bluuebunny/crossref_metadata_2025 huggingface dataset:
Antioxidation effects of polysaccharide fucoidan extracted from seaweeds in skin photoaging
Authors: Ku Mi‐Jeong , Lee Myeong‐ Sook , Moon Hee‐Jung , Lee Yong‐Hwan
Published: June 2021

The skin aging can be divided into two areas, intrinsic aging and photoaging. Damage to human skin is due to repeated exposure to ultraviolet (UV) radiation. Especially UVB causes cutaneous changes such as sunburn, skin aging and skin cancer. UVB irradiation induces formation of reactive oxygen species (ROS) and depletes cellular antioxidant stores. In the present study, using the human skin fibroblast HS68 cell line, the author investigated antioxidation effects of fucoidan through UVB‐induced glutathione (GSH), malondialdehyde (MDA), ROS and DNA fragmentation. Fucoidan, with doses of 10 μg/mL, inhibited (8.4%) the production of MDA. GSH level in HS68 cells was increased (5.8%) at pretreated with fucoidan 1 μg/mL. The levels of intracellular reactive oxygen species (ROS) were reduced by dose‐dependently.

Based upon these results, it was suggested that fucoidan might play an important role in the celluar antioxidation effects against UVB irradiation‐induced photoaging.

*This work was supported by the grant of KICOS
Induction of differentiation in K‐562 leukemia cells by PMA and AD 198
Authors: Barham Whitney Jo , Lothstein Leonard
Published: June 2021

Both the phorbol ester phorbol‐12‐myristate‐13‐acetate (PMA) and the anthracycline N‐benzyladriamycin‐14‐valerate (AD 198) are protein kinase C (PKC) activators. Both act by binding to the C1b domain of PKC. PMA and AD 198 promote apoptosis in cells exposed to their respective cytotoxic doses. PMA promotes differentiation when administered at lower than cytotoxic levels. AD 198 may too induce differentiation when administered at non‐cytotoxic levels.

K‐562 chronic myeloid leukemia cells were exposed to AD 198 at a non‐cytotoxic dose. Other cells were exposed to a non‐cytotoxic dose of PMA. Western blot analysis showed the level of expression of p21 in these samples, revealing significant p21 expression in the PMA treated cells, and no expression in the AD 198 treated cells. Reverse transcriptase polymerase chain reaction (RT‐PCR) was used to probe for differentiated cell specific RNAs including CD61, pdgf, and c‐myc. Significant amounts of both CD61 and pdgf were found in the PMA treated cells, but were not found in those treated with AD 198. C‐myc was absent in all samples. It was concluded that AD 198 does not induce differentiation as PMA does. Since AD 198 and PMA both increase PKC‐ ε expression, but only PMA promotes PKC‐ ε activation and nuclear localization (both implicated in differentiation), differential PKC‐ ε expression may account for this functional difference. Research funded by NIH NCI #CA 100093.
Response of human erythroleukemia cells to heteroarotinoids
Authors: Miller Matthew R. , Hillard Jared M. , Evans Timothy L. , Birckbichler Paul J.
Published: June 2021

All trans‐Retinoic acid (RA) is a naturally occurring molecule that binds to retinoic acid receptors and contributes to proliferation regulation. Heteroarotinoids (Hets) are synthetic structural analogues to RA, defined as systems with one aromatic ring and one heteroatom that exhibit lower toxicity as well as increased biological activity. In this study, human erythroleukemia cells were cultured for 72 h and chemically treated for the latter 48 h with Het 1, Het 4, thapsigargin (TH) for positive apoptosis control, RA for biological activity standard, and no treatment for control. Cells were assayed for viability and growth inhibition using trypan blue exclusion, relative levels of apoptosis induction using Hoechst stain and propidium iodide with fluorescent microscopy, and tissue transglutaminase (tTG) expression. RA showed the least growth inhibition and apoptosis induction relative to Het 1 and Het 4. Het 1 and Het 4 showed approximately equivalent growth inhibition relative to control, three‐fold greater than RA. Het 1 and Het 4 resulted in a 89% and 77% apoptosis induction relative to TH respectively. Het 1 and Het 4 also showed a 4‐5‐fold increase in apoptosis relative to control. Both Het 1 and Het 4 contain sulfur heteroatoms and the increased biological activity of Het 1 is attributed to the presence of a thiourea link. Relative to other modifications, changes to the end of the side chain made virtually no enhancement. Support for this research was supplied by the College of Health, Science and Environment and Faculty/Student Research Grant program of Slippery Rock University.
Effects of ziram on tumor cell binding capacity, cell‐surface marker expression, and ATP levels of human natural killer cells
Authors: Taylor Thyneice Rochelle , Whalen Margaret M
Published: June 2021

Human natural killer (NK) cells lyse tumor cells, virally infected cells, and antibody coated cells. Increased tumor formation and viral infection may occur if NK lytic function is disrupted. Ziram is used in the production of latex rubber and as an antifungal agent in agriculture. Previous studies have shown that ziram exposure decreased ability of NK cells to lyse tumor cells. To examine the mechanism by which ziram exposure decreases NK lytic function, we have assessed the effects of ziram exposure on the tumor‐cell‐binding function, cell‐surface marker expression, and ATP levels of NK cells. Exposure of NK cells to 1 μM ziram for 24 h decreased NK‐binding to tumor cells by 36%. Levels of expression of CD2, CD11a, CD16, CD18, and CD56 were examined and there were no significant decreases in any of these markers after a 24 h exposure to 1 μM ziram. However, a 24 h exposure to 1 μM ziram caused a 46 % decrease in ATP. These studies indicate that a decrease in the ability of NK cells to bind to tumor cells is, in part, responsible for the loss of NK lytic function and that this binding does not appear to be dependent upon any of the cell surface markers that were examined. Further, the depletion of ATP levels in NK cells after ziram exposure may account, in part, for the decrease in the ability of NK cells to bind target cells. Supported by grant S06 GM008092‐32.
Probing the Beta‐Amyloid Structure and Function of Hemolysin A
Authors: Wawrzyn Grayson Thomas , Werren Rebecca
Published: June 2021

