bluuebunny/crossref_metadata_2025 huggingface dataset:
The Salmonella effector SopB affects phospholipid content of Salmonella‐induced membrane ruffles
Authors: Steele‐Mortimer Olivia
Published: June 2021

Salmonella enterica serovars are pathogenic Gram‐negative bacteria that can cause disease in a variety of eukaryotic hosts. Central to pathogenesis is the ability to invade non‐phagocytic cells within the intestinal epithelium, a process that involves massive actin rearrangements and membrane ruffle formation on the plasma membrane. This is absolutely dependent on a type III secretion system (T3SS), which translocates a number of bacterial effector proteins directly into the host cell. One of these effectors, SopB/SigD, is an inositol phosphatase that is absolutely required for activation of Akt/PKB in infected epithelial cells. We have previously shown that the SopB/Akt pathway can protect infected cells against the onset of apoptosis. Here we have further delineated the mechanism of Akt activation in Salmonella‐infected cells. Unlike the canonical Akt pathway this process is Wortmannin‐insensitive and is prolonged, being detectable for at least 8 hrs. Using PH‐domain GFP fusions we have performed semi‐quantitative analysis of the phospholipids present in Salmonella induced ruffles and demonstrate a role for SopB in ruffle formation. This activity is absolutely dependent on the phosphatase activity of SopB since a catalytically inactive SopB mutant is unable to affect the phospholipid content of Salmonella‐induced ruffles.

Conclusions

The T3SS effector protein SopB modulates the phospholipid content of Salmonella‐induced membrane ruffles.
Neuro‐retinal disorders in Trf2 mutant zebrafish
Authors: Islam Asiful , Kishi Shuji
Published: June 2021

By applying ionizing irradiation (IR+) on wild‐type (WT), and Trf2 mutant (both homo and heterozygous mutant) backgrounds, we observed the neurodegenerative phenotypes and neuro‐retinal disorders in the adult zebrafish and their offspring. The offspring from irradiated parental fish revealed several malformations in eyes during early development, suggesting that the integrity of the gametogenic processes is compromised beyond generations. We have identified abnormal neuro‐retinal development in the homozygous Trf2 mutant embryos that also accelerated retinal aging symptoms in adult heterozygous Trf2 mutant fish. The partial loss‐of‐function (decrease‐of‐function) of telomeric protein TRF2 is responsible for late‐onset retinal disorders as well as neuro‐retinal development with losing genomic integrity. A functional link and crosstalk between p53 and TRF2 in neuro‐retinal development has been demonstrated in both embryonic neuro‐retinal development and adult retinal function in zebrafish. The genetic effects of TRF2 and p53 insufficiency in neuro‐retinal systems of IR+ adults and their offspring detected. In the adult Trf2 mutant fish, we observed the development of AMD (age‐related macular degenerative disease)‐like symptoms with retinal dysfunctions. In our preliminary results, adult heterozygous Trf2 mutant fish showed accumulations of autofluorescent drusen‐like material around the retinal pigment epithelium (RPE) in their earlier age than that of WT siblings. The age‐related macular degeneration caused by the accumulation of lipofuscin in RPE revealed in the retinae of Trf2 mutant zebrafish. These studies exposed a new insight into retinal disorders in Vertebrates and established zebrafish as an efficient model organism for studying human retinal abnormalities and stress‐associated neural disease researches.
Origin of the Diverse Lipid and Protein Interaction Properties of C2 Domains
Authors: Stahelin Robert Virgil , Gunasekaran Christopher , Jones Keaton R. , Cullen Sean
Published: June 2021

The C2 domain (∼130 residues) was originally identified as one of two conserved regulatory domains of Ca2+‐dependent protein kinases C. A large number of proteins containing the C2 domain have been identified since, and most of them are involved in signal transduction or membrane trafficking. Early experiments demonstrated C2 domains could bind bulk phospholipids such as phosphatidylserine or phosphatidylcholine with high affinity and specificity, while more recently; C2 domains have been shown to interact with different phosphoinositides and/or sphingolipids in both Ca2+‐dependent and ‐independent manners. C2 domains have also been shown to bind specific proteins, interact with phosphotyrosine, induce membrane curvature changes, participate in intramolecular interactions, and interact with viral proteins. How the multifaceted C2 domains achieve diverse signaling and interaction properties is only beginning to come to fruition. We have begun a comprehensive and rigorous experimental, computational, and structural analysis of the mammalian C2 domain family. We begin to classify these domains with regards to lipid affinity, specificity, membrane penetration, intramolecular interactions, and protein‐protein interactions, which will lead to a better understanding of how these properties manipulate the full‐length proteins’ functions.
TAK1 prevents TRAIL‐induced cell death through an NF‐κB‐independent mechanism
Authors: Morioka Sho , Omori Emily , Kajino Taisuke , Kajino‐Sakamoto Rie , Matsumoto Kunihiro , Ninomiya‐Tsuji Jun
Published: June 2021

TAK1 functions downstream of several cytokines such as TNF, and activates both MAPK and NF‐κB pathways. We have previously determined that TAK1 is essential for preventing TNF‐induced cell death in epithelial tissues such as skin epidermis and intestinal epithelium. Deletion of TNF receptor I (TNFRI) largely rescued cell death caused by TAK1 deletion; however, dead cells in double (TNFRI and TAK1)‐deficient epithelial tissues were still increased compared to TNFRI knockout tissues. Here, we attempted to determine the TNF‐independent mechanism of cell death in TAK1‐deficient cells. TNF‐Related Apoptosis‐Inducing Ligand (TRAIL) belongs to TNF family, and is also known to induce cell death. We found that TAK1 deletion increased sensitivity to TRAIL‐killing. Although NF‐κB is one of the most important cell survival factors, we found that TRAIL did not induce NF‐κB activation even in TAK1 wild type cells, and that NEMO/IKKγ deletion did not increase sensitivity to TRAIL. In contrast, we found that expressions of several antioxidant genes were reduced in TAK1‐deficient cells, and that treatment of exogenous antioxidant butylated hydroxyanisole (BHA) rescued TRAIL‐killing. These results suggest that TAK1 regulates antioxidant gene expression and contributes to TRAIL‐resistance independently of NF‐κB activity. This work was supported by grants from Crohn's and Colitis Foundation of America and NIH (GM68812).
A mutation in the coding region of Phka1 results in transcriptional repression of all phosphorylase kinase genes in the I/LnJ mouse
Authors: Chavarria‐Smith Joseph , Rice Nancy A.
Published: June 2021

