bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of gelatin on proliferation of cord blood derived mesenchymal stem cells
Authors: Kwack KyuBum , Won SeongSik , Cha Yun‐Yi , Park Soo‐Jeong , Kim Hyun Jae , Lee Seung Ku , Kang HyunJun
Published: June 2021

Mesenchymal stem cells (MSCs) are isolated from various tissues including bone marrow (BM), placenta, adipose tissue, and umbilical cord blood (CB). Compared to MSC isolated from BM (BM‐MSC), MSC from cord blood (CB‐MSC) has a short logarithmic growth stage before entering the senescence stage in vitro long‐term culture. Gelatin coated dishes are used to culture for embryonic stem cells, primary cell and clones. To determine whether the usage of gelatin‐coated dishes gave superior effects on efficient isolation MSC from CB and longer proliferation of CB‐MSC in vitro, we cultured mononuclear cells (MNC) isolated from human CB with gelatin‐coated or uncoated dishes. In the results, CB‐MSC in gelatin‐coated dishes revealed higher and longer proliferation than those in uncoated dishes. To unveil the mechanism, we compared gene expression profiles of MSC cultured in gelatin‐coated and uncoated dishes using the microarray technology. Many of genes involved in adhesion molecule induced signaling pathway were identified. Our findings suggest that gelatin‐derived signaling may contribute to the expended proliferation and maintenance of CB‐MSC and confer the chance to study what signals involved to CB‐MSC proliferation.
Imaging the cell motions, lineages and interactions that build embryos
Authors: Fraser Scott E.
Published: June 2021

Reductionistic approaches have yielded unprecedented knowledge of the components involved biological processes, and present the challenge of integrating this into a complete understanding. For example, the revolution in molecular biology has yielded dramatic new insights into the genes and gene products that might guide embryonic development. To answer the basic question of how an embryo develops, we must determine how these molecular processes are assembled into the working macroscopic entities we call organisms. Biological imaging provides a natural solution to such challenges, but must resolve several competing demands, including: the high resolution needed to track single cells; the challenge of imaging cells in their normal positions in vivo; the need for true volumetric imaging; the wide field of view needed to place these images in context. No single technique has offered the needed combination of attributes, making our present challenge to integrate data from different modalities. I will draw on new findings from the Biological Imaging Center using intravital imaging to illustrate the current state of our misunderstandings and of the imaging technologies that may resolve them.
DNA Damage Induced Inhibition of RNA Polymerase II and the Contributions of BRCA1
Authors: Heine George F , Horwitz Andrew A , Parvin Jeffrey D
Published: June 2021

Cellular DNA damage elicits the phosphorylation and ubiquitination of RNA polymerase (RNAPII), leading to the global repression of transcription. Here we show that there are at least two different pathways to transcriptional repression, depending on the type of DNA damage. After H2O2 treatment, transcription was rapidly inhibited and rapidly restored. On the other hand, UV treatment caused a much slower transcriptional inhibition, with a corresponding depletion of unphosphorylated RNAPII. We found that after UV treatment, but not treatment with H2O2, the inhibition of transcription was dependent on both the proteasome and new protein synthesis. In addition, RNAPII activity and ubiquitination were regulated through the phosphorylation of RNAPII by the P‐TEFb kinase. Furthermore, we show that BRCA1 is associated with the ubiquitination of RNAPII in response to UV induced DNA damage. These results highlight that multiple cellular pathways exist to globally repress transcriptional processes that might interfere with the repair of DNA damage.
Role of Integrins in Angiotensin II‐induced Proliferation of Vascular Smooth Muscle Cells
Authors: Bunni Marlene A , Kramarenko Inga I , Walker Linda , Raymond John R , Garnovskaya Maria N
Published: June 2021

Angiotensin II (AII) binds to G protein‐coupled receptor (GPCR) AT1 and stimulates extracellular signal‐regulated kinase (ERK), leading to vascular smooth muscle cells (VSMC) proliferation. Proliferation of mammalian cells is tightly regulated by adhesion to the extracellular matrix (ECM), which occurs via integrins.

To study a crosstalk between GPCR‐ and integrin‐induced signaling, we hypothesized that integrins are involved in AII‐induced proliferation of VSMC. Using quantitative RT‐PCR, we established that messages for α1, α5, αv and β1 integrins are predominant in VSMC. VSMC were cultured on plates coated with ECM or poly‐D‐lysine (which promotes electrostatic cell attachment). AII significantly induced an ERK‐dependent proliferation only in VSMC grown on collagen or fibronectin. VSMC grown on ECM demonstrated ∼400% increase in AII‐induced ERK phosphorylation, whereas VSMC grown on poly‐D‐lysine showed only minor (∼30%) ERK activation, supporting the importance of integrin‐mediated adhesion. AII‐induced ERK activation was reduced by ∼60% by RGD peptides that inhibit α5β1 integrin, and by ∼40% by the KTS peptide specific for integrin α1β1. Silencing of α1, α5, and β1 expression by transfecting VSMC with siRNAs also resulted in decreased AII‐induced ERK activation.

