types. The aim of this work is to study the role of ROS, via NADPH oxidase activation in the mechanism that generates the second intracellular calcium increase. The second calcium signal was inhibited by two drugs (xestospongin B and U73122) that interfere with IP3 pathway. Calcium increase was also blocked by bisindolylmaleimide (BIM), and rottlerin, both inhibitors of protein kinase C. Using the probe 2′, 7′‐dichlorofluorescein diacetate (DCFH‐DA) we showed that insulin induced ROS production in myotubes. Both calcium increase and ROS production were abolished by diphenyl iodonium (DPI), NADPH oxidase inhibitor apocynin and siRNA against p47phox; a regulatory subunits of NADPH oxidase. These results point to a role of insulin‐induced ROS in triggering calcium increase via IP3 receptor. NADPH oxidase is probably activated by a PKC and then ROS modulate IP3 receptors to increase intracellular calcium.

FONDAP 15010006
How arrestin assembles MAP kinases into a signaling complex
Authors: Song Xiufeng , Gurevich Vsevolod V
Published: June 2021

Arrestins interact with GPCRs and numerous non‐receptor partners. We found that all four members of the arrestin family bind protein kinases JNK3a2 and ASK1, but only arrestin3 facilitates JNK3 phosphorylation. Mutant arrestin which does not bind receptors induced even greater JNK3 activation, demonstrating that this is a function of free arrestin. In contrast to arrestin2, arrestin3 has a native nuclear export signal (NES) and is exclusively cytoplamic. We explored the role of subcellular localization and found that arrestin3 with disabled NES activates JNK3 less efficiently. However, even engineered NES+ arrestin2 does not activate JNK3 as strongly as arrestin3. Truncated JNK3 lacking both N‐ and C‐termini does not bind arrestin3. The deletion of only N‐ or C‐terminus of JNK3 reduced binding to arrestin3 compared to full‐length, suggesting that both termini mediate this interaction. To identify arrestin3 elements involved in JNK3 and ASK1 binding, we used nuclear exclusion assay and coimmunoprecipitation. Separately expressed N‐ and C‐domains of arrestin3 interact with both JNK3 and ASK1, demonstrating that each kinase has two binding sites on arrestin. These data suggest a novel model of arrestin‐dependent scaffolding of MAP kinase cascades: arrestin3 assembles the components with both MAPKKK and MAPK positioned along the long axis of the arrestin molecule.

NIH grants GM 77561, EY11500 (VVG)
Isothermal Titration Calorimetric Study of Amyloid b (Ab) Peptide Interaction with Cu2+ and Zn2+
Authors: Jin Lihua , Kaatz Laura , Zbyszynski Pawel , Behbahani Siavash
Published: June 2021

Alzheimer's disease is characterized by extracellular plaques that cause neurodegeneration. The major constituent of the plaque is the fibrillar form of the amyloid β (Aβ) peptides of 39–43 amino acids long. Physiological levels of copper and zinc ions have been shown to cause marked aggregation of Aβ peptides. Spectroscopic studies have identified metal ion coordination sites on Aβ peptides, binding affinity and stoichiometry, although discrepancies exist among various studies. To complement spectroscopic approaches and to obtain binding energetics, we have used isothermal titration calorimetry to examine the interaction of copper and zinc ions with Aβ‐(1–16), the N‐terminal portion of the longer Aβ peptide that is known to contain all the coordination sites for the two ions. Titration orders of metal ion into peptide and peptide into metal ion were carried out at pH 5.5 and 6.5 at several temperatures. Two stoichiometries were obtained for each ion depending on the injection order: two metal ions bound per peptide when the peptide was titrated into metal ion and one metal ion bound per peptide when metal ion was titrated into peptide. Analysis of the result suggests that two different binding modes were employed by each ion depending on the injection order. Copper binds more strongly than zinc. For both ions, binding was enthalpy driven; affinity increases with increasing pH, suggesting deprotonation coupled to binding, a result consistent with histidine participates in metal coordination. It was also found that prolonged incubation of the peptide with copper or zinc induces an additional slow process that is best explained by a conformational change, possibly, aggregation of Aβ‐(1–16) which in turn increased the affinity of the peptide for the two metal ions.
Transfection of Vascular Smooth Muscle Cells with Novel Biodegradable Arginine Based Poly(ester‐amide)s
Authors: Yamanouchi Dai , Wu Jun , Lazar Andrew , Kent K. Craig , Chu Chih‐Chang , Liu Bo
Published: June 2021