In this study we aimed to analyze the structure and function of a truncated form of hemolysin A from Proteus mirabilis using a combination of template‐assisted hemolysis and x‐ray crystallography. The hemolysis assay performed in the presence of both truncated and full‐length hemolysin A noted a cross‐seeded, biphasic activity profile, where the biphasic nature could be eliminated upon pre‐incubation of the protein components in the absence of red blood cells. X‐ray structure analysis of truncated hemolysin A reported a right‐handed parallel beta‐helix fold with three adjoining segments of anti‐parallel beta‐sheet, three buried solvent molecules near the disulfide bond between cysteines 144 and 147, and a unique dry dimeric interface. The disulfide bond stabilizes a unique beta‐arc between beta‐strands 11 and 12, which form two sides of the third full solenoid turn within right‐handed beta‐helix. The unusual dry dimeric interface propagates the monomeric beta‐helix structure forming a fibril appearance. The biochemical and structural results are discussed in reference to the propagation of beta‐amyloid type infectious prion diseases via a template‐assisted model.
Antagonists of ribonuclease inhibitor: Small molecules, dendrimers, and peptides
Authors: Ellis Gregory A , Lavis Luke D. , Johnson R. Jeremy , Raines Ronald T.
Published: June 2021

Recent successes have indicated the feasibility of disrupting protein–protein interactions (PPIs) with small molecules, peptidomimetics, and peptides to modulate cellular processes. Among the tightest of all known PPIs are the interactions between ribonuclease inhibitor (RI) and its ribonuclease ligands, which bind with femtomolar affinity even though these ribonucleases share only ∼30% amino‐acid sequence identity. Due to this high affinity, identifying antagonists of this interaction would be a notable advancement in the area of PPI inhibition.

Multiple strategies are currently being used to identify antagonists of RI. Several preliminary antagonists have been identified through screening of small‐molecule libraries. In addition, poly(amidoamine) dendrimers were screened as ribonuclease mimics and demonstrated moderate affinity for RI. Finally, phage display is being conducted to identify peptide antagonists.

Ribonucleases that evade RI exhibit selective toxicity for cancer cells, and the lack of affinity for RI is considered a primary determinant of that toxicity. Therefore, it is hypothesized that inhibiting the RI–ribonuclease interaction with antagonists will potentiate the cytotoxic activity of modestly RI‐evasive ribonucleases, possibly leading to extremely potent chemotherapeutics.

This work was supported by Grant CA73808 (NIH) and the Wisconsin Alumni Research Foundation.
Using PXR in chemical complementation to engineer new receptors to control transcription
Authors: Shaffer Hally , Doyle Donald
Published: June 2021

Chemical complementation (CC), a three‐component genetic selection system, links the presence of a small molecule to the survival of yeast through the action of a nuclear receptor (NR). CC is used to engineer receptors to activate transcription by a desired small molecule. The retinoid X receptor (RXR) is the only NR used in CC to date. PXR is an attractive NR to use in CC because it is a promiscuous receptor that activates transcription through a variety of ligands (including rifampicin and 17‐β‐estradiol). We expressed PXR in the CC system and found it is constitutively active (i.e. the yeast grow in the absence of an exogenously applied ligand) making PXR unfit for use in CC. To make PXR suitable for use, we are taking several approaches. We have engineered a yeast strain with the ERG24 steroid biosynthetic gene knocked out to remove potential endogenous sterol ligands. We are also testing PXR in mammalian cells to understand the possible mechanism of PXR function through analysis with RXR. We are investigating RXR/PXR heterodimer and PXR/PXR homodimer activation to determine the role of RXR heterodimerization in PXR activity, as well as the role of co‐repressor proteins. Funding provided by NIH.
Proteomic Profiling of Dynamic Palmitoylation
Authors: Martin Brent R. , Cravatt Benjamin F.
Published: June 2021

Protein S‐acylation is an abundant post‐translation modification required for the trafficking, compartmentalization, and membrane‐tethering of numerous key signaling proteins. Traditionally, proteins are identified as targets of palmitoylation by metabolic labeling with 3H‐palmitate, and the specific cysteine sites of palmitoylation are inferred by mutagenesis. A recent method has been developed to replace acyl‐thioesters with affinity tags for enrichment and proteomic identification in yeast, yet this relies on the fidelity of the exchange reaction and is hampered by numerous false positives. We have developed a bioorthogonal palmitoylation probe for metabolic incorporation at in vivo palmitoylation sites in cultured mammalian cells. After enrichment and trypsin digestion, peptides are analyzed using an LTQ ion‐trap mass spectrometer using multidimensional protein identification technology (MudPIT). Hundreds of palmitoylated proteins have been identified with the potential to identify the exact modified cysteines. Various cell types, stimuli and labeling times have been profiled, revealing novel sites of dynamic palmitoylation across complex proteomes. This research was funded by F32NS060559, CA087660, and the Skaggs Institute for Chemical Biology.
Expression and functional analyses of PCA‐1 (ABH3) for molecular‐targeted therapy of prostate cancer
Authors: Koike Kazuo , Tsujikawa Kazutake , Kitae Kaori , Arima Hiroshi , Suzuki Takashi , Masai Seiko , Fusamae Yasuyuki , Konishi Noboru , Yamamoto Hiroshi
Published: June 2021