These data demonstrate specific roles for integrins (most likely α5β1 and α1β1) in AII‐induced proliferation of VSMC.
The TCF‐4 signaling pathway negatively regulates HIF‐1a transcriptional activity in response to hypoxia
Authors: Hao Wei , Chou Chih‐Ling , Xu Wei , Israel Davelene , Hutchinson Anthony , Ji Ruyue , Regan John W.
Published: June 2021

Hypoxia inducible factor‐1α (HIF‐1α) plays an important role in cancer by helping cells survive under low oxygen conditions through the up‐regulation of genes that promote angiogenesis. Dysregulation of the β‐catenin/T‐cell factor (TCF) signaling pathway is also known to occur in several cancers; however, the relationship between up‐regulation of HIF‐1α and activation of TCF signaling has not been previously explored. The present study examines this relationship in a model system using RWPE‐1 prostate cells. HIF‐1α transcriptional activity was assessed using a luciferase reporter gene containing an upstream hypoxia response element (HRE). Cells were incubated under hypoxic conditions (1% O2, 5% CO2, 94% N2) for 24 hr and HRE reporter gene activity was assessed of either alone or following pre‐treatment with a dominant negative mutant of TCF‐4 (DN‐TCF‐4). In response to hypoxia, HRE reporter gene activity was increased relative to cells maintained under normoxic conditions. In cells pre‐treated with DN‐TCF‐4, HRE reporter gene activity was further increased in response to hypoxia as compared with cells exposed to hypoxia alone. These findings suggest that the TCF signaling pathway can negatively regulate the induction of HIF‐1α in cells exposed to hypoxia, which may be of significance to understanding the up‐regulation of HIF‐1 α that occurs in cancer. Supported by EY 11291.
Type 5 Adenylyl Cyclase Disruption Increases Longevity, Food Intake and Exercise Capacity
Authors: Dillinger Jean‐Guillaume , Williams Jeffrey G , Serpillon Sabrina , Barrows Brian , Reinhardt Heather , Ghaleh Bijan , Hittinger Luc , Chess David , Stanely William C , Hintze Thomas H , Vatner Dorothy E , Sadoshima Junichi , Vatner Stephen F
Published: June 2021

Adenylyl cyclase (AC) is the effective enzyme in the beta‐adrenergic receptor signaling cascade coupled with the G‐protein. AC type 5 knockout mouse (AC5KO) is a new mouse model of longevity with increased median lifespan of ~30%. Caloric restriction (CR) is the most established mechanism of longevity, which occurs through modifications in metabolism. We examined these features in AC5KO mice determining their metabolic rate at rest and during exercise. Male AC5KO mice (n=6) and wild type (WT) mice (n=6) were exercised on a treadmill. Food intake was greater in AC5KO than WT, 3.7±0.1 vs 3.1±0.1g/day (p<0.05), respectively, whereas their body weight (BW) tended to be lower (29±1 vs 33±2g, p<0.05). Oxygen consumption (VO2)and CO2 production (VCO2) were similar in both groups (56±2 vs 51±3ml/kg/min; 43±2 vs 38±3ml/kg/min). AC5KO mice ran significantly longer (834±107 vs 578±42m; p=0.05) and performed more work than WT (38±3 vs 26±2m.kg; p<0.05). RERmax, an indicator of metabolic substrate utilization, decreased in the AC5KO group (0.92±0.02 vs 0.96±0.02; p<0.05). Thus, AC5KO not only results in increased longevity, but also enhanced exercise performance. This occurs in the face of increased food intake, and reduced body weight, suggesting that a common feature of this model of longevity and the CR model is an alteration in metabolism.
Activation of Apoptotic Markers by Inositol Polyphosphates in MC3T3 Cells
Authors: Agarwal Rakhee , Ali Nawab
Published: June 2021

Exogenous InsPs inhibit cellular growth by inducing apoptosis in certain cancer cell lines. Cellular levels of higher InsPs change during apoptosis. However, the molecular mechanism of action of InsPs is not well known. We studied InsPs induced apoptosis and its signaling pathways in mouse osteoblastic MC3T3 cells. Exogenously added InsP6, InsP5 and InsP4 dose dependently induced apoptosis. InsP6 was more potent than InsP5 and InsP4. We used NaF and antimycin A to change cellular levels of InsPs. NaF is known to increase higher InsPs by inhibiting InsP phosphatase, a process that is reversible by antimycin A by inhibiting InsP kinase as a result of depletiion of cellular ATP. NaF dose and time dependently induced apoptosis; 50% apoptosis was at 1 mM NaF in 8 hrs. Prior treatment with antimycin A significantly reduced NaF‐induced apoptosis indicating that NaF induced apoptosis could be due to the increased levels of InsPs. NaF also activated caspase‐3, caspase‐9, and phosphorylated Akt. These effects were reduced by prior treatment with antimycin A. Further, NaF and etoposide affected cellular levels of InsPs;InsP1 and InsP2 levels slightly increased, whereas InsP3 and InsP4 levels drastically decreased indicating that inositol polyphosphates play an important role in apoptosis. Supported by ASGC and Kathleen Thompson Foundation.
Regulation of Human Inducible Nitric Oxide Synthase by Protein‐protein Interaction
Authors: Rashid Mohammad Bazlur , Challawala Amama , Chakravarty Deepavali , Curtis Leigh , Eissa N Tony
Published: June 2021

Nitric oxide (NO) is a potent cell‐signaling molecule that plays important roles in diverse range biological processes within various tissues. Almost in every cell of our body, NO is generated by certain enzymes called nitric oxide synthases (NOS). NO is synthesized constitutively in nervous systems and endothelial cells, whereas in some other cells NO production is induced. Excessive NO production is linked to carcinogenesis, septic shock, asthma, stroke, etc. On the other hand, insufficient NO is involved with hypertension, impotence, arteriosclerosis, etc. So, there are dual roles for NO molecule at different tissue level. Since three isoforms of NOS responsible for NO production, an isoform‐specific inhibitor(s) needs to be designed that would not interfere with another NOS. In this regard, we have employed BD‐Clontech made Yeast Two‐Hybrid System to identify the putative proteins that would specifically bind iNOS protein for regulation. An N‐terminal iNOS‐bait protein has been utilized to identify a mammalian cDNA library. Employing all sorts of negative and positive controls necessary to interpret Yeast Two‐hybrid data, we could identify a number protein that shows possible protein‐protein interaction with iNOS. The probable role of these proteins in the regulation or modulation of iNOS function will be discussed.
Estimating the magnitude of PDE4 activity near the plasma membrane of HEK‐293 cells
Authors: Rich Thomas C , Zhu Bing , Xin Wenkuan
Published: June 2021