Phosphorylase kinase (PhK) is a hexadecamer composed of four different subunits each encoded by a separate gene located on individual chromosomes. Muscle‐specific α and γ subunits are encoded by Phka1 and Phkg1; Phka2 and Phkg2 encode the liver variants of these subunits. The β and δ subunits are ubiquitously expressed from Phkb and calmodulin genes, respectively. To understand the transcriptional regulation of PhK, we have determined the developmental expression profile for each gene in muscle, liver and brain of both wild‐type (C57BL6) and Phka1 mutant mice (I/LnJ) by quantitative real‐time PCR and subsequently, identified novel genes by microarray analysis that may be involved in temporal and/or spatial coordination of PhK expression. Tissues were isolated at 1, 7, 14, 28 and 56 postnatal day (pnd) and Phka1, Phka2, Phkb, Phkg1 and Phkg2 transcripts were quantified. Subsequently, RNA from pnd 7, 28 and 56 from muscle and liver was analyzed on a whole mouse genome microarray. Our results indicate that: all Phk genes are developmentally regulated and increase in expression over the early postnatal period; Phka1, Phkb, and Phkg1 are expressed ~ 100‐fold higher in muscle than corresponding levels of Phka2, Phkb, and Phkg2 in liver and brain; and all Phk genes are repressed in the I/LnJ mice when compared to the same gene in the C57BL6 mice with muscle and liver expression being repressed more than brain transcription. Phka1 expression is the most variable with a 39.1‐, 7.7‐ and 4.4‐fold decrease in muscle, liver and brain respectively at pnd 56. No compensatory upregulation of Phka2 was found in any tissue. Preliminary microarray results indicate > 200 differentially expressed genes at all time points and tissues assayed between the I/LnJ and C57BL6 mice. This work was supported by the National Institute of Health grant 2PORR16481.
cPKC isozyme specific substrates in murine embryonic stem cells.
Authors: Costa Helio Miranda , Andrade Alexander , Labate Carlos , Krieger José Eduardo , Mochly‐Rosen Daria , Schechtman Deborah
Published: June 2021

PKCs are intracellular signalling enzymes that participate in embryonic stem cells (ES) biology and most likely modify targets associated with key processes including self‐renewal and differentiation. We first identified the PKC isozymes that are present in ES and used an exploratory approach to select potential substrates using cell based assays and phosphoproteomic. Murine ES express mainly PKCs α, βI, βII, δ, ε and ζ/λ. PMA or cPKC peptide modulators were used to identify cPKC substrates, which were resolved in 2D gels and developed with phospho‐specific dye. Upon treatment with cPKC‐selective activator peptide, ψβRACK, 41‐unique protein spots were identified, 9 increased phosphorylation. In contrast, treatment with the cPKC inhibitor, βC2‐4, resulted in a decreased phosphorylation of 51 spots and disappearance of 6. Furthermore, 27 spots disappeared upon treatment with βIPKC‐selective inhibitor, βIV5‐3, and 11 had a decrease in phosphorylation, whereas 10 spots disappeared with βIIPKC‐specific inhibitor, βIIV5‐3, and 11 ‐ decreased phosphorylation. Nine spots increased their intensity with the cPKC activator and inhibited with either beta PKC=specific inhibitor peptide and 8 spots were inhibited by more than one inhibitor peptide. Preliminary data using MS/MS revealed an increased phosphorylation of splicing factor 1 following treatment with either PMA or cPKC activator peptide while elongation factor 1 δ increased phosphorylation with PMA, but decreased with cPKC inhibitor treatment. Taken together, we began the identification of substrates of specific PKC isozymes in mES using isozyme‐selective inhibitors and activators, which deserve to be investigated in the future.
Compartment‐dependent modulation of Ras ubiquitination
Authors: Terrillon Sonia , Bar‐Sagi Dafna
Published: June 2021

Ras GTPases are key regulators of cell growth, differentiation and transformation. Ubiquitination of Ras modulates its intracellular trafficking and signaling output. Yet, detailed knowledge about the in vivo dynamics of this process is limited. Here, we took advantage of the Bioluminescence Resonance Energy Transfer (BRET) technology to monitor HRas ubiquitination in living cells and to investigate its relationship to membrane compartmentalization. We demonstrate that HRas ubiquitination is dynamically altered upon growth factor stimulation. The growth factor‐dependent modulation of HRas ubiquitination is temporally coupled to changes in HRas microdomain localization as determined by BRET analysis of HRas/Caveolin1 interactions. Significantly, an HRas chimera that is unable to interact with Caveolin1 is refractory to ubiquitination, indicating that HRas ubiquitination requires an interaction with Caveolin1. Consistent with this observation, disruption of lipid microdomains by membrane cholesterol depletion inhibits HRas ubiquitination. Furthermore, Caveolin1 expression levels are tightly correlated with HRas ubiquitination. Taken together, our data demonstrate that HRas ubiquitination is dynamically regulated and requires the integrity of caveolae and their scaffolding protein Caveolin1.