A novel family of synthetic biodegradable poly(ester amide)s (Arg‐PEAs) were synthesized using arginine (Arg), diols and diacids as building blocks. The biosafety and functional properties of these polymers were tested in rat vascular smooth muscle cells, a major cell type participating in vascular diseases. Arg‐PEAs showed high binding capacity for plasmid DNA with a trend that was reversibly related to the number of methylene groups in the diol segment. All Arg‐PEAs transfected smooth muscle cells with an efficiency that was comparable to the commercial transfection reagent Superfect® according to the results from luciferase and GFP plasmid transfection. But unlike Superfect®, Arg‐PEAs, over a large range of doses, had minimum effects on cell morphology, viability or apoptosis in MTT and cell death detection assay. Using rhodamine labeled plasmid DNA, we demonstrated that Arg‐PEAs were able to deliver DNA, through an active endocytosis mechanism, into close to hundred percent of cells under optimal polymer‐to‐DNA weight ratios. However, a large portion of delivered DNA was trapped outside of the nucleus, in acidic endocytotic compartments, and subsequently was not expressed. These results suggest that Arg‐PEAs, with further modifications to enhance endosome escape, are good candidates for non‐viral gene carrier systems owning to their high cellular uptake nature and reliable biocompatibilities.
Bivalent Mannose 6‐Phosphate‐Based Ligands for the Mannose 6‐Phosphate/Insulin‐like Growth Factor II Receptor as Potential Anti‐Tumor Agents
Authors: Connelly Christopher M. , Grosely Rosslyn , MacDonald Richard G.
Published: June 2021

The mannose‐6‐phosphate/insulin‐like growth factor II receptor (M6P/IGF2R) binds both IGF‐II and M6P‐based ligands. Bivalent M6P ligands stabilize the receptor's dimeric structure and thereby accelerate IGF‐II internalization and degradation. This key observation motivates our goal to design bivalent M6P‐based ligands as novel anti‐cancer agents to suppress growth of IGF‐II dependent cancers. To test this concept, a panel of synthetic ligands consisting of pentamannosyl 6‐phosphate (PMP) chemically linked to proteins and peptides of decreasing molecular size was prepared and evaluated for binding to the M6P/IGF2R. Ligand binding was measured by displacement assays using a series of ligand concentrations with 125I‐PMP‐BSA as tracer and wild‐type or mutant M6P/IGF2R coupled to a resin source. The IC50 values for the glycoprotein ligands were in the 13–21 nM range, whereas tripeptide ligands bearing two or more PMP groups had IC50 values of 50–100 nM. These data indicate that both types of synthetic ligands bound with high affinity, most likely by bivalent cross‐bridging of the dimeric receptor, in contrast to M6P, a monovalent, low‐affinity ligand with an IC50 of 72 μM. In conclusion, we have prepared several novel pseudoglycoprotein and peptide ligands that exhibit high‐affinity, apparently bivalent binding to M6P/IGF2R. These ligands will now be tested for the ability to enhance IGF‐II internalization.
The Synthesis of Polypeptide Analogues used as Molecular Transporters
Authors: Roper Latoya A. , Olson David , Danowski Barbara , Kehlbeck Joanne D.
Published: June 2021

Molecular transporters are compounds noted for their unique ability to traverse the cell membrane while transporting cargo into the cell. One such macromolecule studied is the naturally occurring HIV‐1 transactivator of transcription (TAT). A nine amino acid sequence rich in arginine residues is responsible for cellular uptake (R49KKRRQRRR57). According to Rothbard and colleagues, the polarizability and potential for bidentate hydrogen bonding of arginine with cell surface proteoglycans like heparin sulfate allows for effective cell penetration.

To test this proposal will we have synthesized polypeptide analogues called polypeptoids. Peptoids are constitutional isomers of peptides and differ in the connectivity of the side chain. While the side chain of the amino acid residue in a peptide is attached to the alpha carbon, the side chain of a peptoid is attached to the nitrogen. Polypeptoids serve as excellent mimics of polypetides and are stable to proteolysis. In particular the synthesis of a novel peptoid compound containing guanine group in the side chain will be described. The side chains of this molecule have increased polarizability and the potential to participate in bidentate hydrogen bonding. The cellular uptake properties of this compound will be compared to that of polypeptoids with arginine‐like side chains in live cell assays.