The importance of subcellular localization of phosphodiesterases (PDEs) has been clearly demonstrated. However, with the exception of sensory neurons, little is known about the subcellular concentrations of these enzymes. We have previously used cyclic nucleotide‐gated (CNG) channels as biosensors to measure near‐membrane cAMP signals. We have used this approach to demonstrate that PDE4 is primarily responsible for the hydrolysis of cAMP near the plasma membrane of HEK‐293 cells. Here, we use a similar approach in addition to measurements of total cellular PDE activity and mathematical modeling to estimate the concentration of PDE4 near the plasma membrane. Currents through CNG channels were measured in order to monitor the time‐course and magnitude of cAMP dialysis from the whole‐cell patch pipette to the plasma membrane. We found that PDE4 inhibitors had a profound effect on the magnitude of cAMP‐induced currents at cAMP levels ranging from 2 to 200 μM. In separate experiments, we estimated that PDE4 was responsible for ∼50% of the total cAMP PDE activity, with a maximal hydrolysis rate of ∼0.5 μM/s and a Km of ∼3 μM. We are using these data to constrain our previously described compartmental model (Rich et al., J. Gen. Physiol., 2000, 116:147), and estimate the magnitude of PDE4 activity in the near‐membrane compartment. This work was supported by NIH awards R01HL074278 and K02HL081463.
Glycyrrhizin Induces Apoptosis In Prostate Cancer Cell lines DU‐145 and LNCaP
Authors: Munirathinam Gnanasekar , Sivasakthivel Thirugnanam , Xu Lipeng , Kalyanasundaram Ramaswamy
Published: June 2021

Over 2 million Americans are currently living with prostate cancer. Current treatment strategies are inefficient in controlling the disease. This has made it necessary to identify newer strategies for treating prostate cancer. Use of active principles from medically important herbs has proven to be effective in treating various forms of cancers. Glycyrrhizin, a triterpene compound isolated from roots of licorice is shown to exhibit potent in vitro cytotoxic activity against several human cancer cell lines. No information is available for prostate cancer. Therefore, in this study, we evaluated the effects of glycyrrhizin on the in vitro viability of two human prostate cancer cell lines LNCaP (hormone‐dependent) and DU‐145 (hormone‐independent). Cell viability assay using MTT showed that Glycyrrhizin inhibited prostate cancer cell proliferation in a time and dose dependent manner. The decreased viability of prostate cancer cells was found to be due to cell death. Annexin‐V FITC staining and flow cytometry confirmed significant (P<0.01) numbers of prostate cancer cells were undergoing apoptosis following exposure to glycyrrhizin compared to untreated cells. Immunocytochemistry also confirmed significant Annexin‐V staining on the surface of the glycyrrhizin‐treated cancer cells. Subsequent studies showed that glycyrrhizin also inducedDNA damages in both DU145 and LNCaP cells in a time dependent manner. In addition, caspase‐3 and caspase ‐8 activities were also significantly elevated in glycyrrhizin treated prostate cancer cells compared to untreated control cells. Taken together, these studies suggest that glycyrrhizin is a potent inducer of apoptosis in prostate cancer cells as reported with other cancer cells. This study is funded by EAM.
Structure‐based inhibitor design of AccD5/AccD6, essential carboxyltransferases of Mycobacterium tuberculosis
Authors: Lin Ting‐Wan , Nguyen Tan , Kurth Daniel , Swamidass Joshua , Baldi Pierre , Gramajo Hugo , Tsai Sheryl
Published: June 2021

Mycolic acids and multimethyl‐branched fatty acids are found uniquely in the cell envelope, which are essential for the survival, virulence and antibiotic resistance of Mycobacterium tuberculosis. Acyl‐CoA carboxylases (ACCases) commit acyl‐CoAs to the biosynthesis of these unique fatty acids. Unlike other organisms, Mycobacterium tuberculosis contains six ACCase carboxyltransferase domains, AccD1‐6. Previous studies indicate that AccD4, AccD5 and AccD6 are important for cell envelope lipid biosynthesis and its disruption leads to pathogen death. We have determined the structures of AccD5 and AccD6, whose sequences, structures, and active site are highly conserved to each other. However, both crystals structure and kinetic assays indicate that AccD5 prefers propionyl‐CoA as its substrate and produces methylmalonyl‐CoA. Contrary to AccD5, AccD6 prefers acetyl‐CoA (versus propiony‐CoA) to generate malonyl‐CoA, the extender unit for fatty acids. We also conduct extensively in‐silico screening and identify inhibitors NCI‐65828 (Ki=13 μ M for AccD5) and NCI‐172033 (Ki=1.8 μ M for AccD6). Moreover, NCI‐172033 also inhibited growth of several pathogenic species of Mycobacterium, including MDR‐TB. Our results pave the first step towards understanding the biological roles of key ACCases, in addition to providing a new target for structure‐based development of anti‐tuberculosis therapeutics.
Physical models of bacteriophage lytic enzymes: 2008 Pingry School S.M.A.R.T. Team Project
Authors: Hata Tommie Shintaro , Fischetti Vincent , Herman Tim , Ciriello Jonathan , Cordrey Mac , Cosentino Danielle , Jones Calvin , Krakora Becky , Lisko Bozhena , Nabar Yamini , Pinke Will , Srivastava Neha
Published: June 2021