Supported by Human Frontier Science Program (S.T.) and NIH grant CA055360 (D.B.‐S.)
Novel probes of acetylcholinesterase inactivation
Authors: Guo Lilu , Suarez Alirica I. , George Katie , Thompson Charles M.
Published: June 2021

Organophosphates (OPs) react with acetylcholinesterases (AChEs) to form a covalent bond at a specific serine residue in the active gorge, thereby providing a highly precise modification of this enzyme and offering unique explorations of protein structure, function and mechanism. The long‐range goal of this study is to show how covalent modification of AChEs by chromophore‐linked OPs can be used to probe the protein and its local environment. Click chemistry was used as a key transformation method to prepare an array of chromophores linked to a reactive fluorophosphonate (FP) head group. Successfully synthesized chromophore‐FPs varying in length and chromophore structure were docked in FlexX (Sybyl 7.0) to visualize and calculate positioning of the chromophore‐FPs relative to the protein active site/gorge/peripheral site. The inhibition rates of chromophore‐FPs against rMoAChE and EEAChE were determined via colorimetric assay. In addition, the binding effects of chromophore and FP part to protein were evaluated by measuring the inhibition rates of chromophores and FP parts, respectively. Results demonstrated that the chromophore structure and the ligand length mutually affect the inhibition potency. To differentiate AChE stereoselectivity, asymmetric analogs of the chromophore‐linked phosphonates will be synthesized and tested as anti‐AChEs.

Supported by NIEHS UO1 ES016102 and NCRR COBRE RR15583
Quantitation and characterization of low molecular weight iron complexes in mammalian cells in conditions of iron overload and deficiency that may comprise components of the ‘labile iron pool’
Authors: Rojas Ben R , Ton‐Nu Lena , Planutyte Lina , Thon Vireak , Mason Andrew Z , Linder Maria C
Published: June 2021

We have determined that when cultured mammalian cells are exposed to 59Fe‐labeled 1–2 uM Fe(II)‐ascorbate for 3h, and cell lysates are applied to native PAGE, two radiolabeled peaks are shown. One is ferritin, the other of low molecular weight and filterable through a 10 kDa membrane. In the studies reported here, we determined the proportion of 59Fe in the unknown relative to ferritin in several cultured cell lines with and without 24h pretreatment with ferric ammonium citrate (10 ugFe/ml), or desferrioxamine (DFO) to increase and decrease iron availability. The ratio of 59Fe in the unknown to that in ferritin varied inversely with iron status. The unknown peak was present not just in cytoplasm but also in the lysosomes. After elution from native PAGE gels, two components were separated by size exclusion HPLC on Biosep 2000 coupled with ICP‐MS. This was also the case on an open Biogel‐P2 column, where Mr values of 1.8 and 0.6 kDa were obtained. Treatment of filtrates with proteinase K caused a shift in elution of the 1.8 kDa component to 0.6 kDa (detectable in native PAGE), indicating presence of peptide. Lectin analysis demonstrated a lack carbohydrate. Iron in the unknown components was directly available to DFO upon cell lysis, and could be chased out with non‐radioactive Fe. We hypothesize that these low molecular weight iron‐peptide complexes are part of the 'labile iron pool' in mammalian cells.
Characterization of copper binding to Riboflavin Binding Protein
Authors: Benore‐Parsons Marilee , Russ Kristen , Bencze Krisztina , Stemmler Tim L. , Smith Sheila R
Published: June 2021

Riboflavin Binding Protein (RBP), abundant in both the yolk and white portions of avian egg, has been shown to bind copper in a 1:1 molar ratio under dialysis conditions. Binding forms a well‐defined Type II site with g values that indicate an oxygen rich environment for the copper. XAS studies indicate a mixture of copper(I) and copper(II) in the XANES spectrum; spin quantitation by cw‐EPR suggests that all of the copper(I) observed by XANES cannot be accounted for by photo‐oxidation during XAS, although no external reducing agents were added and dialysis was performed aerobically. We propose a possible role for RBP in copper transport and storage in avian embryo.
Characterization of the Metal and DNA Binding Properties of an Unusual Zinc Finger from Poly(ADP‐ribose) Polymerase‐1 (PARP‐1)
Authors: Zhou Ying , Liu Hung‐wen
Published: June 2021

Poly(ADP‐ribose) Polymerase‐1 (PARP‐1) plays a crucial role in DNA repair, replication, transcription, and cell death. It recognizes damaged DNA by two unusually long zinc fingers of the form CX2CX28/30‐WHX2C. In this study, we investigated the properties of various divalent metal ions binding to the zinc finger to understand the potential inhibition of PARP. Results of electrophoresis mobility shift assays (EMSA) and southwestern blots showed that, compared to the first zinc finger (FI), the second zinc finger (FII) plays a more important role in binding to different DNA substrates. Metal coordination of FII was studied by competition assays using UV‐vis spectroscopy. Zn2+ and Cd2+ were found to have comparable binding constants of 1013.0±1.0 M−1, and Co2+ and Pb2+ showed a 1000‐fold lower binding affinity. Circular dichroism and 1H‐NMR suggested that Co2+ and Cd2+ could induce the protein fold in a similar manner to that of the holo‐protein, while the Pb2+ complex is less structured. To study the effect of ligand substitution on metal binding, CCCC and CCHH zinc finger mutants were designed and distinct UV‐vis spectra were observed for Co2+ titration. Our results represent the first detailed study of metal binding to the unusual PARP‐like zinc fingers.
Nuclear Factor‐kB‐mediated cytokine expression in adipocytes is reduced by curcumin and resveratrol
Authors: Gonzales Amanda M , Orlando Robert A
Published: June 2021