Funding for research provided by Union College Chemistry Department and Union College Future Professors Program.
Expression and Function of the Non‐phagocytic NADPH Oxidase, Duox1 in T Lymphocytes
Authors: Williams Mark S , Kwon Jaeyul , Burdette Kristen , Miot Franciose
Published: June 2021

The family of NADPH oxidase enzymes serve to generate reactive oxygen species (ROS), and the prototypic phagocyte‐type NADPH oxidase produces large amounts of ROS that serve a host defense role. Fibroblasts, B lymphocytes and platelets also express NOX2/gp91phox protein which leads to ROS production. Recent discovery of multiple homologues of gp91phox (e.g., NOX1, NOX4, Duox1/2) has broadened the NADPH oxidase activity in “non‐phagocytic” cells. In non‐phagocytic cells and tissues, NOX family members produce lower levels of ROS and participate in regulatory and signaling roles. We have recently shown that T cells express the phagocyte‐type NADPH oxidase which regulates T cell responses. Our new data indicate that T cells also express mRNA and protein for the calcium‐dependent, non‐phagocytic oxidase Duox1 and Duox1 produces ROS early in TCR signaling. Downregulation of Duox1 expression in primary and cultured T cells with siRNA resulted in selective changes in T cell receptor induced signal transduction. While phosphorylation of early mediators of TCR signaling (ZAP‐70, PLC‐g) was largely unaffected, TCR‐induced activation of ERK was strongly inhibited and secretion of multiple cytokines including IL‐4, IFN‐g, and TNFa was also reduced. Thus, T cells express multiple NADPH oxidases and Duox1 serves to regulate early TCR signal transduction. Supported by NIH R01 AI070823.
Increased TLR Responses in Dendritic Cells Lacking the Itam‐Containing Adapters Dap12 and FcRγ
Authors: Hamerman Jessica A , Yu Yen‐Ling , Shen Kuan‐Yin , Lowell Clifford A , Lanier Lewis L , Chu Ching‐Liang
Published: June 2021

The inhibitory effect of DAP12 on macrophages has been revealed by examining myeloid cells from DAP12‐deficient mice. In this report, we demonstrate that both DAP12 and the FcεRγ ‐chain (FcRγ) are required for negative regulation of TLR responses in bone marrow‐derived dendritic cells (DCs). Loss of both DAP12 and FcRγ enhanced the pro‐inflammatory cytokine production and maturation of DCs after TLR stimulation, resulting in a greater percentage of DCs that produced IL‐12 p40, TNF, and IL‐6, and expressed high levels of MHC class II, CD80, and CD86. Whereas DCs lacking only DAP12 showed some increased TLR responses, those lacking only FcRγ had a greater enhancement of maturation and cytokine production, though to a lesser extent than DCs lacking both DAP12 and FcRγ Additionally, antigen‐specific T cell activation was enhanced by DAP12−/−FcRγ −/− DCs relative to wild‐type (WT) DCs after maturation. Similar to DAP12−/−FcRγ −/− DCs, Syk‐deficient DCs also had increased inflammatory cytokine production, maturation, and antigen presentation. These results confirm the inhibitory effect of ITAM signaling in myeloid cells and show that DCs and macrophages differ in their dependence on the ITAM‐containing adapters DAP12 and FcRγ for negative regulation of TLR signaling.
Role of Human Skin Resident T Cells in Wound Healing
Authors: Toulon Antoine , Jameson Julie , Bhavsar Dhaval , Tenenhaus Mayer , Rudolph Ross , Gueniche Audrey , Breton Lionel , Havran Wendy
Published: June 2021

Cutaneous wound healing is a complex process requiring constant communication between cells in the form of cytokine release, cell‐to‐cell contacts and cell‐to‐matrix interactions. Chronic wounds are a serious clinical problem affecting numbers of elderly and diabetic patients. Skin γδ T cells have been shown to play unique roles in tissue homeostasis and during tissue repair factors in mice. Since the distribution of epidermal resident T cells (γδ vs αβ) is different in mice and humans, we aimed to characterize the role of epithelial resident T cells in wound repair in humans. We investigated the ability of T cells isolated from normal epidermis, acute wounded skin and chronic wounds to secrete Insulin‐like Growth Factor‐1 (IGF‐1), a growth factor that has been shown to be critical for wound healing. We found that epidermal resident T cells freshly isolated from normal skin produce IGF‐1 upon in vitro stimulation. Interestingly both αβ and γδ T cells were activated in acute wounds and secreted IGF‐ at an early stage of the wound healing process. In contrast, in chronic wounds neither T cell subset produced IGF‐1 or was able to respond to in vitro stimulation, supporting a crucial role for human skin T cells in tissue repair. Further characterization of the functional activity of resident T lymphocytes in acute wounds should allow the development of new therapeutic strategies to treat chronic wounds.