The 2008 Pingry School S.M.A.R.T. Team (Students Modeling A Research Topic) has been working with the Fischetti laboratory at The Rockefeller University to design and produce accurate, three‐dimensional physical models of bacteriophage lysins using rapid prototyping (RP) technology. Lysins are bacterial cell wall hydrolases used by bacteriophage during progeny release. These enzymes in their pure form can be applied directly to Gram‐positive bacteria to quickly induce hypotonic lysis from without. This rapid activity shows promise as a novel antibacterial agent to both prevent and treat infections. Discussions with members of the Fischetti laboratory allowed the students to use RP‐RasMol to design models of lysins to highlight their structural and functional characteristics. These designs were used to direct RP machines to build physical models of these enzymes. Along with a Jmol web tutorial (www.pingrysmartteam.com), these physical models serve as “communication tools” used to enhance the understanding of lysins and their applications among the scientific and academic community. By contributing this new tool to the Fischetti laboratory research team, the students have the unique opportunity to experience and participate in the activities of a research laboratory. This work is supported by a grant awarded to Tim Herman by the NIH NCRR SEPA program and the HHMI Precollege Science Education Program.
Molecular Analysis of T‐lineage Commitment: a possible role for Bcl11b
Authors: Li Long , Rothenberg Ellen
Published: June 2021

Hematopoietic stem cells (HSC) commit to the T‐lineage by gradually losing developmental plasticity and gaining T cell identity. A recently discovered transcription factor Bcl11b is expected to be a key regulator in this process. We have found that its mRNA level increases dramatically as HSCs begin to undergo T‐lineage specification. Our data so far has shown that there are a variety of Bcl11b transcripts expressed in immature T cells. While full‐length Bcl11b is growth inhibitory, one noncanonical transcript of Bcl11b may stimulate T cell development in vitro. Transduction of hematopoietic precursors with this variant isoform appeared to allow Thy‐1+ c‐Kit+ CD25+ cells to develop in vitro even in conditions where the Notch ligand DL1 was absent. Thus, certain isoforms of Bcl11b may promote T cell development and may even have a novel relationship to Notch. The expression pattern of Bcl11b is unique, because it is the only gene we have found so far that showed such developmental stage‐specific as well as T‐lineage specific expression. We have mapped DNA methylation of the promoter region in Bcl11b by using bisulfite DNA sequencing. These analyses show a cell type specific unmethylated window from −900 to −300 bp in Bcl11b expressing P2C2 cells. Understanding of how Bcl11b is regulated will dramatically increase our knowledge of T‐lineage commitment.
Trans‐presentation of Interleukin‐15 by different bone marrow‐derived antigen presenting cells (APC) has distinct effects on CD8 T cells in vitro
Authors: Frasca loredana , Stonier Spencer w , Overwijk Willem , Schluns Kimberly S
Published: June 2021

IL‐15, crucial in maintaining memory CD8+ T cell development and homeostasis, mediates its functions mainly via a mechanism called trans‐presentation. Trans‐presentation is the phenomenon by which IL‐15Rα, the high affinity IL‐15 binding protein, presents IL‐15 to opposing cells expressing the IL‐15Rβ/γC signaling components. Our group has previously shown that memory CD8+T cell development requires IL‐ 15α expression by the hematopoietic cell compartment. However, the identity of the hematopoietic cell types mediating IL‐15 trans‐presentation to memory CD8+ T cells is presently unclear. We attempted to mirror homeostatic proliferation of memory CD8+ T cells in vitro, by co‐culturing memory CD8+ T cell lines expressinga single T cell receptor with syngeneic bone marrow (BM) derived APC, in the absence of foreign antigen. We used this model system to compare the capacity of different dendritic cells (DC) subtypes, monocytes, and macrophages obtained either from normal mice, or mice lacking IL‐15α, to trans‐present IL‐15. The results show different outcomes of IL‐15‐mediated proliferation, survival and memory markers expression on CD8+T cells, depending on the cell type and activation status of the IL‐15 trans‐presenter. The different capacity to trans‐present IL‐15 by various hematopoietic cells may help localize the niche in which CD8+T cell memory is maintained.
Significant IFNγ production by early effector T cells signifies development into memory
Authors: Ellis Jason Scott , Bell J. Jeremiah , Guloglu F. Betul , Zaghouani Habib
Published: June 2021

The mechanism underlying transition of T cells from effector to memory remains largely undefined. It is believed that this transition occurs as a result of exposure to residual suboptimal antigen found in lymphoid tissues as the effector phase wanes. This led to the interpretation that memory arises from slightly activated late effectors producing reduced amounts of IFNγ. Herein, an in vivo model suitable for tracking memory transition was developed and shows that highly activated early effector CD4 T cells transit to memory. Moreover, effector T cells that have undergone 4 divisions expressed significant IL‐7R, produced IFNγ and yielded rapid and robust memory responses. Cells that divided three times which had marginal IL‐7R expression and no IFNγ raised base level homeostatic memory while those which have undergone only two divisions and produced IFNγ yielded conditioned memory despite low IL‐7R expression. These findings indicate that highly activated early effectors generated upon exposure to optimal antigen in vivo develop into memory and such transition is dependent on a significant production of the cell's signature cytokine, IFNγ.

Research supported by grant 2RO1AI48541‐08 from the NIH. JSE and JJB were supported by training grant DHHS2T32GM08396‐16A1 from the NIGMS.
Hypoxia inhibits the maturation and function of murine bone marrow‐derived dendritic cells
Authors: Zhang Lining , Wang Qun , Liu Chunmei , Wang Xiaoyan , Zhu Faliang , Sun Wensheng
Published: June 2021

Hypoxic is a prominent characteristic of inflammatory tissue lesions and solid tumor microenvironment. Under hypoxia condition, the changes of phenotype and function of infiltrated immune cells will affect the disease development and prognosis. It has been suggested that hypoxia might inhibit the production of matrix metalloproteinases (MMP) and the migration of human monocyte‐derived dendritic cells. However, the effects of hypoxia on murine bone marrow‐derived DC (BM‐DC) remain unclear. The present study is designed to compare the effects between hypoxic and normoxic conditions on the maturation and function of murine BM‐DC. We show here that hypoxia down‐regulated the expression of MHC II molecules and costimulatory molecules CD80, CD86 and CD137 on murine BM‐DC stimulated with LPS compared to that treated with normoxic condition, indicating that hypoxia inhibited the maturation of DC. Furthermore, hypoxia suppressed the expression of pro‐inflammatory cytokines (IL‐1, IL‐6, IL‐12 and TNF‐γ) and cell migration‐related gene (MMP9) in murine BM‐DC, but increased the levels of chemokine receptor (CCR7). Importantly, the hypoxia‐treated BM‐DC had reduced ability to stimulate the allogeneic CD4+T cell proliferation compared to normoxia‐differentiated BM‐DC. Thus, hypoxic microenvironment suppresses the maturation and function of murine BM‐DC demonstrated by downregulation of MHC Π and co‐stimulatory molecules and pro‐inflammatory cytokines, suggesting hypoxic DC as a possible target for tumor therapy.