Adipocytes express inflammatory mediators that contribute to low‐level, chronic inflammation found in obese subjects and is linked to the onset of cardiovascular disorders and insulin resistance. We examined if curcumin or resveratrol can inhibit NF‐κB activation in adipocytes and in doing so reduce expression of inflammatory mediators. Both preadipocytes and adipocytes express the genes for TNFα, IL‐6, and COX‐2, key mediators of the inflammatory response. Preadipocytes were found to express IL‐1β; however, IL‐1β expression was absent in differentiated adipocytes. TNFα treatment activated NF‐κB signaling in adipocytes by inducing IκB degradation and NF‐κB translocation to the nucleus. This treatment increased IL‐6 (6‐fold) and COX‐2 (2.5‐fold) mRNA levels. TNFα also activated IL‐1β gene expression in differentiated adipocytes, but had no effect on TNFα mRNA levels. Curcumin or resveratrol treatment inhibited NF‐κB activation resulting in a reduction of TNFα, IL‐1β, IL‐6, and COX‐2 gene expression (IC50 ∼ 2μM) and a reduction in secreted IL‐6 and prostaglandin E2 (IC50 ~20μM). These data establish the antiinflammatory properties of curcumin and resveratrol in adipocytes and suggest these natural products may provide a novel and safe avenue to reduce or inhibit the chronic inflammatory properties of adipose tissue. This research was supported by a Grant‐in‐Aid from the American Heart Association.
Three Tropical Plants with Hepatoprotective properties
Authors: Iroanya Onyekachi Ogbonnaya , Okpuzor Joy Elizabeth , Mbagwu Herbert O
Published: June 2021

The use of medicinal plants is becoming increasingly popular among all social classes in our society. The lower socioeconomic class cannot afford the high cost of orthodox treatment; the only alternative to remain in good health is the use of medicinal plants which abounds in their surrounding to treat all types of diseases. The study aims to substantiate the claims made by the traditional healers on the use of a mixture of Gongronema latifolia, Ocimum gratissimum and Vernonia amygdalina in the treatment of liver problems.

The mixture showed no toxicity when administered orally or intraperitoneally to Wistar albino rats. Aqueous extract of the mixture was evaluated against Acetaminophen induced acute hepatotoxicity in Wistar rats. The animals were divided into six groups with each group containing ten animals. Three doses of the extract (2000, 4000, 8000 mg\kg) were fed to the rats for four days at 24 hours interval.

At a dose of 8000 mg\kg body weight there was a significant change in the level of Alkaline Phosphatase (21.3± 3.8), Aspartate Aminotransferase (23.3±2.3) and Alanine Aminotransferase (18 ±1.1) when compared to positive (19.7 ± 0.3, 23.7 ± 1.2 and 23.7 ± 1.2) and normal (25 ± 2.1, 22.3 ± 1.9 and 23.7 ± 1.2) control values. These results indicate a significant hepatoprotective effect against liver damage induced by Acetaminophen.

We therefore, provide a scientific rationale for the traditional use of the mixture in the management of liver diseases.
Modulation of Dendritic cell response by human CD8 T cells in the presence of a microbial stimulus
Authors: Chong Shu Zhen
Published: June 2021

Dendritic cells (DCs) play a central role in the induction of immune responses by priming CD8 T cells for effector and cytotoxic functions. However, little is still known about the priming effects of CD8 T cells on DCs in the event of a microbial infection. Here, we show that human CD8 T cells are capable of modulating DC cytokine production early during the priming process. We showed that activated CD8 T cells were consistently better in activating DCs for cytokine production. Activated CD8 T cells enhanced the cytokine production of IL‐12p40, TNF‐α and GM‐CSF by DCs in the presence of a microbial stimulus. Notably, IL‐1 and IL‐12p70 required the absolute presence of both CD8 T cells and LPS. Interestingly, IL‐1 is produced by DCs with CD8 T cells but not with IFN‐g, indicating the involvement of other signals. CD8 T cells were also able to prime DCs for the production of chemokines such as RANTES and IP‐10. This study demonstrates the helper role of CD8 T cells in the enhancement of DC activity and its possible implications in immune modulation.

This work is funded by the Immunology Programme (Grant no. C182‐002‐002‐001). Chong SZ is supported by the NUS Graduate School for Integrative Science and Engineering Scholarship
Dendritic cells play a key role in immune regulation by type II NK T cells
Authors: Halder Ramesh , Maricic Igor , Kumar Vipin
Published: June 2021

Here we describe regulation of type I NK T (iNK T) cells following cellular interactions between dendritic cells (DC) and type II NK T cells. A subset of type II NK T cells recognize a self‐glycolipid, sulfatide, bound to murine CD1d. Using glycolipid/CD1d‐tetramers and cytokine responses we have shown that activation of type II NK T cells leads to upregulation of costimulatory molecules on hepatic plasmacytoid DC (pDC), but not conventional DC (cDC). This is associated with rapid recruitment of iNK T cells into liver in an IL‐12‐ and MIP‐2‐dependent fashion. However, iNK T cells are not activated and are anergic. On adoptive transfer hepatic DCs can induce anergy in the recipient's iNK T cell population. Recent experiments suggest that pDC and cDC play distinct roles in anergy induction. Our data describe a novel pathway of DC‐mediated immune regulation by type II NK T cells that can be exploited for intervention in inflammatory diseases. (Supported by grants from the NIH to VK).
Rapid & Marked Upregulation of B7‐H1 on Macrophages
Authors: Bucknum Amanda , Gonzalez Diana , Silberman Daniel , Bogineni Anup , Jenifer Theron , Riggs James E.
Published: June 2021