Funded by L’Oreal and NIH AI36964
DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B cells and Idiotype Positive Marginal Zone B cells Before and After Activation
Authors: Kin Nicholas Wayne , Crawford Dianna M. , Liu Jiabin , Kearney John F.
Published: June 2021

Marginal Zone (MZ) B cells play an important role in the clearance of blood borne bacterial infections via rapid T‐independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ‐specific genes that are regulated to allow for this rapid response, resting MZ and Follicular (FO) B cells were sort‐purified and analyzed via DNA microarray. We identified 181 genes that were significantly different between the two cell populations. 92 genes were more highly expressed in MZ B cells while 81 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, idiotype positive and negative MZ B cells were sort‐purified before (0 hour) or after (1 hour) i.v. immunization with R36A, heat killed Streptococcus pneumoniae, and analyzed via DNA microarray. We identified 189 genes specifically up‐regulated (> 5‐fold) and 192 genes specifically down‐regulated (> 10‐fold) by 1 hour after immunization in the idiotype positive MZ B cells. These data provide unique insight into the gene expression pattern in resting MZ vs. FO B cells and idiotype positive MZ B cells following activation, providing new candidate genes and pathways to explore.
Regulation of the Germinal Center Response by T Regulatory Cells
Authors: Alexander Carla‐Maria , Waldschmidt Thomas
Published: June 2021

Previous work from our laboratory demonstrated the splenic germinal center (GC) reaction to display traits of a steady state, especially in regards to the ratio of IgM+ to switched GC B cells. The GC is a site of intense proliferation, class switching, apoptosis and selection, and as such must have strict regulation. Studies in our laboratory have shown that T regulatory cells (T‐regs) play a pivotal role in regulation of the GC, as steady state dynamics were disrupted when T‐regs were depleted or dysregulated by anti‐CD25 and anti‐GITR, respectively. Current studies are exploring the mechanism by which T‐regs exert their control within the GC. Based on previous reports studying the means by which T‐regs suppress their targets, we are examining whether Granzyme B, Perforin, Fas or IL‐10 affect GC dymanics. This is being accomplished using either Ab neutralization or genetically targeted mice. Of the various molecules tested to date, results have suggested Fas‐Fas ligand to play a role in maintaining steady state conditions within the GC.
The role of glucose energy metabolism in B lymphocyte survival responses
Authors: Dufort Fay J. , Blair Derek , Bleiman Blair , Gumina Maria R. , Chiles Thomas C.
Published: June 2021

Naïve B lymphocytes must acquire and metabolize external glucose in order to support the bioenergetics associated with maintaining cell volume, ion gradients, and basal macromolecular synthesis. Interleukin‐4 (IL‐4) is a pro‐survival cytokine that prevents death of naïve B lymphocytes, in part, through the up‐regulation of anti‐apoptotic proteins, such as Bcl‐xL. We show that IL‐4 stimulates glucose utilization in the form of glucose uptake and glycolytic catabolism in ex vivo splenic B cells; this is accompanied by increased cellular ATP. IL‐4‐induced glycolysis occurs normally in B cells deficient in insulin receptor substrate‐2/phosphatidylinositol 3‐kinase signaling. By contrast, Stat6‐deficient B cells exhibit impaired IL‐4‐induced Glut1 expression and glycolysis. In keeping with this, inhibition of B‐cell antigen receptor signaling following co‐cross‐linking of FcgammaRIIb results in decreased glucose uptake and glycolytic metabolism and lower cellular ATP. Moreover, mitochondrial homeostasis is significantly impaired as evidenced by decreased oxygen consumption and FcgammaRIIb‐induced mitochondrial membrane depolarization. These results suggest that glycolysis is necessary to maintain cell survival in either naïve B lymphocytes or in response to negative signaling by the FcgammaRIIb.

This work was supported by USPHS grant AI60896 and AI49994.
Listeriolysin O (LLO) as a Vaccine vehicle
Authors: Vivanco Hector , Unanue Emil R.
Published: June 2021

Specific Aim: In this project we aimed to test antigen presentation to Listeriolysin O (LLO), the major virulence factor of Listeria monocytogenes (LM). The purpose was to examine the immunogenicity of LLO and its possible use as a carrier of antigens for immunization purposes.