This work was supported by grants from the Natural Science Foundation of China (No. 30628015) and (No. 30671976) and National “973” program (No. 2006CB503803).
Differential expression of Stat1 regulates opposing B cell responses to IFNγ
Authors: Wesemann Duane Robert , Gongora Rafael , Stephan Robert P. , Cooper Max D.
Published: June 2021

Interferon‐γ can inhibit the lymphopoiesis process, while paradoxically enhancing B lymphocyte responses. Activation and proliferation of mature B cells is essential for effective immunity, however, inappropriate stimulation of immature B cells may lead to autoimmunity and cancer. The present studies address the mechanism of how B cells of different maturity have opposing responses to IFNγ. Using primary mouse B cell progenitors in an ex‐vivo B cell development model, we show that IFNγ induces pro‐B cell death. However, the same cytokine paradoxically induces survival and proliferation in pro‐B cells deficient in Stat1. RNA expression analysis uncovered widely different Stat1 expression levels among B cell subpopulations. Germinal center B cells, which are poised for activation and proliferation, showed the lowest amount of Stat1, whereas, pro‐B cells express much higher levels of Stat1. Additionally, IFNγ enhances proliferation of the class‐switched, low Stat1‐producing A20 B cell line. But, A20 cells that are engineered to express higher levels of Stat1 are inhibited by IFNγ. Taken together, these results suggest that IFNγ induces simultaneous, but opposing stimulatory and inhibitory signals in B lineage cells, and that the level of Stat1 expression may dictate the ultimate cellular response to this cytokine. This work was supported by NIH AI39816 (to M. D. C.).
VHJ558 transgenic mice: A model to study the development of polysaccharide specific B cells and the antibody response to gram‐negative bacteria
Authors: Foote Jeremy Bruce , Kearney John F
Published: June 2021

To investigate development of the antibody response to α 1→3‐dextran (DEX), expressed by Enterobacter cloacae, we made transgenic mice expressing a VHJ558.3‐DSP2.2‐JH1 heavy chain (TG) that pairs with λ1 light chains to generate specificity for DEX (DEX+). DEX+ B cells, identified with anti‐idiotypic reagents, enrich in MZ and B1b B cell subsets in a B cell receptor dependent process. TG mice immunized intravenously (IV) with E. cloacae generate a DEX specific antibody response, which peaks at 3 days and persists at 10% of peak titers for 28 days before returning to preimmune levels. Analysis of DEX+ plasma cell kinetics by Brdu incorporation indicated that persistent antibody titers were maintained through de novo plasma cell generation. After immunization, B lymphopoiesis is temporarily abolished and DEX specific B cell production curtailed, yet, DEX specific B cells begin repopulating the MZ niche a week after their initial depletion in a manner that appears to be the result of DEX induced peripheral B cell clonal expansion. This model provides an example of selection of carbohydrate specific B cells into the MZ and B1b B cell subsets and illustrates the rapid, persistent nature of DEX specific antibody production. Furthermore, repopulation of the MZ by DEX+ B cells in the temporary absence of B lymphopoiesis indicates a role for peripheral B cells in the reconstitution of this niche.
Influence of commensal bacteria on the induction of UEA‐1+ NKM‐16‐2‐4+ cells in small intestine
Authors: Gotoh Yoshiyuki , Obata Takashi , Igarashi Osamu , Terahara Kazutaka , Nochi Tomonori , Kunisawa Jun , Satoh Shintaro , Sakamoto Mitsuo , Matsuki Takahiro , Umesaki Yoshinori , Benno Yoshimi , Kiyono Hiroshi
Published: June 2021

Antigen‐sampling M cells in the follicle‐associated epithelium (FAE) of Peyer's patches take up various antigens from intestinal lumens for initiation of antigen‐specific immune responses. Our recent finding showed that villous M cells also act as alternative antigen‐sampling cells. However, the influence of commensal microflora on the M cell mediated antigen‐sampling system in the intestinal FAE and villi have not been elucidated. Our recently established M cell‐specific monoclonal antibody (NKM 16‐2‐4) and lectin Ulex europaeus agglutinin 1 (UEA‐1), which posses the specificity to M cell‐specific carbohydrate molecule of α(1,2) fucose, specifically react with both of PP‐associated and villous M cells. Whole mount examination of small intestine using UEA‐1 and NKM16‐2‐4 revealed higher numbers of UEA‐1+NKM16‐2‐4+ double positive (DP) cells were observed in ileum than duodenum of naïve mice. In contrast, low numbers of DP cells are found in germ‐free mice, even in ileum. Subsequent metagenomic analysis revealed that segmented filamentous bacteria are predominant in mucus and epithelial layers in ileum than duodenum. These results suggest that distribution of specific commensal bacteria influences on the induction and maintenance of the mucosal antigen‐sampling system.
Commensal‐dependent regulation of the IL‐17 cytokine family governs susceptibility to intestinal inflammation
Authors: Zaph Colby , Du Yurong , Kobuley Dmytro E. , Nair Meera G. , Taylor Betsy C. , Perrigoue Jacquleine G. , Saenz Steven A. , Troy Amy E. , Guild Katherine J. , Kastelein Robert A. , Cua Daniel J. , Yu Yimin , Artis David
Published: June 2021