Prostate cancer is the most common malignancy in men and the second leading cause of cancer death in Western countries. Many studies found out a number of genetic changes in prostate tumorigenesis, but it is not sufficient to understand the molecular mechanism of prostate tumorigenesis. We have recently identified and cloned a new gene named prostate cancer antigen‐1 (PCA‐1), which shows higher expression of both mRNA and protein in prostate cancer than in normal prostate tissue. Database analysis indicated that PCA‐1 has high similarity to Escherichia coli AlkB, a DNA and RNA alkylation damage repair enzyme, and belongs to the ABH (AlkB homologue) family as ABH3. PCA‐1 localized in cytoplasm as well as nuclei in some specimens of prostate cancer and in transfected HeLa cells, supporting that PCA‐1 might repair alkylated DNA and RNA in mammalian cells. To investigate the function of PCA‐1, we used the RNA interference technique to specifically deplete PCA‐1 mRNA in the prostate cancer cell lines DU145 and PC3. Transfection with PCA‐1 siRNAs causeda dramatic reduction in PCA‐1 at both mRNA and protein levels (to less than 20% of the levelsseen in control siRNA transfectants for 48 h culture). Interestingly, down‐regulation of PCA‐1 by siRNAs markedly inhibited cell proliferation and induced cell death by apoptosis. These results suggest that PCA‐1 may be a new molecular‐target for the treatment of human prostate cancer.
Teaching scientific literacy in an undergraduate program through a combination of reading and graded presentations
Authors: Bloom Timothy J
Published: June 2021

To be successful at communication, students must learn how to receive as well as transmit information. An important early skill in receiving scientific information is identifying the critical content of primary literature, which allows for evaluation of that content. It is also important for students to become comfortable with sharing information, particularly in the format of an oral presentation. My department uses a two‐course series to develop these information skills in undergraduate students. In the first course students learn to identify the information present in a scientific paper during small group discussions, and how to summarize that information by abstracting several papers of their choice. The students’ mastery of information identification, retrieval and transfer is assessed with an oral presentation of a scientific paper to their peers and department faculty members. Grades for the presentation are based on scoring by the faculty, the class and the presenting student after watching a video of their presentation. The second course includes two more oral presentations. Over the two semesters, student's scores for their own presentations move from higher than those of the faculty to the same as those of the faculty, suggesting that in addition to improving their communication skills they have improved their ability to accurately assess their effectiveness at oral presentations.
Executing RasMol Scripts in PyMOL
Authors: Mottarella Scott , Wischmeyer Corey , Hanson Brett , Westin Charles , Bernstein Herbert , Craig Paul A.
Published: June 2021

There are many freely available molecular visualization programs used by scientists to view macromolecules. Each application has advantages. However, each of these applications also has its own scripting language. For scientists to utilize multiple molecular visualization applications, they must learn each of those scripting languages. To unite these applications, it is necessary to create tools to interconvert these scripts according to the conventions of each program. We have created a plugin that can read scripts from RasMol and execute the equivalent commands in PyMOL. Challenges have included different sets of commands and object‐oriented vs. simple text formats. It is the goal of the Structural Biology Extensible Visualization Scripting Language project to create an interface that will enable use of scripts from one molecular visualization application in a variety of other applications. This will enable users to run multiple programs while only needing to develop expertise in the scripting language for one of those programs. The project is funded in part by NIGMS grant #1R15GM078077.
Applications of Chemotaxis in the Classroom
Authors: Burke Teresa Rose , Cruikshank William
Published: June 2021

Cells of the immune system migrate toward specific chemical signals generated by an inflammatory response. During inflammation, specific cells are responsible for secreting chemokines. Chemokines are small cytokines that promote the chemotaxis (cell migration) of specific target cells that display the corresponding protein receptors. Many of these target cells regulate the immune system by either enhancing or suppressing the reaction.

In this laboratory experience, mouse models were used to demonstrate chemotaxis of human T‐cells toward two specific chemokines, IL‐16 and MIP. Human primary lymphocytes were labeled with an internal fluorescent marker and injected into immunocompromised SCID mice. The chemoattractants were then intratracheally instilled into the lungs of the mice. After 2–4 days, lung and lymph node tissue was extracted and cell migration was analyzed using a flow cytometer.

The curriculum designed from this experiment is geared towards an Advanced Placement Biology course. The main concepts that this unit will cover are cell communication, membrane structure, protein receptors, and immune system response. Students will use column chromatography to separate an “unknown” lymphocyte concentration from an imitation human blood suspension. The Ochterlony technique will be used to analyze the composition and the chemotaxis of the “unknown” lymphocytes towards a specific chemokine, either IL‐16 or MIP. Results will be analyzed based on the formation of precipitates in the agar.
Knowledge of carbohydrate consumption does not alter exercise‐induced lymphocyte apoptosis following an acute bout of high‐intensity aerobic exercise
Authors: Navalta James Wilfred , McFarlin Brian Keith , Lyons Thomas Scott , Arnett Scott Wesley , Callahan Zachary Joseph
Published: June 2021