Results

Peptides were made to segments of LLO molecule known to induce CD4 responses. C57Bl/6 mice were immunized with the peptides and their CD4 T cells were isolated, made into hybridomas and used to examine the presentation of LLO ex vivo. The CD4 T cell response was extraordinarily potent when LLO was used as an exogenous antigen testing different Antigen presenting cells (APC). The response to LLO was about 10,000 times more efficient than that for peptide. Looking for a safer molecule without the detrimental effects at higher concentrations, we generated a LLO mutant, the LLOW492A, which has a 90% reduced lytic activity but still maintains the binding capacity to the cell membranes. When we tested this LLO mutant ex vivo the magnitude of the response at low doses of antigen was as strong as that for the wild type LLO. We generated a recombinant LLO protein by attaching a CD4 epitope to the NH2 terminus region. We used a known epitope (45–65 epitope) of the hen egg white lysozyme (HEL) molecule. When we tested the response of a known T cell to 45–65 epitope of HEL linked to the amino terminus region of LLO, the response was about 100 fold stronger than to the peptide alone.

Conclusions: We have identified an extraordinarily potent LLO‐specific CD4 T cell response when the whole molecule or a mutant derivative is used as an exogenous antigen. Peptides linked to the protein are likewise highly immunogenic. LLO is a potentially powerful vehicle for immunization.
Cholesterol regulates the loading of foreign antigens onto MHC class II in dendritic cells
Authors: Berger Adam Craig , Roche Paul A.
Published: June 2021

In immature DCs, MHCII resides in cholesterol‐rich intracellular lysosomal compartments. Upon activation, intracellular MHCII binds antigenic peptides and traffics to the plasma membrane. In this study, we have investigated the role of membrane cholesterol in MHCII transport through the use of the cholesterol transport inhibitor U18666A. Pretreatment of immature DCs with U18666A had no effect on the LPS‐induced expression of either the DC maturation marker CD40 or cellular levels of total MHCII and only a slight effect on CD86. However, U18666A treated DCs had a reduced capacity to stimulate naïve antigen‐specific T cells. Similar results were found using DCs from mice lacking NPC1, a protein involved in lysosomal cholesterol egress. Pretreatment of DCs with U18666A resulted in a 4‐fold reduction in the surface and cellular expression of MHCII‐peptide complexes in immature DCs and almost completely inhibited the LPS‐induced appearance of MHCII‐peptide at the plasma membrane of mature DCs. Microscopic analysis of U18666A treated DCs shows that MHCII‐peptide complexes are not found in cholesterol engorged lysosomal antigen processing compartments, suggesting that there are fewer compartments that are competent for antigen loading. We conclude that lysosomal cholesterol regulates MHCII‐peptide loading but not MHCII transport from lysosomes to the plasma membrane in DCs.

This work is supported by the NCI.
Maturation and Activation of Antigen Presenting Cells in Young and Aged Mice by a Novel Adjuvant Derived From Human Anaphylatoxin C5a
Authors: Morgan Brandon N , Morgan Edward L , Sanderson Sam , Phillips Joy A , Thoman Marilyn L
Published: June 2021

Maturation and Activation of Antigen Presenting Cells in Young and Aged Mice by a Novel Adjuvant Derived From Human Anaphylatoxin C5a

Human anaphylatoxin C5a is known to enhance humoral and T cell mediated immunity and inflammation. We have developed an analog peptide based on the C‐terminal region of human C5a [EP67: YSFKDMP(MeL)aR which preferentially enhances immune function. We hypothesize that enhanced immune function occurs via the recruitment and activation of antigen presenting cells. EP67 induces the activation and maturation of splenic adherent and bone marrow derived cells from both aged and young mice in vitro. Supernatants derived from these cultures contained IL‐6, TNF‐α, and INF‐γ, representing a pro‐inflammatory cytokine profile. EP67 also promotes the expansion and maturation of CD11b+, CD11b+ CD11x+, and CD11b+ CD11c+ CD80+ CD86+ cells derived from young and aged bone marrow. These results suggest that these adjuvant peptides promote the activation of antigen presenting cells in the enhancement of humoral and cell‐mediated immune function. This work was supported by Grants AG09948–09, AG031496, and AI065712
Reconstitution of functional status of cytotoxic T lymphocyte in chronic infection of hepatitis B virus by mimogen
Authors: Wu Yuzhang
Published: June 2021

Cytotoxic CD8+ T lymphocytes (CTL) play a crucial role in controlling both viral replication and liver damage. We recently showed that HBV‐specific CTLs could be easily detected in peripheral blood of active chronic HBV infections. However, those results suggested that the existence of HBV‐specific CTLs were not directly correlated to hepatocyte injury, and the frequencies of HBV‐specific T cells were not determinant of immune‐mediated protection in chronic HBV infection. We now report that the detailed analysis of the function and differentiation state of epitope‐specific CTLs in chronic HBV infections by using tetramers combined intracellular cytokine staining (ICCS) and phenotypic markers. We found that HBV‐specific CTLs were skewed to an early central memory phenotype, were enriched for CD27+/CD28+/− expressing cells with low levels of perforin and IFN‐γ, reflecting an early intermediate stage of differentiation. Our results suggested that failure immune control in human HBV viral infection might be a result of impaired CTLs maturation into fully differentiated effector T cells. In Phase IIa clinical trial by using mimogen‐based therapeutic vaccine, We found the mimogen could upregulate the the CTLs frequency and drive the impaired functional status to functional status.
The Role of SDF‐1/CXCR4 axis in Th17 migration to bone marrow
Authors: Zheng Mingquan , McKinly Laura , Kolls Jay
Published: June 2021