The lower intestinal tract is a major site of colonization by commensal bacteria. This bacterial population is acquired shortly after birth, is estimated to number 1014 organisms, and is remarkably diverse, being composed of at least 500–1000 individual species. The host‐commensal relationship is the product of millions of years of coevolution and alterations in the composition of this bacterial population can influence susceptibility to multiple inflammatory diseases in animal models of asthma, arthritis, multiple sclerosis, obesity and inflammatory bowel disease (IBD). However, the mechanisms through which commensal bacteria regulate intestinal immune homeostasis and disease susceptibility remain undefined. Here we show that the size of the large intestinal CD4+ T helper‐17 (TH17) cell pool is regulated by commensal bacteria. Commensal‐dependent expression of the IL‐17 family member IL‐25 inhibits the expansion or survival of TH17 cells in the intestine by inhibiting expression of accessory cell‐derived IL‐23. We propose that acquisition of–or alterations in–the intestinal flora after birth directly influences the IL‐25/IL‐23/IL‐17 axis, and that dysregulation of this pathway results in increased susceptibility to intestinal inflammation.
Gastric Th Cells Express CD39 and CD73 Leading to Adenosine Accumulation that Suppresses Helicobacter‐induced Gastritis by Modulating Th Cell Function
Authors: Alam Mohammad Samiul , Kurtz Courtney C. , Reuter Brain K. , Crowe Sheila E. , Ernst Peter B.
Published: June 2021

Background. Helicobacter pylori is a life‐long infection in humans that causes chronic gastritis and gastric cancer. Adenosine is an anti‐inflammatory mediator that limits tissue damage during inflammation. The surface enzymes CD39 and CD73 synthesize adenosine. We studied expression of CD39 and CD73 in human Th cells and evaluated the role of CD73 in regulating Helicobacter felis‐induced gastritis.

Methods. Human Th cells from blood and gastric tissue were assayed for expression of CD39/CD73 mRNA and protein. The function of CD73 was investigated by examining its effects on proliferation and cytokine production as well as its effect on H. felis infection in CD73−/− mice.

Results. CD73 was expressed on Treg from peripheral blood and gastric tissue, usually in association with CD39. Activation of Th cells increased CD73 with little effect on CD39 which remained associated with Foxp3+ Th cells. Gastric Treg had highest expression of CD39/CD73. CD73 antagonists partially reversed Treg suppressive ability in vitro and increased IFN‐γ production. H. felis infection exacerbated gastritis, increased production of pro‐inflammatory cytokines and reduced bacterial load in CD73−/− mice.

Conclusion. These data indicate that CD73 expression by Th cells contributes to adenosine accumulation and attenuates gastritis. Adenosine accumulation may impair immunity to Helicobacter infection and favor persistence.
CCL5 modulates mucosal immunity against chlamydial infection
Authors: Lillard James W , Singh Udai P , Sakthivel Senthilkumar K , Dove Terri M , Singh Shailesh , Igietseme Joseph U
Published: June 2021

Following genital chlamydial infection, an early T helper type 1 (Th1)‐associated immune response precedes the activation of recruitment of specific Th1 cells bearing distinct chemokine receptors, subsequently leading to the clearance of Chlamydia. We have shown that CCR5, a receptor for CCL5, is crucial for protective chlamydial immunity. Our laboratory and others have also demonstrated that CCL5 deficiencies found in man and animals can increase the susceptibility and progression of infectious diseases by modulating mucosal immunity. These findings suggest the CCR5‐CCL5 axis is critical for chlamydial immunity. We hypothesized CCL5 is required for protective humoral and cellular Chlamydia immunity. The present study revealed that CCR5 and CCL5 mRNAs are elevated in the spleen, iliac lymph nodes (ILNs), and genital mucosa following Chlamydia challenge. Antibody (Ab)‐mediated inhibition of CCL5 during genital chlamydial infection suppressed humoral and Th1 > Th2 cellular responses by splenic‐, ILN‐, and genital mucosa‐derived lymphocytes. Antigen (Ag)‐specific proliferative responses of CD4+ T cells from spleen, ILNs, and genital organs also declined after CCL5 inhibition. The suppression of these responses correlated with delayed clearance of the chlamydial infection. These results indicate that chlamydial immunity is mediated by Th1 immune responses driven in part by CCL5.
a4b7/MAdCAM‐1 Interactions Play an Essential Role in Transitioning Cryptopatches into Isolated Lymphoid Follicles, but not in Cryptopatch Formation
Authors: Wang Caihong , McDonough Jacquelyn , McDonald Keely G , Newberry Rodney
Published: June 2021

α4β7 α4β1 and their ligands, mucosal vascular addressin cell adhesion molecule (MAdCAM‐1) and vascular cell adhesion molecule (VCAM‐1), have prominent roles in the formation of secondary lymphoid tissues at early and later time points. In this study, we evaluated the role of α4 integrins in the development of inducible mucosal lymphoid tissues, isolated lymphoid follicles (ILFs). We observed that diverse cellular populations express α4β7 and α4β1, including the LTi‐like cells and lymphocytes, while ILF stromal cells and high endothelial venules express VCAM‐1 and MAdCAM‐1 respectively. Evaluation of β7 deficient mice and mice given blocking antibodies to α4β7, MAdCAM‐1, or VCAM‐1 did not identify a role for α4 integrins in the localization or clustering of the LTi‐like cells, or cryptopatch (CP) development. However, these studies demonstrated that α4α7 and MAdCAM‐1 were absolutely required for the transitioning of CP into ILFs. Bone marrow chimeric mouse studies confirmed that β7 expression by lymphocytes was essential for their entry into the developing ILF. Collectively these findings demonstrate a role for α4β7/MAdCAM‐1 interactions at late stages in ILF development corresponding to the influx of mature β7 expressing lymphocytes, and implicate distinctions between the role of early β7 events leading to ILF and secondary lymphoid tissue development.
Defining the mechanism(s) of protection by cytolytic CD8 T cells against a cryptic epitope derived from a retroviral alternative reading frame
Authors: Rutkowski Melanie R , Ho On , Green William R
Published: June 2021