Carbohydrate consumption during strenuous aerobic exercise has been reported to reduce the amount of lymphocyte cell death compared to placebo. It is not known whether actual carbohydrate consumption or merely the knowledge of carbohydrate consumption mediates this alteration. The purpose of this investigation was to determine if knowledge of carbohydrate beverage could result in an alteration of apoptotic lymphocytes, independent of actual carbohydrate intake. Endurance trained males and females (N = 10) were randomly assigned to either a correct or false knowledge of carbohydrate intake. For instance in the false group, subjects were informed that they were receiving the carbohydrate beverage, but actually received the placebo beverage. Participants completed a 60‐min ride on a cycle ergometer at 80% VO2peak. Venous blood samples were collected at rest (PRE) and immediately after exercise (POST), and used to determine plasma glucose concentration and lymphocyte apoptosis. We did not find a significant effect of knowledge of drink type on lymphocyte cell death (P = 0.43). There was a significant effect of exercise on lymphocyte apoptosis (PRE = 6.3±3%, POST = 11.6±3%, P<0.01). Knowledge of drink type does not appear to alter immunity in terms of lymphocyte cell death following exercise.
TNF α and IGF‐I levels in Down Syndrome (DS)
Authors: Mehta Pankaj D , Patrick Bruce A , Dalton Arthur J
Published: June 2021

TNF α a proinflammatory cytokine, and IGF‐I, the pleiotropic hormone have been involved in the pathogenesis of neurodegenerative diseases. Persons with DS have a number of abnormalities in their immune system. Studies showed that the neuropathology of Alzheimer disease (AD)is always present in individuals with DS 40 years of age and older. We examined levels of TNFα and IGF‐I in plasma from 41 persons with DS (45±8 years old) and 28 age‐matched healthy controls. TNFα levels were higher in DS than controls (p ≤ 0001). Total IGF‐I levels were simlar between DS and controls. However free IGF‐I levels were lower in DS than controls (p ≤ 05). The combined measurements of TNFα and IGF‐I might be useful to monitor anti‐inflammatory or neurotrophic drug effects in DS. It will be useful to determine whehter the levels are useful as biomarkers to predict early signs of AD.
Elucidation of the role of chemokine receptors in the control of lung metastasis of breast cancer cells.
Authors: Biragyn Arya , Anderson Robin L , Olkhanud Purevdorj B
Published: June 2021

Chemokines regulate trafficking of cells acting through differentially expressed chemokine receptors. They have been implicated in migration of malignant cells, although their direct role in the organ metastasis remains unresolved. The major obstacle is that chemokine receptors on tumor cells are often not detectable by use of conventional surface staining techniques. We have hypothesized that this is due to the heterogeneity of cancer cells that would “hide” a minor subset of cells that could express chemokine receptors in their mist. To associate organ metastasis with expression of chemokine receptors, the hypothetical chemokine receptor‐expressing cells were depleted using chemotoxins (chemokines that specifically kill the respective receptor‐ expressing cells). As a result, we have generated a number of 4T1 clones that only differed by the loss of expression of a particular chemokine receptor. This enabled us to also demonstrate that CCR4 is the key receptor that controls lung metastasis 4T1 cells. Thus, our data not only indicate that expression of a single chemokine receptor is sufficient to induce lung metastasis, but also clearly demonstrates that organ homing pattern is only determined by the chemokine receptor‐expressing subset of cancer cells.
Endothelial CD47 interaction with SIRP‐γ is essential for T cell transendothelial migration in vitro
Authors: Stefanidakis Michael , Parkos Charles A , Luscinskas Francis W
Published: June 2021

6006‐AAI

Leukocyte transendothelial migration (TEM) is a critical event in inflammation and requires sequential action of several families of adhesion molecules. CD47 or Integrin‐Associated Protein (IAP) is a 50kD transmembrane protein that belongs to the IgSF and is highly expressed on platelets, leukocytes, endothelial and epithelial cells, and neurons. CD47 is known to bind two members of the signal regulatory protein family, SIRPα and SIRPγ. So far, little is know about the role of endothelial CD47 in T cell TEM under physiological flow conditions in vitro or in vivo. FACS and Western blot analysis show that CD3+ T cells express SIRPγ but not SIRPα. Confocal microscopy showed that CD47 was enriched at endothelial junctions, suggesting a role for CD47 in T cell TEM. To determine the role of endothelial CD47 in T cell TEM, function blocking mAb to CD47 or SIRPγ were used in an in vitro flow model of TEM. mAb to CD47 or SIRPγ inhibited T cell TEM by 65 % and 75 % (n=4), respectively, but had no effect on adhesion. Both blocking mAb also decreased T cell apical migration velocity. Strikingly, the TEM of murine T helper 1 cells through TNF‐α activated murine CD47 deficient endothelium was inhibited by 80 % (n=2). In summary, these data indicate that interactions between endothelial CD47 and SIRPγ play an important role in T cell TEM under flow conditions. Support by grants from the American Heart Association and NIH HL36028.
Chronic ethanol (EtOH) feeding delays Langerhans cell (LC) migration by altering the kinetics of chemokine receptor (CCR) and adhesion molecule (AM) expression
Authors: Parlet Corey Patrick , Schlueter Annette J.
Published: June 2021