The chemokine receptor CXCR4 and its unique ligand, stromal‐derived factor 1 (SDF‐1, also called CXCL12), play critical roles in the recruitment and retention of hematopoietic cells within bone marrow and in their mobilization into the circulation. Its interactions directboth the recruitment of different infiltrating cell types tothe lung and the production of inflammatory mediators that intensifypulmonary damage. During lung infection, the T helper cells (Th)17 produce and release the proinflammatory cytokine IL‐17, IL‐17F and IL‐22 which controls CXC chemokine, G‐CSF and antimicrobial protein production in the lung. We hypothesized that Th17 cells are also recruited to sites of granulopoiesis and that CXCR4 signaling is required for the migration of IL‐17 producing T‐cells to these sites. Widetype(WT) mice were treated with anti‐CXCR4, anti‐SDF‐1, or control Ab followed by challenge with K. pneumoniae(KP). Ouctome measures included survival, complete blood counts in peripheral blood, serum and lung cytokines and chemokines, and Th17 precurosr frequncey by Elispot. CXC4 is express on approximately 5% of in vitro polarized Th17 cells grown in Th17 condition media. WT mice showed an increase in Th17 cells by Elsipot in both the lung and bone marrow 18 hours after Kp infection. Administration of anti‐SDF‐1 or anti‐CXCR4 ablated Th17 cells in the bone marrow but did not affect Th17 precursor frequency in the lung. This was also associated with significant reductions in serum G‐CSF levels as well as a reduction in the increase in absolute neutrophils counts observed in the peripheral blood of infected animals given control antibody. The SDF‐1/CXCR4 axis is activated by KP lung infection and is required for Th17 cell recruitment to the bone marrow as well as controlling serum G‐CSF and increases in neutrophil counts. We postulate that Th17 cell use CXCR4 to home to sites of granulopoiesis.
IL‐27‐IL‐27R interactions regulate homeostasis of the TH17 pool and limit intestinal inflammation
Authors: Troy Amy E , Saris Christiaan J M , Hunter Christopher A , Artis David
Published: June 2021

IL‐27 can suppress production of IL‐17 by TH17 cells both in vitro and in vivo. While TH17 cells are abundant in the lamina propria of the intestine under steady‐state conditions, whether IL‐27 regulates homeostasis of these cells is unknown. Using IL‐27R−/− mice, we demonstrate that IL‐27‐IL‐27R interactions limit the size of the intestinal TH17 pool. To determine whether IL‐27 could limit intestinal TH17 responses under inflammatory conditions, IL‐27R−/− mice were exposed to DSS in an acute model of colitis. IL‐27R−/−mice exhibited significantly increased CD4‐derived IL‐17 and developed rapid and severe colitis compared to wild‐type controls. To address the role of IL‐27‐IL‐27R interaction on innate immune cells, Rag−/−/IL‐27R−/− mice were exposed to DSS. These mice exhibited increased severity of disease compared to Rag−/− controls, indicating that IL‐27‐IL‐27R interactions can regulate innate cell function during DSS colitis. rIL‐27 decreased transcription of pro‐inflammatory cytokines by activated neutrophils in vitro; this may contribute to its ability to inhibit innate cell–driven inflammation. Together these results identify a role for IL‐27‐IL‐27R interactions in controlling the size of the intestinal TH17 cell pool under steady‐state conditions and in limiting intestinal inflammation through its effects on innate and adaptive immune cells.
A method for enrichment and detection of viable mouse IL‐17‐secreting T cells
Authors: Richter Anne , Foerster Anna , Assenmacher Mario
Published: June 2021

IL‐17 is discussed to be a key player in the protection against certain infections, but it is also involved in the pathogenesis of autoimmune diseases. We have developed an IL‐17 secretion assay, a tool for the detection and enrichment of viable mouse IL‐17‐producing cells.