The goal of this study was to determine the functional properties of protective cytolytic CD8 T cells (CTL) specific for an MHC class I cryptic epitope, SYNTGRFPPL, during infection with the LP‐BM5 murine retrovirus. Disease‐susceptible CD8−/ − BALB/c mice reconstituted with SYNTGRFPPL‐specific CTL were able to clear the retrovirus and were therefore protected from disease. Since the mechanism(s) involved in the protective response against this alternative epitope were uncertain, perforin (pfp) −/ −, IFNγ−/ −, pfp−/−/IFNγ−/ − (DKO) and wild‐type (WT) BALB/c mice were infected with LP‐BM5 and assessed for disease. A subset of infected pfp−/ − mice had reduced splenomegaly and immunosuppression, compared to CD8−/ − mice. However, IFNγ−/ − and DKO mice were resistant to disease. SYNTGRFPPL‐specific CTL were generated from all strains of mice by priming with a Vaccinia construct expressing LP‐BM5 gag followed by in vitro restimulation with SYNTGRFPPL peptide. Effector CTL were then transferred into CD8−/ − mice following infection with LP‐BM5. Compared to CD8−/ − recipients protected with WT CTL, CD8−/− mice receiving DKO CTL had moderate disease whereas CD8−/− mice receiving Pfp−/ − CTL developed more severe disease. CD8−/− mice receiving IFNγ−/− CTL were fully protected from disease. These results indicate pfp, but not IFNγ, as a critical component of the CTL response against cryptic epitope SYNTGRFPPL.
T cell migration and memory differentiation within the mouse intestinal mucosa in response to infection
Authors: Masopust David , Vezys Vaiva , Wherry E. John , Ahmed Rafi
Published: June 2021

Analyses in mice have revealed that the phenotype and function of pathogen‐specific memory CD4 and CD8 T cells vary considerably between gut and other anatomic locations. Understanding T cell migration and differentiation within the gut is of particular interest, given the predisposition of this tissue to infection from a variety of pathogens and the observation that the gut is an immediate and critical target of HIV. We have found that T cell migration to the small intestinal epithelium occurs only during the first few days after T cell activation, regardless of the route of infection, and provide evidence that memory T cells within this compartment do not recirculate. We will present data demonstrating that the unique qualities of memory CD8 T cells within the intestinal mucosa are partly regulated in situ by tissue‐specific factors. We are currently dissecting the developmental cues that regulate expression of the gut homing machinery by activated T cells. These studies may provide new strategies for establishing cellular immunity preferentially within the intestinal mucosa.
T‐cell responses to influenza vaccination among patients with heart failure
Authors: Moran John Joseph McDonald , Hayney Mary S. , Sweitzer Nancy K. , Vardeny Orly
Published: June 2021

Purpose: The objective of this study was to measure T cell response in patients with heart failure (HF) compared to healthy controls (HC) following influenza vaccination using the trans vivo delayed‐type hypersensitivity (DTH) assay, which mimics physiologic conditions.

Methods: Eighteen HF subjects and 16 HC were immunized with influenza vaccine. Peripheral blood mononuclear cells (PBMC) were isolated from blood drawn 4–6 weeks post immunization. PBMC were mixed with influenza vaccine antigens (H1N1, H3N2, and B), and injected into the footpads of SCID mice. The resulting swelling is an index of human T cell sensitization. To detect a difference of 50 μm (α=0.05 and power = 95%) 15 per group were needed.

Results: All subjects mounted an antibody response to the influenza vaccine. T cell responses to the B antigen were similar between the HF and HC groups (75±8 vs. 66±9 μm; p=0.49). Responses to the newest vaccine antigen, H3N2, were less vigorous in the HF participants compared with the HC response (mean 65±8 vs. 89±6 μm; p=0.031; t test), and there was a trend toward a reduced response to the H1N1 antigen by the HF group (mean 58±7 vs. 81±10 μm; p=0.06).

Conclusions: HF pts demonstrated reduced T cell responses to influenza vaccine, due to lower response to the newest vaccine antigen compared with HC. Lower T cell responses may be a reason that HF patients are at increased risk for influenza and its complications.

Funded by the NIH.
Towards a new generation of potent and multifunctional anthrax vaccines
Authors: Wang Julia Y , Roehrl Michael H
Published: June 2021

The recent use of anthrax as a lethal biological weapon has prompted renewed research interest in the anthrax field. The disease consists of three critical components, i.e., spore, bacillus, and toxin. Current remedies rely on postexposure antibiotics to eliminate bacilli and pre‐ and postexposure vaccination to target primarily toxins. The currently available vaccine is based on protective antigen (PA), a central component of anthrax toxin. Vaccination with PA raises no direct immune response against the bacilli and, being a natural toxin component, PA might be hazardous to use immediately following exposure to Bacillus anthracis. We therefore sought to develop a new generation of potent and effective anthrax vaccine that can confer protection against multiple components of anthrax and that is also that safe for postexposure administration. Towards this goal, we first developed a dually active anthrax vaccine that is effective against both toxin and bacilli by chemically conjugating the protective antigen of anthrax toxin with the capsular polyglutamate of B. anthracis. We then developed an improved version of the conjugate vaccine and an improved anti‐toxin vaccine based on non‐toxic PA mutant that is safe for postexposure immunization. These newly developed vaccines and our strategies will be presented.
Anti‐JCV TCR‐based chimeric immune receptors create functional designer T cells for PML therapy
Authors: Junghans Richard Paul , Yang Wen , Bais Anthony , Beaudoin Esther
Published: June 2021