Alcoholism is associated with increased susceptibility to and severity of microbial infection. LCs play a critical role in initiating immune responses to cutaneous infection. Using a murine model, we previously reported that migration of LCs into the regional lymph node (RLN) was delayed in mice maintained on a regimen of chronic EtOH feeding. Optimal migration of LCs from the skin to the RLN requires that LCs acquire a pro‐migratory phenotype (PMP). LCs acquire the PMP in two broad phases. First, they down‐regulate the CCRs and AMs needed for retention in the epidermis. Second, they up‐regulate other CCRs and AMs required for migration through the afferent lymphatics. To determine if EtOH alters LC migration by impeding acquisition of the PMP, epidermal cell suspensions and skin explants from age matched control and EtOH fed mice were cultured +/− the addition of pro‐migratory stimuli (e.g. CPG/CCL21) and receptor expression was evaluated by flow cytometry. Chronic EtOH feeding was found to reduce LC migration out of skin explants and delay the down‐regulation of E‐cadherin, CCR1 and CCR6. Thus, the inefficient down‐regulation of surface molecules mediating epidermal retention may provide a mechanism for the migratory defects exhibited by LCs in EtOH mice. Evaluations of EtOH induced alterations in lymphatic migration & lymphangiogenesis are in progress. This work was supported by NIH RO1 AA014405 and AA014406.
Recombinant Activated Protein C Regulates Integrin‐Mediated Neutrophil Migration
Authors: Elphick Gwendolyn F. , Chen LiiFeng , Morin Nicole A. , LeBlanc Brian , Lavigne Liz M. , Rezaie Alireza R. , Biffl Walter , Reichner Jonathan S. , Kim Minsoo
Published: June 2021

Leukocyte migration to infected tissue is necessary to control pathogenic infections. Migration is mediated through integrin/ligand interactions between immune cells and the blood vessel endothelium or tissue matrix. Migration is modulated by a host of factors present in the bloodstream and the tissues during infection, such as cytokines, chemokines, and bacterial debris. Recombinant human activated protein C (rhAPC) has been shown to modestly increase patients survival during severe sepsis, although the mechanism underlying this protective effect remains unclear. During sepsis, tissue injury results, in part, from excessive infiltration and sequestration of neutrophils in part caused by dysregulation of cell surface adhesion molecules such as integrins. We show that rhAPC binds directly to β1 and β3 integrins and inhibits human neutrophil migration. We found that human APC possesses a RGD sequence, critical for intergrin interactions. Mutation of this sequence (RGD to RGE) abolished integrin binding and inhibition of neutrophil migration. Thus, rhAPC decreases neutrophil recruitment into tissues by blocking neutrophil integrins, providing a potential mechanism by which rhAPC may protect from sepsis. These findings point to a novel target through which neutrophil migration is effected.
Characterization of a NZM2328 derived recombinant congenic strain which has an 8Mb C57L/J fragment on distal chromosome 1
Authors: Ge Yan , Jiang Chao , Morris Amy , Gaskin Felicia , Sung Sun‐Sang , Fu Shu Man
Published: June 2021

NZM2328 mice develop severe proteinuria and glomerulonephritis (GN) with a gender bias towards females and are used as a spontaneous murine SLE model. Previously, a recombinant line NZM2328.C57L/Jc1 was characterized in which a 26Mb C57L/J chr. 1 fragment was introgressed onto NZM2328. These female mice have no ANA, anti‐dsDNA Ab's, or GN. We have further generated a novel recombinant congenic strain R27, in which an 8Mb C57L/J chr. 1 fragment was introgressed onto NZM2328 background. Severe proteinuria and ANA were inhibited in R27 female mice at 12 months of age. However, R27 sera stained normal kidney sections, indicating the presence of Ab's against the kidney. Immune complex (IC) deposits were detected in kidneys of 12‐month‐old as well as 5‐month‐old R27 female mice. Kidney histology showed acute GN without chronic GN. In addition, mononuclear cell infiltration into glomeruli was detected. These results support the hypothesis that acute GN need not proceed to chronic GN. Acute and chronic GN may be caused by two distinct mechanisms. Also, it is apparent that autoantibodies other than those for dsDNA are responsible for the IC deposits. Thus, R27 strain is a valuable model to study the pathogenesis and genetics of SLE, suggesting that some of the genes within this 8Mb region may be important for resistance to end organ damage and that these genes may be distinct from those promoting innate and adaptive immunity.
Polymorphisms in murine histamine receptor H1 leading to differential cell surface expression influence autoimmune disease progression
Authors: Noubade Rajkumar , Saligrama Naresha , Spach Karen M , Rio Roxana del , Kantidakis Theodoros , Milligan Graeme , Rincon Mercedes Rincon , Teuscher Cory
Published: June 2021

Structural polymorphisms (L263P, M313V and S331P) in the third intracellular loop of the murine histamine receptor H1 (H1R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis (EAE) and experimental allergic orchitis (EAO). The P‐V‐P haplotype is associated with increased disease susceptibility (H1RS) whereas the L‐M‐S haplotype is associated with less severe disease (H1RR). Here we show that selective reexpression of the H1RS allele in T cells fully complements EAE susceptibility and production of disease associated cytokines while selective reexpression of the H1RR allele does not. Mechanistically, we show that the two H1R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H1RR allele being retained within the endoplasmic reticulum (ER). Moreover, we show that all three residues (L‐M‐S) comprising the H1RR haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G‐protein couple receptor in T cells regulates immune functions and autoimmune disease susceptibility.
The Role of IL‐21 In Lymphopenia‐Induced Homeostatic Proliferation
Authors: Datta Shrimati , Sarvetnick Nora
Published: June 2021