Although cells essential for immunity exist within tumors they often fail to prevent cancer progression. T cell activation is often suppressed in tumors. Our model mimics this aspect of tumors. As seen when tumor macrophage (Mf) numbers are increased, we find high Mf:T cell ratios foster suppression. Mf catabolism of ARG and TRP contributes to reduced T cell activation as IDO and iNOS inhibitors permit T cell activation. Using FACS we assessed expression of costimulatory ligand‐receptor combinations to determine if their aberrant expression correlated with suppression. The MFI of Mf Class II expression was reduced in suppressed cultures. In contrast, CD80 and CD86 MFI increased. The greatest change was a 20X increase in B7‐H1 expression. Marked up‐regulation was evident by 24 hrs for C57BL/6 but not BALB/c mice. This reflects the Th1/Th2 dichotomy of these strains with IFNg driving more rapid B7‐H1 expression on C57BL/6 Mfs. Mfs lacking the IFNgR failed to suppress and expressed less B7‐H1. Mf‐like B‐1 B cells constitutively expressed B7‐H1. As the Mf:T cell ratio was reduced, T cell activation returned, Class II MHC and CD80 expression increased, and CD86 and B7‐H1 expression decreased. These data reveal targets to curb the suppression evident in tumors with aberrant Mf:T cell ratios. Supported by NIH AREA R15‐AI060356‐01.
STRESS, CONTROL AND IMMUNE REGULATION IN AN EXPERIMENTAL MODEL OF ASTHMA
Authors: Deshmukh Aniket Gunwant , Jones Harlan P
Published: June 2021

Asthma is becoming more prevalent in today's world, particularly prevalent among children. Africans American children are more to susceptible to asthma than white Americans. It is becoming increasingly clear that stress is a major risk factor for asthma. Though asthma and stress has a very strong relationship, we still lack a clear understanding the of underlying mechanisms through which stress influences disease pathogenesis. Asthma has been described as psychosomatic disorder. As the pulmonary immune system is under the control of the Central Nervous system (CNS) and endocrine system, dys‐regulation in this system may be a contributing factor in immune mediated exacerbation of asthma. Our current focus is to understand how signaling events between brain, an immune privileged organ can control disease severity along the respiratory tract. To investigate the effects of stress on asthma severity, ovalbumin sensitized mice were exposed to an experimental stress paradigm involving control and uncontrolled stress conditions. Our preliminary data has shown that significant changes in behavioral patterns between the conditions that corresponded with immune dys‐regulation that corresponded immune mediated severity of asthma. We also demonstrate cortocotrophin Realeasing Hormone (CRH) expression level in the lung is associated with differences in the disease severity of asthma. Such differences suggest that stress may be a contributing factor in regulation of Antigen Presenting Cells, the key player in innate immune system. We have seen accumulation and of these cells in the lungs. We hypothesize that stress induced alteration in APC function will dictate pro‐asthmatic immune response. This link can lead us to functioning of these immune cells in the case of asthma. These findings can have the implications for treatment of Th2 mediated disease such as asthma.
The evolutionary conserved HoxC4 homeodomain protein induces AID expression and regulates immunoglobulin class switch DNA recombination and somatic hypermutation
Authors: Park Seok‐Rae , Pal Zsuzsanna , Al‐Qahtani Ahmed , Mai Thach , Xu Zhenming , Zan Hong , Casali Paolo
Published: June 2021

The regulation of activation induced cytidine deaminase (AID) gene (aicda) expression and the modulation of immunoglobulin gene class switch DNA recombination (CSR) and somatic hypermutation (SHM) remain to be defined. We found that the HoxC4 homeodomain protein is preferentially expressed in germinal center B cells and upregulated by stimuli that induce AID expression and CSR. In HoxC4‐deficient mice, the mutation frequency in the intronic JH‐iEμ region of IgH DNA and the serum IgG1 levels were decreased by about 60%. Accordingly, the high affinity anti‐NP response was significantly compromised in hoxC4−/− mice, and CSR to IgG1, IgG2a, IgG2b, IgG3 and IgA in in vitro stimulated hoxC4−/− B cells was impaired. This was not due to altered hoxC4−/− B cell proliferation or germinal center formation in hoxC4−/− mice. Rather, it resulted from a defective induction of AID expression. Indeed, by performing in vitro promoter bashing experiments using luciferase gene reporter assays and ChIP, we showed that HoxC4 activates aicda by binding to an evolutionarily conserved HoxC4/Oct‐binding ATTT(GCAT) motif in the promoter for this gene. Enforced expression of AID in HoxC4‐deficient B cells restored CSR. Thus, these findings show that by regulating AID expression through direct activation of the aicda promoter, HoxC4 plays a critical role in modulating CSR and SHM.

Supported by NIH grants, AI 45011, AI 60573 and AR 40908.
CD70 Expression on Spodoptera Frugiperda (Sf9) Cells by Baculovirus to Induce CD27 Stimulation in Mouse B Cells
Authors: LEE KYUNG‐HO
Published: June 2021

Members of the tumor‐necrosis factor receptor (TNFR) superfamily of molecules have a variety of important roles in the regulation of activation and apoptosis of many cell types, but they are particularly important for cells of the immune system as expressed on B cells and T cells. CD27 is expressed by a subpopulation of B cells, and it is thought to be a marker of memory cells. Although signals delivered by CD27 overlap with those delivered by CD40, they seem to be most important in the late stages of the humoral immune response. For achievement of selective TNF‐TNFR stimulation without ¡°noise¡± background stimulation, insect cell line based expression system was adopted. In this study, CD70, which is the ligand of CD27 on B cells and T cells, was expressed on Spodoptera frugiperda (Sf9) cells with Baculovirus expression system to induce CD27‐CD70 interaction. CD27 stimulation by mouse CD70 expressed Sf9 cells, upregulated CD14 and CD80 expression on A20, mouse B cell line. Interestingly, levels of CD40 and IgG expression was decreased after CD27 stimulation on A20 cells. We can apply this insect cell lines to induction of physiologic condition of receptor‐ligand interaction between immune cells.
Induction of Humoral and Cell‐Mediated Immune Responses by a Novel Adjuvant Derived From HumanAnaphylatoxin C5a
Authors: Morgan Edward L , Morgan Brandon N , Sanderson Sam D , Thoman Marilyn L , Phillips Joy A
Published: June 2021