Mouse T cells were either directly ex vivo activated with PMA/ionomycin or beforehand stimulated and expanded using CD3/CD28 antibodies. Secreted IL‐17 was detected on the surface of IL‐17‐producing cells after labeling first the cells with a bispecific CD45‐IL‐17 antibody affinity matrix. During culturing IL‐17 was secreted and specifically caught by the affinity matrix of IL‐17‐producing cells. Subsequently IL‐17‐secreting cells were fluorescently stained with a second IL‐17 antibody or additionally labeled with magnetic beads. We detected IL‐17 secretion by CD4 T cells, CD8 T cells and γδ T cells. Already after 1–3h of stimulation the frequency of IL‐17‐secreting cells was maximal. Costaining of cells for secreted IL‐17 and intracellular IL‐17 confirms the specificity of our labeling method. After magnetic enrichment we yield populations of IL‐17+ T cells with purities of more than 90–95%.

The IL‐17 secretion assay allows the detection and provides highly enriched populations of viable IL‐17‐producing cells to investigate the role of IL‐17 in host defense and disease development.
Translational Development in University Settings: Moving University Innovation Towards the Development and Commercialization of Biomedical Products
Authors: Dahms A. Stephen , Watson John , Schuster Sheldon , Holmes Edward , Roth Duane
Published: June 2021

University‐ and research institute‐based discoveries and innovation in the biomedical arena ultimately have to reach the commercial marketplace if they are to benefit mankind and if they are to further justify the substantial taxpayer investment in capitalizing fundamental basic biomedical research. This session will address new, university‐ and research institute‐based approaches that facilitate the movement of exciting biomedical innovation and intellectual property (IP) towards the commercial marketplace. The challenges facing commercialization enablement immediately post‐basic research are extensive and expensive, and they can only be met with investment of capital at the early and highly risk‐prone stage, a stage that the investment community refers to as the “Valley of Death”, a region that the investment community and companies scrupulously avoid due to the considerable uncertainty of success surrounding raw and unimproved early stage IP. New models of commercialization enablement in university and research institute settings are appearing, falling into the category of “translational research” and, more importantly, the newly identified category of “translational development”, an area that is outside of the traditional university and research institute technology transfer process and experience. This session will deal particularly with the challenges that academic scientists face in commercializing their ideas. The speakers will address the newly‐developed, applied research and development endeavors and novel solutions that are now being deployed inside entrepreneurial universities and research institutes to reduce the hurdles facing the ultimate commercialization of their biomedical IP, innovative solutions that improve that IP and move it up the value chain to the point where the further risk of development is substantially reduced and where the net valuation is substantially increased…and thus made far more attractive to investors and commercial licensees due to the increased valuation and the substantially reduced front‐end risk. The net effect on the academic scientist inventors and the universities or research institutes (i.e., the licensors) is that potential financial returns and royalty rates can be greatly increased. The speakers will address not only the essential actors and events involved in moving molecular life science innovation towards the marketplace, but will also provide needed glimpses for basic research‐focused faculty, post‐docs and students into the reality of the industrial biomedical therapeutic and product development and specification world, a funnel through which all biomedical innovation must flow if that innovation is to achieve social and financial success.
IL‐6 modulates Tgf‐Beta expression in skin
Authors: Luckett‐Chastain Lerin
Published: June 2021

Interleukin‐6 deficient mice display delayed wounding healing characterized by decreased collagen deposition/granulation tissue formation. The Tgf‐β family of proteins have been linked to nearly every aspect of wound repair. Although it is clear that Tgf‐β and IL‐6 play important roles in wound repair, a link between these two cytokines remains to be identified. Unwounded skin from C57/BL6 mice showed significantly increased Tgf‐β1 and βR2 mRNA expression levels as compared to IL‐6KO mice but declined upon wounding. However, expression levels increased 3 days post wounding in both mouse types. Immunohistology revealed that Tgf‐β1, Tgf‐β3, and Tgf‐βR2 expression was present in discrete locations and each isoform has a unique localization pattern. Manipulation of the Jak/Stat and Mapk/Erk pathways found that IL‐6 modulates Tgf‐β through a Mapk/Erk dependent mechanism. Inhibition of Jak/Stat showed a significant increase in IL‐6 induced Tgf‐β1 expression, and inhibition of Mapk/Erk displayed a dose‐dependent decrease in Tgf‐β1 expression, indicating possible cross‐talk between the two pathways. Interestingly, IL‐6 seemed to modulate total Erk protein expression in dermal fibroblasts. These studies indicate that IL‐6 has the ability to modulate the expression of Tgf‐β1 and Tgf‐βR2 to varying degrees in skin. This work was funded by NIH/NIGMS grant R01 GM067745
TNFRSF25 promotes Th2 polarization after CD28 costimulation
Authors: Wolf Dietlinde , Deyev Vadim , Adkins Becky , Podack Eckhard R.
Published: June 2021