Non‐obese diabetic (NOD) mice have been characterized as lymphopenic and undergo homeostatic proliferation, leading to the generation of auto‐reactive T cells. In addition, previous work has shown that NOD mice have increased expression of the common γ‐binding cytokine, IL‐21, and its receptor, IL‐21R. To investigate whether IL‐21 is responsible for inducing the development and progression of diabetes, we generated IL‐21R knockout NOD mice. Our data indicate that NOD mice lacking IL‐21R are protected from disease. Further analyses show that IL‐21R knockout NOD mice are not lymphopenic and exhibit decreased homeostatic proliferation. In addition, IL‐21R knockout NOD mice support increased T cell survival and reduced T cell effector function. Our results suggest IL‐21 produces an unstable T cell population that drives lymphopenia and homeostatic expansion, generating autoimmunity. Interestingly, non‐autoimmune mice deficient in IL‐21R have an increased lymphoid pool size associated with decreased homeostatic proliferation, increased T cell survival and an increased resting T cell population. Taken together, our results suggest IL‐21 regulates T cell homeostasis in autoimmune as well as normal mice. Based on our study, IL‐21 blockade could represent an important therapeutic target to control T cell survival and homeostatic expansion, leading to novel treatment strategies for autoimmune diseases.
Expansion of a unique T cell subset Th40 describes a commonality in type 1 diabetes and MS
Authors: Wagner David H , Waid Dan M , Vaitaitis Gisela M , Pennock Nathan D , Wagner Becky , Corboy John , Gottlieb Peter
Published: June 2021

We have described a unique T cell subset defined by CD4loand CD40 expression (Th40) that is significantly expanded in peripheral blood of T1D but not control or T2D subjects. Because Th40 cells may be a sub‐population where pathogenic T cells may arise, we examined Th40 cells in other disease models including Multiple Sclerosis (MS). Etiology of the autoimmunity in animal models suggests a prominent role for CD8+ and CD4+ T cells. We compared Th40 cells from T1D, MS, T2D and non‐autoimmune controls. Th40 levels from T1D subjects were significantly (p = 10−17) elevated compared to T2D or non‐autoimmune controls. Interestingly as in T1D, Th40 levels were significantly elevated in MS subjects compared to controls including healthy siblings. Th40 expansion occurs in T1D subjects carrying the high risk HLA DR3 or DR4 haplotypes but, T1D subjects who do not carry either DR3 or DR4 haplotypes still have an expanded percentage of Th40 cells. A portion of Th40 cells from T1D subjects proliferates when exposed to diabetes‐associated self‐antigens. Th40 cells from T1D produce pro‐inflammatory cytokines including IFNγ, TNFα and IL‐6. When Th1‐associated chemokine receptors, CXCR3 and CCR5 are examined, Th40 cells from T1D preferentially express CXCR3, while Th40 cells from MS subjects do not. In contrast, T1D Th40 cells do not express CCR5 but Th40 cells from non‐autoimmune subjects do. These data demonstrate demonstrable differences in Th40 cells between autoimmune and non‐autoimmune subjects and importantly within distinct autoimmune diseases.
Characterization of Islet Infiltrating Lymphocytes in NOD mice
Authors: Young Ellen , Hess Paul , Arnold Larry , Tisch Roland , Frelinger Jeffrey
Published: June 2021

Nonobese diabetic (NOD) mice are a major animal model for the study of Type 1 diabetes (TID). However, the lymphocytes infiltrating the islets are not well characterized because of low cell numbers. A detailed analysis of the islet infiltrating lymphocytes was carried out using 9 color flow cytometry. Interestingly, up to 30% of the CD4+ T cells and 40% of the CD8+ T cells infiltrating the islets were CD62Lhi and CD44lo indicating a naïve phenotype. In contrast, islet infiltrating CD8+ T cells with specificity for islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein (IGRP) had an effector phenotype. As the number of CD4+T cells per islet increased the relative number of CD8+ T cells also increased. The percentage of naïve CD4+T cells increased from less than 10% to 30% of the CD4+ T cell population and the percentage of IL‐4+ CD4+ T cells fell, although the number per islet increased linearly with the increase in CD4+ T cells per islet. We concluded that infiltrates with the same number of cells per islet had the same composition regardless of source, i.e. male or female NOD mice. There was a significant difference in the time to reach the same cell number, with females accumulating cells at an earlier age.

Research support ‐ NIAID
Different clinical course of experimental autoimmune encephalomyelitis (EAE) in neurointermediate pituitary lobectomized (NIL) plus adrenalectomized (ADX) Lewis and Wistar rats
Authors: Quintanar‐Stephano Andrés , Organista‐Esparza Alejandro , González‐Delgado Evangelina , de Jesús García‐Gallegos Diego , Campos‐Tenorio Beatríz , Reyes‐Echeverría María Cruz , Kovacs Kalman , Berczi Istvan
Published: June 2021

We have described that NIL Lewis rats develop mild signs of acute EAE with no changes in the HPA axis activity. Lewis and Wistar rats develop EAE, but differences in the HPA axis response to EAE exist between them. This suggests a different strain‐specific role of corticosterone (CO) in the immunoregulation of the EAE. Here we study the effects of CO and neurohypophyseal (NH) hormones deficiency (NIL) on the EAE. Rats from both strains were sham operated (SHAM) or NIL. Three weeks after surgeries rats were immunized with an encephalitogenic emulsion, and clinical course of EAE registered before and after ADX (performed at 27 day post‐immunization (DPI)). SHAM animals of both strains developed similar transient paralytic scores of EAE (10–20 DPI). In both strains, NIL induced decreased scores of the EAE. In Wistar SHAM ADX and NIL ADX groups no relapses of EAE occurred, whereas in Lewis SHAM ADX and NIL ADX groups a prompt and worse relapse of the EAE occurred, similar to the observed in the acute paralysis.