C5a enhances both humoral and cell‐mediated immunity as well as inflammation. We have developed two molecular adjuvant (MA) analogs based on the C‐terminal region of human C5a [EP67: YSFKDMP(MeL)aR and EP54: YSFKDMPLaR] that trigger immunity. When conjugated to either peptide or whole protein antigen, these analogues enhance both humoral and cell‐mediated immunity in vitro and in vivo. The antibody response induced by a MA‐peptide antigen construct differs from that of a conventional adjuvant (CFA) in that the response is mainly IgG2a and IgG2b. Protein antigen conjugated to EP67 induces a high‐titer mix of IgG1, IgG2a, and IgG2b antibodies. In contrast, OVA induces an IgG1 antibody response. In vitro stimulation of spleen cells derived from EP67‐antigen injected mice produced an enhanced T cell proliferation response compared to mice immunized with antigen alone. This work was supported by Grants Ag09948‐09, AG031496, and AI065712
Hsp90 associates with N‐terminal extended peptides and is required for direct antigen presentation
Authors: Garg Manish , Callahan Margaret K , Srivastava Pramod K
Published: June 2021

Antigen presentation by MHC I molecules requires the generation of peptides in the cytosol, ferrying of peptides from cytosol to endoplasmic reticulum, loading of MHC I with peptides, and transport of MHC I‐peptide complexes to the cell surface. Hsp90 is a cytosolic chaperone known to associate with peptide and peptide precursors of MHC I epitopes. We report here that treatment of a variety of cells with hsp90 inhibitors leads to generation of “empty” MHC I molecules on the cell surface, due to inhibited loading of MHC I with peptides. Inhibition of hsp90 does not inhibit protein synthesis in general, nor synthesis of MHC I in particular, nor does it affect the activity of proteasomes. Hsp90‐inhibited cells, like lactacystin‐treated cells, are poor stimulators of T lymphocytes. The role of hsp90 in presentation of an ovalbumin epitope is shown to be at a post‐proteasomal step: hsp90 associates with N‐terminally extended precursors of the SIINFEKL epitope, and such peptides are depleted from hsp90 preparations in inhibitor‐treated cells. These results argue for a substantial role for hsp90 in chaperoning of antigenic peptides in direct presentation.

This work was supported by NIH grant CA 084479 to P.K.S.
Establishing the molecular mechanisms of B cell receptor internalization
Authors: McCabe Kathleen O. , Yelton Allison , Drake Lisa , Drake James R.
Published: June 2021

The B cell receptor (BCR) is comprised of ligand binding (IgH/L) and signaling (CD79a/b) subunits and mediates the processing/presentation of bound antigen. Internalization of the BCR had been reported to occur both through clathrin‐coated pits (CCPs) and lipid rafts (LRs), although it is unclear which pathway predominates. Use of a previously described model, consisting of a CD32‐CD79b fusion protein, demonstrates that the cytoplasmic tail of CD79b contains one or more functional endocytosis motifs. Internalization is crosslinking‐dependent, recapitulating the behavior of the native BCR when ligated with an antibody against the variable region of the IgH/L subunit. CD79b contains an ITAM with two Yxx? motifs that may bind the clathrin adaptor AP‐2, suggesting CCPs may be involved in internalization. However, there is also an increased association between the BCR and LRs upon receptor crosslinking, implying that under certain circumstances, LR localization of the BCR may lead to internalization subsequent to signaling. Current work focuses on mutating the conserved ITAM residues of CD79b to disrupt these potential AP‐2 binding sites. Additionally, by visualizing the BCR in morphologically identifiable CCPs by electron microscopy or in LRs by sucrose fractionation, the primary mechanism of BCR internalization will be further established. Supported by NIH grant AI‐065773 to JRD.
Fetal/neonatal ethanol exposure irreversibly alters dendritic cell function in adult mice
Authors: Fan Ji , Schlueter Annette
Published: June 2021

Children born to alcoholic mothers are known to have increased incidence and severity of infections.We have developed a model of fetal/neonatal alcohol exposure (FAE) in which female mice are maintained on 10% EtOH in the drinking water during breeding and gestation, and then on 12% EtOH during lactation.Pups have successfully been weaned and showed no significant difference in weights from controls through 12 weeks of age.These pups have been studied at 20 d (weaning) and 12 weeks (young adult) for DC function.Splenic DC numbers did not change. FAE decreased the ability of these DC to stimulate allogeneic T cells,changes that persisted following cessation of EtOH exposure.In addition, antigen‐specific Foxp3+ regulatory T cells were induced following antigen presentation by FAE DC at a higher rate than by control DC. IL‐10 production by OTII T cells increased and IL‐2 production decreased following stimulation with OVA peptide‐pulsed FAE DC relative to control DC.These results indicate that mice born to EtOH‐consuming mothers have deficiencies in DC function that are persistent following cessation of EtOH exposure, and provide a mechanism, which could at least partially explain the increased incidence and severity of infection in children of alcoholic mothers.
Francisella tularensis elicits a soluble factor that induces ubiquitin‐dependent MHC class II endocytosis and degradation in macrophages
Authors: Wilson Justin E , Drake James R
Published: June 2021