TNFRSF25 (TNFR25) is a member of the TNF‐receptor family; its ligand is TL1A (TNFSF15). TNFR25 is expressed constitutively on murine CD4+ T cells and up‐regulated after T cell activation. We have shown previously that genetic blockade of TNFR25 signals in CD4+ T cells interferes with Th2 polarization in vivo and in vitro. Investigating the molecular mechanism, we now show that CD28 co‐stimulation during primary activation causes significant up‐regulation of TNFR25 on CD4+ T cells. TNFR25 signals, elicited with an agonistic anti‐TNFR25 antibody (4C12) during secondary stimulation, mediate significant up‐regulation of Th2 cytokine production. In dominant negative (DN) TNFR25 transgenic CD4+ T cells, 4C12 does not up‐regulate Th2 cytokine production despite up‐regulation of DN‐TNFR25 and endogenous TNFR25 expression. We conclude that TNFR25 signals are supportive and may be required for up‐regulation of Th2 cytokine production during secondary stimulation of Th2 polarized effector/memory CD4+ T cells.
A small subset of dendritic cells controls the Th2 bias of neonatal immunity through an interplay between IL‐12 cytokine and IL‐13Rα1 expression
Authors: Hoeman Christine Marie , Lee Hyun‐Hee , Hardaway John , Zaghouani Habib
Published: June 2021

It is known that neonates are susceptible to microbial infection and allergic reactions. This is likely due to the lack of Th1 but excess of Th2 cells. The mechanism underlying this Th1/Th2 unbalance has not been clearly elucidated. When exposed to antigen (Ag) on the day of birth, both Th1 and Th2 cells develop in the primary response but Th1 cells up‐regulate IL‐13Rα1 chain which associates with IL‐4Rα to form an IL‐4Rα/IL‐13Rα1 heteroreceptor. During rechallenge with Ag, IL‐4 from Th2 cells utilizes this heteroreceptor to drive apoptosis of Th1 cells, hence creating a secondary Th2 immunity bias. Herein, it is shown that day 6 after birth represents a point at which the neonate acquires the ability to develop Th1 responses and overcome the Th2 bias. This transition lies on delayed developmental maturation of the CD8α+CD4‐ dendritic cell (DC) subset. Indeed, during the first 6 days of birth the neonates had minimal CD8α+CD4‐ DCs and were unable to produce significant IL‐12 which led to up‐regulation of IL‐13Rα1 expression on primary Th1 cells and formation of IL‐4Rα/IL‐13Rα1 heteroreceptor. By day 6 accumulation of CD8α+CD4‐DCs reaches optimal level leading to significant IL‐12 that opposes IL‐13Rα1 up‐regulation on Th1 cells and minimizes IL‐4Rα/IL‐13Rα1 expression.

This research was supported by grants 2RO1AI48541 and R21AI06279 from NIH. CMH is supported by training grant GM08396‐16A1 from NIGMS.
Autocrine CCR7 signaling is mediated by NF‐kappaB in SCCHN
Authors: Mburu Yvonne , Wang Jun , Walker William H. , Ferris Robert L.
Published: June 2021

The chemokine receptor CCR7 is a seven‐transmembrane G‐protein coupled receptor that is expressed by leukocytes and facilitates their chemotactic migration to the lymph nodes. However, aberrant CCR7 expression has been described in a number of human malignancies and is linked to metastatic behavior, pro‐survival, and invasive pathways. In squamous cell carcinoma of the head and neck (SCCHN), we have previously described the selective upregulation of CCR7 in metastatic, but not primary tumors. Strikingly, the metastatic SCCHN tumor cells also secrete and respond to the CCR7 ligands CCL19 and CCL21, suggesting that an autocrine signaling mechanism may promote self‐sufficiency of growth signals. Based on reports that the nuclear transcription factor NF‐kappaB regulates inflammatory genes involved in chemokine‐mediated tumorigenesis, we hypothesized that NF‐kappaB is a key mediator of autocrine CCR7 signaling in SCCHN. We identified four NF‐kappaB binding sites in the 900bp promoter region upstream of the CCR7 gene, and confirmed their activity using luciferase, EMSA and ChIP assays. Our findings argue that NF‐kappaB is important for the induction of CCR7 expression as well as for the propagation of downstream signaling pathways. The observed autocrine signaling and subsequent downstream NF‐kappaB activation appear to be important for the evolution and progression of metastatic SCCHN tumors.