Conclusions: 1) In NIL Wistar and Lewis groups, decreased severity of the EAE is under the NH hormones control, 2) In Wistar rats CO‐independent mechanisms protect against the EAE relapses, 3) In Lewis rats CO is required to protect against the EAE relapses, 4) Although NH hormones and CO participate in the immunopathology of the EAE it seems that they work through different mechanisms.
CTLA‐4 expression regulation and its functions in systemic lupus erythematosus (SLE)
Authors: Zhang Zheng , Kyttaris Vasilieos C , Zhu Chen , Wu Henry Y , Tsokos George C
Published: June 2021

Cytotoxic T lymphocyte Antigen 4 (CTLA‐4) is an important inhibitory signaling molecule of T cell. There are 4 splice variants in human and mice suggesting its functional importance. We characterized the CTLA‐4 splice variant expression regulation and their biological significance in both human and mouse SLE T cells. The residual expression of the full length (FL) CTLA‐4 mRNA was increased in resting T cells of SLE patients (p < 0.01) and lupus‐prone mouse strains MRL/lpr and C57B6/lpr. However, following activation, the peak expression of FL CTLA‐4 mRNA was lower than control T cells. Additionally, mRNA degradation increased by 2.7 fold in human SLE T cells compare to healthy T cells. Furthermore, we found a 2.4 fold increase in T cells of MRL/lpr compared to control T cells of MPJ mice. The short splice variant of CTLA‐4 (encoding exons 1 and 4) showed exactly opposite kinetics than the FL CTLA‐4. It was decreased in resting T cell and displayed decreased mRNA degradation. Protein expression pattern was the same as mRNA expression for both splice variants. These findings suggest that CTLA‐4 short splice variant may act differently to the FL CTLA‐4 in SLE T cell activation. This inverse relationship between short splice variant and FL CTLA‐4 may have a profound effect on effector CD4+ T cell activation and signaling.
Monocytes differentiate into osteoclasts in the presence of RANKL and M‐CSF expressing natural killer cells
Authors: Soderstrom Kalle , Comenero Paula , Purath Ulrich , Müller‐Ladner Ulf , Zhang Angel L , Tarner Ingo H , Engelman Ed
Published: June 2021

Osteoclasts are responsible for physiological bone resorption. These cells develop from monocytic precursor cells in the presence of macrophage colony stimulating factor (M‐CSF) and receptor activator of NF‐kappa‐B ligand (RANKL). Pathologic bone resorption such as that seen adjacent to inflamed joints of patients with rheumatoid arthritis (RA) is also due to the action of osteoclasts but the mechanism responsible for the appearance of osteoclast at this site is uncertain. We show here that natural killer (NK) cells derived from the synovial fluid RA patients, as well as CD56bright NK cells present in PB of healthy donors, express RANKL and M‐CSF and induce osteoclast formation from monocytes. This process is dependent on cell contact and IL15, an abundant cytokine in RA joints that upregulates the expression of RANKL and M‐CSF on NK cells. These results indicate that NK cells may play a critical role in the destruction of bone associated with inflammation.
Isoaspartyl Posttranslational Modification In Drug‐Induced Lupus
Authors: Yang Mei‐Ling , Mamula Mark
Published: June 2021

Autoimmune responses, such as those in lupus, rheumatoid arthritis, and multiple sclerosis, frequently target self proteins that undergo post‐translational protein modifications. We have previously identified one modification, termed isoaspartyl (isoAsp), that triggers autoimmunity in murine models of lupus. Isoaspartyl modification is a non‐enzymatic, spontaneous event that occurs at physiologic pH and temperature in all cells. We have demonstrated that intracellular levels of isoAsp modified proteins are significantly higher and accumulate with age in spontaneous MRL lupus‐prone mice. IsoAsp modification contributes to T cell hyperproliferation via the Erk 1/2 pathway and is coincident with the onset of autoantibodies and pathology in the MRL mouse. Within cells, aberrant isoAsp self‐proteins are normally repaired by protein L‐isoaspartyl methyltransferase (PIMT) via methylation reactions that utilize S‐adenosyl‐methionine (SAM) as a methyl donor. Our recent studies suggest that isoAsp modification may also be important in the onset of drug‐induced lupus. The well‐known lupus‐inducing drugs, procainamide and hydralazine, both are inhibitors of DNA methylation similarly utilize SAM in the cell. We examined the effects of lupus‐inducing drugs on PIMT repair, isoaspartyl protein accumulation, and lymphocyte functions. We now demonstrate that PIMT repair activity is inhibited by hydralazine in dose‐dependent manner leading to increased isoAsp protein modification and T cell hyperproliferation similar to that observed in the MRL mouse. These data suggest that the balance between isoAsp formation and its repair by PIMT is also relevant to T cell function in drug‐induced lupus. [Grant support: Arthritis Foundation Postdoctoral Fellowship (MLY) and NIH‐AI‐48120 (MJM)]
New triggers for autoimmunity: Novel lipids from commensal bacteria promote autoimmune disease
Authors: Housley William , Nichols Frank , Wohlfert Elizabeth A. , Wu Shuang , Clark Robert B.
Published: June 2021