The intracellular bacterium Francisella tularensis avoids degradation and replicates within macrophages (MØ), ultimately killing the host cell. Resolution of infection requires generation of IFN‐γ and development of adaptive immunity through presentation of F. tularensis antigens to CD4+ and CD8+ T cells. This laboratory has reported that F. tularensis induces MØ prostaglandin E2 (PGE2) production, leading to skewed T cell responses. PGE2 has been shown to downregulate MHC class II on MØs, suggesting that F. tularensis infection may alter T cell responses via inhibition of class II expression. To test this hypothesis, the effect of soluble factors produced by F. tularensis infected MØs on class II and CD86 expression by activated MØs was determined. Exposure of MØs to infection supernatants results in reduced surface class II and CD86. The factor eliciting class II downregulation is distinct from PGE2 and heat‐sensitive in nature. Moreover, biochemical analysis revealed a ubiquitin‐dependent mechanism of class II internalization and degradation within intracellular acidic compartments, and revealed that blocking this class II ubiquitination prevents surface clearance. These results suggest a mechanism for the modulation of MØ antigen presentation and T cell recognition during F. tularensis infection. Supported by NIH grant AI‐056320.
Substance P Enhances Chemokine Expression in Murine Macrophages via ERK/p38 MAPK‐NF‐κB Pathways
Authors: Jia Sun , Ramnath Raina , Bhatia Madhav
Published: June 2021

In the current study, we aim to investigate the role of substance P (SP) in enhancing the inflammatory chemokine response of macrophages. Using the murine macrophage cell line RAW 264.7 as well as isolated primary macrophages, we show that SP (10 nM) elicits selective chemokine production from macrophages. MIP‐2 and MCP‐1 are two major chemokines synthesized by macrophages in response to SP. Moreover, SP strongly induces the classical pathway of I B‐dependent NF‐κB activation and enhances DNA‐binding as well as transactivation activity of the transcription factor. SP‐evoked transcriptional induction of chemokines is neurokinin‐1 receptor‐specific. Furthermore, SP stimulation of macrophages activates the ERK1/2 and p38 MAPK. Blockade of the two MAPK pathways with specific inhibitors abolishes SP‐elicited NF‐κB‐driven chemokine production. The attenuation of NF‐κB response by ERK1/2 inhibitor does not involve impairment of DNA binding of the transcription factor but correlates with diminished Ser276‐phosphorylated status and transactivation potential of the p65 subunit. The p38 MAPK controls NF‐κB‐responsive chemokine expression by regulating both the DNA binding and transactivation activity of the transcription factor. Collectively, our data demonstrate that SP enhances selective inflammatory chemokine production by murine macrophages via ERK/p38 MAPK‐mediated NF‐κB activation.
Maturation of bone marrow‐derived dendritic cells stimulated with collagen is induced via DDR2‐ mediated NF‐kB activation
Authors: Lee Ji‐Eun , Lee Sun‐Hee , Lee Young‐Mi , Kim Bum‐Tae , Moon Woo‐Sung , Kim Dae‐Ki
Published: June 2021

Dendritic cells (DC) are critical immune sentinels with the unique ability to initiate both innate and adaptive immunity. The ability of DC to activate naïve T cells is tightly linked to their maturation state. The mechanism by which a collagen receptor discoidin domain receptor‐2 (DDR2) controls the maturation of DC remains undefined. In this study, we investigated a signaling mechanism of DDR2, which is one of collagen receptors, to induce the functional maturation of murine bone marrow‐derived DC. The expression of surface molecules, including MHC class II and CD86, on DC were upregulated by DDR2 engagement to collagen, in comparison with unstimulated DC. DDR2 activation significantly increases the production of IL‐12 by DC, as well as proliferation of allogeneic T cells. Moreover, IL‐12 production of DC by collagen was increased through NF‐kB activation to induce and suppressed by pretreatment of NF‐kB and PKC inhibitors. Also, the pretreatment of DC with inhibitors of NF‐k B and JNK signal pathways inhibited collagen‐induced up‐regulation of surface molecules, such as MHC class II and CD86. Our results demonstrated that collagen stimulation could induce the functional maturation of DC via DDR2 mediated‐NF‐kB, PKC and JNK signal pathways.

Supported by KSEF Basic Research Program [R01‐2005‐000‐10942‐0]
Neutrophil adhesion under flow conditions induces zyxin nuclear translocation in endothelial cells
Authors: Bowley Erin Nicole , Patel Kamala , Colarusso Pina
Published: June 2021

High shear stress causes zyxin translocation from focal adhesions (FA) to stress fibers. We previously showed that leukocyte adhesion to stimulated endothelial cells (EC) activated mechanosensitive signaling pathways in ECs at low shear stress, which in turn modulated leukocyte transmigration. In this study, we examined the remodeling of the FA protein zyxin following neutrophil (PMN) adhesion under shear stress and determined if downregulating this protein altered PMN transmigration. Zyxin localized to FAs and stress fibers under static conditions. Buffer perfusion across TNFα‐stimulated ECs did not change zyxin localization at lower shear stresses, but resulted in modest nuclear translocation of zyxin at a shear stress of 4 dyne/cm2. In contrast, PMN recruitment to TNFα‐stimulated ECs resulted in a loss of zyxin from FAs and translocation of zyxin to the nucleus at shear stresses as low as 1 dyne/cm2. Translocation specifically occured in ECs with adherent PMNs and did not occur in ECs distant from regions containing adherent PMNs. Perfusion of fixed PMNs, which roll but to do not adhere or transmigrate, did not induce changes in zyxin localization. Finally, zyxin downregulation in ECs attenuated PMN transmigration. These data suggest that PMN trafficking across stimulated ECs leads to focal adhesion remodeling and that EC zyxin nuclear translocation during this process may play a potential role in gene transcription.

