bluuebunny/crossref_metadata_2025 huggingface dataset:
Caffeine and Multiple Sclerosis: Is protection in your coffee cup?
Authors: Mills Jeffrey H , Thompson Linda F , Mueller Cynthia , Waickman Adam T , Bynoe Margaret S.
Published: June 2021

The formation of extracellular adenosine during an inflammatory response serves as an immunosuppressive negative‐feedback signal to prevent excessive inflammation. Since CD73 is a cell surface enzyme that catalyzes the breakdown of AMP into adenosine, we investigated the role of CD73 (and extracellular adenosine) in controlling inflammation in experimental autoimmune encephalomyelitis (EAE), the animal model for the inflammatory autoimmune central nervous system (CNS) disease multiple sclerosis (MS). While we predicted that cd73−/− mice would develop severe EAE due to their inability to produce extracellular adenosine to modulate CNS inflammation, surprisingly, cd73−/− mice were resistant to EAE. In addition, cd73−/− mice had fewer CNS infiltrating lymphocytes compared to wild type mice following EAE induction. This EAE protection in cd73−/− mice cannot be attributed to a T cell functional deficiency since adoptively transferred CD4+ T cells from cd73−/− mice can potentiate severe EAE in tcrα −/− mice (which are normally resistant to EAE due to their lack of T cells). Moreover, while adenosine receptors (ARs) are expressed in the CNS, blockade of AR signaling with caffeine protects wild type mice from EAE development. Thus, we conclude that CD73 and AR signaling are required for the efficient entry of lymphocytes into the CNS during EAE, and quite possibly MS, development. AI 18220 (LFT) and AI 57854 (MSB)
Effect of genetic A2B adenosine receptor ablation on proinflammatory actions of adenosine in mast cells
Authors: Ryzhov Sergey , Zaynagetdinov Rinat , Goldstein Anna E. , Biaggioni Italo , Feoktistov Igor
Published: June 2021

We have previously shown that A2B adenosine receptors (AR) play a pro‐inflammatory/pro‐angiogenic role, upregulating production of Th2 cytokines and angiogenic factors in human HMC‐1 mast cells.1,2 However, the pro‐inflammatory role of A2bAR in mast cells was recently questioned by a study that found an enhanced antigen‐induced degranulation of A2bAR knockout (A2BKO) mouse bone marrow‐derived mast cells (BMMCs).3 The authors interpreted this as evidence of anti‐inflammatory actions of A2BAR in BMMCs and suggested that A2BAR antagonists with inverse agonist activity could promote activation of mast cells. Here, we revisited these conclusions by studying the effect of genetic A2BAR ablation on adenosine‐dependent regulation of BMMCs. Our results show that genetic ablation of A2BAR had no effect on adenosine‐dependent potentiation of antigen‐induced degranulation, but abrogated adenosine‐dependent stimulation of IL‐13 and VEGF secretion. A2BAR antagonists with inverse agonist activity inhibited A2BAR‐dependent secretion, but did not mimic the enhanced antigen‐induced degranulation observed in A2BKO BMMCs. Thus, our study confirmed the pro‐inflammatory role of mast cell A2BAR and demonstrated anti‐inflammatory actions of A2BAR antagonists.

Supported by NIH.
Chimeric NKG2D receptor expressing T cells require host immune cells for anti‐tumor efficacy
Authors: Barber Amorette , Zhang Tong , Sentman Charles L.
Published: June 2021

We have developed a chimeric NKG2D receptor (chNKG2D) consisting of NKG2D fused to the CD3ζ chain of the T cell receptor and have shown that chNKG2D therapy can be an effective treatment in murine models of lymphoma and ovarian carcinoma. In this study, we demonstrate the key mechanisms that chNKG2D T cells require and a role for the host immune system in tumor elimination. Treatment of B6 mice with chNKG2D T cells led to long‐term, tumor‐free survival in a murine ovarian cancer model. ChNKG2D T cells were required to produce perforin, IFNγ, and GM‐CSF for complete efficacy in vivo. Additionally, chNKG2D T cell treatment led to an increased number and activation of host NK cells at the tumor site. Inhibition of tumor growth was limited in host mice that lacked perforin or T and B cells (Rag1‐/‐), indicating that host immune mechanisms were involved. There was no role for CD1‐dependent NKT cells or GM‐CSF, as mice deficient in these were able to clear tumors as well as wildtype B6 mice. In summary, treatment with chNKG2D T cells led to long‐term survival of mice with established ovarian cancer, and chNKG2D T cells required cytotoxicity, cytokine secretion, and the participation of host immune cells for complete anti‐tumor efficacy. This work was supported in part by grants from the Norris Cotton Cancer Center and National Institutes of Health (AI 07363).
GENE THERAPY: CCL21 (SLC) INHIBITS PRIMARY PROSTATE TUMOR GROWTH AND METASTASES
Authors: Yousefieh Nazita , Hahto Suzanne , Ciavarra Richard Peter
Published: June 2021