Autoimmune diseases can result from an imbalance between autoreactive T cells and regulatory mechanisms, but the factors that trigger this imbalance are unclear. While infections with pathogenic bacteria have been suggested as promoting autoimmunity, this concept is controversial. In contrast, we now propose that a unique class of dihydroceramide lipids derived from commensal bacteria can be such a trigger. These lipids, distinct from LPS, are produced by the gingival bacteria P. gingivalis and by GI and vaginal tract bacteria. We have previously identified these lipids in human carotid atheromata, suggesting a role in human inflammatory processes. We now report that in experimental autoimmune encephalomyelitis, the murine model of MS, these lipids significantly enhance the severity of both primary and T cell clone‐mediated disease even when administered in very small quantities (250 ng−2.5 ug) and at sites distant from both the sites of immunization and inflammation. Mechanistically, one component of these lipids acts as a TLR‐2 ligand inducing dendritic cell activation and IL‐6 production. Our findings suggest that novel lipids from commensal bacteria may be newly identified triggers promoting the onset and severity of autoimmune diseases and may be targets for therapeutic intervention. (National MS Society, #PP1322).
MicroRNAs Mediate Autoimmune Disease Pathology
Authors: Smith Kristen , Liu Chang‐Gong , Williams Jessica , Kithcart Aaron , Gienapp Ingrid , Shawler Todd , Song Fei , Whitacre Caroline
Published: June 2021

Experimental autoimmune encephalomyelitis (EAE), studied as a model for multiple sclerosis (MS), is mediated by autoreactive CD4+ T cells. MicroRNAs (miRNAs) are a class of small, non‐coding RNAs that regulate gene expression posttranscriptionally. Aberrant expression of miRNAs has been implicated in various disease processes. Recent evidence indicates that miRNAs are central to immune function and play a role in T cell responsiveness to antigen stimulation. We hypothesized that certain miRNAs would be differentially expressed in mice with EAE, and that these miRNAs represent critical mediators of autoimmune disease pathology. We utilized microarray technology, followed by PCR verification, to evaluate miRNA expression level at varying times during the EAE disease course. RNA was obtained from the lymph nodes and spleen during the acute and effector phases of EAE, and compared to animals receiving adjuvant alone. We found that several miRNAs are up‐ and downregulated during EAE and expression is temporally distinct. These data indicate that miRNAs represent a critical component of EAE pathology, and may be useful therapeutic targets or biomarkers in MS and other autoimmune diseases. Supported by NIH grants T32 GM068412, NS 48316 and AI 064320.
Brief exercise increases circulating myeloperoxidase in boys with maturational influences observed at baseline
Authors: Schwindt Christina Danell , Zaldivar Frank , Cooper Dan
Published: June 2021

Rationale: Circulating myeloperoxidase (cMPO) is elevated in asthmatics suggesting leukocytes are inordinately activated in these individuals and may play a role in wheezing. Intriguingly, MPO is increased in response to exercise in non‐asthmatic adults, and exercise is a major trigger for bronchoconstriction in asthmatics. We hypothesized that exercise would increase cMPO in children and the response to exercise would be greater in asthmatics.

Methods: Thirty‐one boys [9‐asthmatic late pubertal (AL), mean age 16.1 years; 8‐asthmatic early pubertal (AE), mean age 10.6 years; and 14 healthy control late pubertal (CL), mean age 17.0 years] performed 6–8 minutes of moderate exercise on a cycle ergometer. Blood was sampled at pre‐exercise (PE) and end‐exercise (EE).

Results: CMPO did not differ between groups at PE (AL 8.0±1.6 ng/mL, AE 9.8±1.7 ng/mL, and CL 5.8±1.3 ng/mL); however, in 3 asthmatics PE cMPO was >2SD above normal. All groups demonstrated a significant increase from PE to EE [AE Δ =3.9±1.5 ng/mL, p=0.013; AL Δ =4.5±1.4 ng/mL, p=0.0032; and CL Δ =5.0±1.1 ng/mL, p=0.0001] without a significant difference among groups. There was no correlation (|r|<0.4) in PE cMPO or ΔcMPO to leukocytes, lung function, or fitness (BMI%, max VO2, VO2/kg). There was a significant negative correlation (r=0.54, p<0.02) between PE cMPO and age in controls.

Conclusions: Baseline cMPO may be influenced by maturational status. Brief exercise led to significant increases in cMPO in all children. The magnitude of difference was not related to asthma. We speculate that exercise‐induced asthma may result from an exaggerated response to leukocyte activation that occurs normally in children even with brief exercise.
A critical role for VEGF secreted by Dendritic cells (DCs) in priming T helper 2 (Th2) development in response to specific stimuli
Authors: Krishnamoorthy Nandini , Oriss Timothy , Paglia Melissa , Fei Mingjian , Ray Anuradha , Ray Prabir
Published: June 2021

Asthma is an inflammatory disease of the small airways caused by an exaggerated CD4 Th2 response. We have shown that OVA coupled with cholera toxin (CT) when administered intranasally into mice recapitulate the hallmarks of asthma. In vitro, CT ‐treated Dcs prime naïve T cells towards a Th2 response. We undertook a microarray approach to dissect the mechanism of CT‐ induced Th2 response. Our microarray data revealed upregulation of expression of vascular endothelial growth factor (VEGF) by CT in DCs. Recently, a novel role for VEGF as a mediator of Th2 immune responses has been reported. In accordance with our microarray data, CT induced secretion of VEGF in tissue and bone marrow DCs. Interestingly, we have found that allergens selectively induce VEGF expression in DCs despite their common ability to promote allergic diseases. Aspergillus fumigatus, the causative agent of allergic bronchopulmonary aspergillosis, induced VEGF production from DCs but house dust mite did not. We also found that VEGF secreted by DCs was critical for the differentiation of Th2 cells. We show that iNOS and NF‐kappa B enhance the production of VEGF from DCs, in contrast to inhibition by IL‐12. We also show the Th2‐ priming ability of DCs to be compromised in iNOS−/−mice. In summary, our study underscores the complexity of Th2 responses by highlighting a previously unknown but essential pathway via DC‐derived VEGF in response to specific stimuli.