Progressive Multifocal Leukoencephalopathy (PML) is a lethal brain disease caused by JC polyomavirus (JCV) that arises in immunodepressed HIV+ patients and in rare individuals who are treated with natalizumab (Tysabri) for multiple sclerosis or Crohn's disease. Chimeric immune receptors (CIRs) are engineered from JCV specific TCR á and â chains cloned from a PML survivor and fused to the transmembrane (TM) and cytoplasmic (cyt) domains of CD3æ providing Signal 1. These form functional á/â chain heterodimers, induce retrovirally transduced expressor T cells to secrete cytokines and exhibit cytotoxicity against JCV+ cells. In further experiments, these TCR‐based CIRs were engineered to create 2nd generation products with added CD28, providing Signal 1+2, containing an extracellular (EC) region and TM and cyt domains of CD28 and cyt of CD3æ. The 2nd generation anti‐JCV designer T cells are intended to resist AICD and proliferate on antigen contact, with resistance to regulatory T cell and other suppressions, and function in the absence of CD4 T cell help. Here, we demonstrate that under the hypothesis of several variations of constructs with either CD28, CD3æ or hybrid TM domains that removal of the CD28 EC region is required for effective expression, heterodimerization and designer T cell function. These products are being prepared for clinical application in HIV+ or natilizumab treated PML patients.
A soluble Fc receptor in channel catfish, Ictalurus punctatus, binds IgM
Authors: Nayak Deepak K. , Stafford James , Miller Norman W. , Wilson Melanie , Bengten Eva
Published: June 2021

An Fc‐receptor (FcR) homolog, IpFcRI, representing the first such receptor from an ectothermic vertebrate, has been identified in the channel catfish. It consists of three immunoglobulin (Ig) domains (D1–3) but lacks transmembrane (TM) and cytoplasmic regions. Due to this lack of a TM, IpFcRI is suggested to be a soluble FcR. The soluble IpFcRI is readily detected in catfish serum and quantitative PCR analyses demonstrate that kidney leukocytes express high levels of IpFcRI message. Recombinant (r) IpFcRI immobilized onto latex beads for Ig binding studies was found to bind both native catfish IgM and IgM‐immune complexes. In addition, rIpFcRI was also found to recognize reduced catfish IgM heavy (H) chain and Ig from other species, including rainbow trout, mouse, rabbit, goat, and chicken. To identify the IpFcRI domain(s) involved in IgM binding, various combinations of rIpFcRI domains were expressed. Recombinant IpFcRI proteins containing two domains, D1–2 or D2–3, reacted with reduced catfish IgM H chain, while the individual D1, D2 and D3 domains did not. Furthermore, the IpFcRI‐binding site appears to be localized on Cμ3 and Cμ4 domains. Taken together, the IpFcRI‐IgM binding studies suggest an immunoregulatory role for IpFcRI. Supported by NIH grant R01 AI19530.
Activation of lymph node derived bovine NK cells by CD244 and CD16
Authors: Mayer Sandra Vanderli , Capinos Scherer Charles F. , de Aguiar Juliana B. , Estes D. Mark
Published: June 2021

Natural Killer cells (NK) are important in both antiviral, bacterial and tumor defense and constitute a major effector population of the innate immune defense. Activation of NK cells by specific cell surface expressed proteins in infected or tumor cells can induce the production of IFN‐gamma and cytotoxicity through release of cytotoxic granules such as perforin and granulysin. Bovine NK cells were recently phenotypically characterized but selective activation via cell surface receptors is not fully understood. Treatments using either inhibitory or stimulatory receptors that activate NKs can be used as a target for vaccine formulation against important bovine and human pathogens. In the present study we used lymph node‐derived bovine NK cells that were activated with different conditions including activation via CD244 AND CD16 resulting in increasing IFN‐gamma and Perforin. Our studies demonstrate that specific in vitro activation conditions of purified bovine NK cells results in a rapid response via secretion of IFN‐gamma and cytotoxic granules. Insight into selective NK functional activities can be used in the development of better vaccines against rapidly replicating pathogens such as foot and mouth disease virus.
OX40 Signals Determine the Fate of Memory CD8+ T cells following Listeria monocytogenes Infection
Authors: Mousavi Seyed Fazlollah , Soroosh Pejman , Yoshikai Yasunobu , Shen Hao , Sugamura Kazuo , Ishii Naoto
Published: June 2021

A TNF receptor super‐family molecule OX40 on T cells regulates several T cell responses including expansion, survival and cytokine secretion. Accumulating evidences have demonstrated the importance of OX40 on generation of memory CD4+ T cells. Although the significance of OX40 signals in CD8+ T cell responses to tumors and acute viral infection have been reported, the precise function of OX40 in the generation of memory CD8+ T cells is still unclear

We carried out several types of adoptive transfer experiments using Ova‐specific OT‐I‐TcR transgenic T cells and recombinant Listeria monocytogenes expressing OVA (Lm‐Ova). To generate antigen specific effector and memory CD8+ T cells, OT‐I or OT‐I/OX40‐KO T cells were transferred into congenic wild‐type mice following infection with Lm‐Ova. Our preliminary results demonstrated that presence or absence OX40 signals have a negligible effect on the frequency and effector function of donor CD8+ T cells during priming phase. In contrast the frequency of OX40‐deficient memory CD8+ T cells and their effector function were dramatically reduced. In addition, the lack of OX40 signals in the donor OT‐I cells significantly suppressed the re‐expansion of the memory CD8+ T cells during recall responses. Ongoing study has revealed that blockage of OX40‐OX40L interaction during the priming phase seems to be sufficient to inhibit generation of long‐lived memory CD8+ T cells.