Funding: Canadian Institutes of Health Research, Alberta Heritage Foundation for Medical Research.
Endothelial cell paxillin is remodeled during leukocyte adhesion and is essential for transmigration
Authors: Roccamatisi Dawn Louise , Zhang Hong , Colarusso Pina , Patel Kamala
Published: June 2021

The focal adhesion protein paxillin plays a critical role in focal adhesion remodeling and signaling and as such was examined within endothelial cells (EC) during in vitro neutrophil transmigration. We perfused isolated human neutrophils across TNF‐treated EC in a parallel plate flow chamber and investigated paxillin remodeling by immunostaining. Immunofluorescence data showed that there was a localized loss in EC paxillin staining following neutrophil adhesion under flow conditions. Imaging exogenous paxillin‐YFP in EC, we observed endothelial paxillin loss and remodeling in real‐time. In flow experiments in which we perfused fixed neutrophils or a neutrophilic cell line, both of which adhere but do not transmigrate, we found that paxillin loss was transmigration‐dependant. A functional role for EC paxillin was examined by utilizing siRNA. Using flow cytometry and western blotting, we showed more than ninety percent transfection efficiency and down regulation of paxillin. Downregulation of EC paxillin had no effect on the total number of neutrophils recruited to TNF‐stimulated EC; however, transmigration was blocked by more than 40%. This was not due to changes in expression of E‐selectin, IL‐8, or ICAM‐1 in paxillin downregulated EC. These data suggest that focal adhesion remodeling occurs during leukocyte recruitment and is essential for maximal transmigration.

Research funded by The Canadian Institutes of Health Research.
E‐selectin induction by IL‐1 and IFN‐ γ in the pathogenesis of type III immune hypersensitivity
Authors: Lee Delphine J , Li Huiying , Carbone Ryan J , Damoiseaux Robert , Rea Thomas H , Eisenberg David , Modlin Robert L
Published: June 2021

Erythema nodosum leprosum (ENL), a reaction in lepromatous leprosy (L‐lep), is a prototypical type III hypersensitivity reaction characterized by immune‐complex deposition and a neutrophilic infiltrate. We investigated the differential gene expression profiles of L‐lep and ENL lesions; both supervised and unsupervised analyses indicated that the different patient groups express a distinct lesional gene profile. The cell adhesion gene cluster, including e‐selectin (E‐sel), was the most overrepresented biological process increased in ENL compared to L‐lep. E‐sel protein was expressed on blood vessels, and was comparatively higher in ENL than L‐lep lesions. IL‐1 and IFN‐γ induced high levels of E‐sel expression on endothelial cells and induced neutrophil adhesion. Thalidomide, an effective treatment for ENL, diminished IL‐1 and IFN‐γ induced E‐sel expression on endothelial cells and diminished neutrophil binding. These data suggest a role for IL‐1 plus IFN‐γ induction of E‐sel on endothelial cells in mediating neutrophil recruitment in the pathogenesis of ENL and type III hypersensitivity reactions.
Involvement of PKCδ in SP‐induced chemokine production in pancreatic acinar cells
Authors: Ramnath Raina , Sun Jia , Adhikari Sharmila , Bhatia Madhav
Published: June 2021

Pancreatic acinar cells are known to synthesise chemokines, which play critical roles in acute pancreatitis. We have previously investigated the ability of substance P (SP) to produce chemokines MCP‐1, MIP‐1α and MIP‐2 in pancreatic acinar cells, however the exact mechanism by which SP stimulates the synthesis of these pro‐inflammatory mediators remain undetermined. The aim of the current study is to look into the participation of protein kinase C (PKC) in SP‐induced chemokine production in an in vitro model of isolated pancreatic acinar cells. Our results show that SP, interacting with neurokinin 1 receptor (NKIR), stimulated phosphorylation of the PKC isoform PKCδ in pancreatic acinar cells. This was followed by an increased activation in MAPKK kinase MEKK1, MAP kinases ERK and JNK as well as transcription factors, NFκB and AP‐1, driven chemokine production. Inhibition of PKCδ with the specific PKCδ inhibitor rottlerin, dose dependently decreased SP‐ NK1R induced PKCδ, MEKK1, ERK, JNK, NFκB and AP‐1 activation. Moreover, rottlerin significantly inhibited SP‐NK1R induced secretion of MCP‐1, MIP‐1α, and MIP‐2 in a concentration dependent manner in pancreatic acinar cells. The present study demonstrates the role of PKCδ in SP‐induced chemokine production in pancreatic acinar cells and hence PKCδ may be critically involved in the pathogenesis of acute pancreatitis.
Short‐term depletion of regulatory T cells converts self‐limiting arthritis into a chronic disease
Authors: Kamradt Thomas , Bruns Lisa , Frey Oliver , Reichel Andreas , Morawietz Lars
Published: June 2021

Background: we have established a new arthritis model, induced by immunization of normal mice with the glycolytic enzyme glucose‐6‐phosphate isomerase (G6PI) (Schubert et al. J. Immunol 2004;172:4503). Arthritis occurs in > 90% of the animals from d9 after the immunization and is usually self‐limiting within 30–40 days. We examined if regulatory T cells (Treg) were involved in the resolution of arthritis.

Results: Injection of anti‐CD25 antibody on d‐11 and –8 before immunization resulted in a massive yet transient reduction of CD25+FoxP3+ Treg. The percentage of Treg was normal at d12 after immunization i.e. early during the course of arthritis. This transient depletion affected the disease course profoundly, resulting in chronic‐destructive arthritis with histologically active inflammation over the whole follow‐up period (80d). Exacerbated arthritis was accompanied by increased T‐cell proliferation and increased numbers of IL‐17 producing T cells.

Conclusions: CD25+FoxP3+ Treg may not be effective in the resolution of ongoing arthritis. Rather, they seem to act in the induction phase of the disease by controlling the extent of T cell activation and differentiation.