TRAMPC2 (transgenic adenocarinoma of mouse prostate) tumors are heavily infiltrated with myeloid but not lymphoid (T and B) cells that may reflect the accumulation of immature dendritic cells (DCs) in TRAMPC2 tumors. Secondary lymphoid tissue chemokine (CCL21, SLC) is chemotactic for T cells and DCs and may serve as co‐localization signal for these cells during early phases of the cellular immune response to prostate tumors. We proposed in this study that modification of tumor microenvironment (TME) by expression of CCL21 in TRAMP tumor would induce infiltration of DCs and T cells and minimizes the myeloid cell infiltrate that appears to be responsible for disruption of T cell signaling complex. We generated TRAMPC2 cell lines with regulated expression of CCL21 using the tetracycline responsive promoter, so that we could activate the transgene at selected times during tumor progression (tet on system). Orthotopic tumor volumes were smaller in tetracycline treated mice and these mice lived longer than control mice. Lymph node (LN) metastasis was detected in all 3 control mice (1 found dead) whereas only 2 out of 4 treated mice had LN metastasis. 2 out of 3 untreated mice but only 1 out of 4 treated mice had lung metastasis. Our data indicates that expression of CCL21 in the prostate TME inhibits tumor growth and prevents metastasis and may represent a novel approach to modulate anti‐tumor immunity and tumor growth.
STAT3 phosphorylation induced by vascular endothelial growth factor is associated with regulation of immune responses by dendritic cells
Authors: Zhang Wenguang , Hauke Ralph
Published: June 2021

OBJECTS

The expression of phosphorylated signal tranducer and activators of transcription 3 (p‐STAT3) is correlated with expressions of vascular endothelial growth factor (VEGF) in some carcinomas. The mechanism of the immunomodulatory effects of VEGF on human dendritic cells (DCs) is not clearly understood. This study seeks to investigate the effects of VEGF on STAT3 phosphorylation and its correlation with immune regulation by human DCs.

METHODS: Monocytes‐derived immature DCs were incubated with VEGF. We detected expression of p‐STAT3 in DCs induced by VEGF with different concentrations and for different effect times by immunohistochemistry and Western blot. The cytotoxicity of DC‐activated T cells against LNCaP prostate cancer cells was determined by lactate dehydrogenase (LDH) activity assay.

RESULTS: A concentration of 100 ng/ml of VEGF was enough to increase phosphorylation of STAT3 and showed nuclear translocation of p‐STAT3 from the cytoplasm to the nucleus. The cytotoxicity of T cells was significantly decreased after co‐culture with VEGF‐treated DCs.

CONCLUSIONS: VEGF plays a key role in immunoregulation of human DCs. VEGF‐induced phosphorylation and nuclear translocation of STAT3 is correlated with DCs activation decreased, suggesting that VEGF decreased DC‐activated T cells cytotoxity activation by phosphorylation of STAT3. Supported by: UNMC Internal Medicine Research Support.
Novel regulation of CD8 T cell‐specific OX40 expression via an IL‐2 and JAK3‐dependent mechanism
Authors: Redmond William L , Weinberg Andrew D
Published: June 2021

In addition to TCR stimulation, engagement of co‐stimulatory receptors provides critical signals that help to augment T cell survival and differentiation. In particular, engagement of the TNFR family member, OX40 (CD134), during T cell activation has been shown to greatly augment both CD4 and CD8 T cell responses and enhance T cell‐mediated anti‐tumor immunity. Although TCR stimulation was thought to be sufficient for inducing OX40 expression on activated T cells, in the current study we demonstrate that TCR signaling is necessary, but not sufficient to promote OX40 expression. Rather, OX40 is induced on activated CD8 T cells through a dual TCR and IL‐2 dependent mechanism. Further analysis revealed that OX40 induction via IL‐2‐dependent signaling required activation and phosphorylation of JAK3. Taken together, these data demonstrate that OX40 is induced on CD8 T cells via an IL‐2 and JAK3‐dependent mechanism.
CD40 Ligation Enhanced Naïve Tumor Antigen Specific CD8+T Cell Responses in Spontaneous Osteosarcoma Model
Authors: Tagaram Hephzibah Rani S. , Rolin Oliver , Avella Diego , Garcia Luis , Kimchi Eric , Schell Todd , Tevethia Satvir S. , O‐Carroll Kevin F. Staveley
Published: June 2021

Introduction: The 501 transgenic mice express the transforming T antigen (Tag) of SV40 virus causing spontaneous Tag expression in bone and salivary gland of mice. T ag expression as a self‐antigen in osteosarcoma mice leads to progressive loss of CTL responses to T ag epitopes.

Objective: 1.To determine the fate of naïve TCR‐I cells in tumor bearing osteosarcoma mice that express T antigen as a tumor antigen. 2. The effect of CD40 ligation on the above response.

Experiment: T antigen epitope I‐specific CD8+ T cells from Tcell receptor transgenic mice (TCR‐I cells) were transferred into tumor‐bearing 501mice or syngeneic normal C57BL/6 mice. Agonistic anti‐CD40 was given day before and day after adoptive transfer(AT), seven days later spleens and cervical lymphnodes, tumors and salivary glands were analyzed for Interferon‐γ, epitope‐1 specific tetramer, T cell infiltration, and apoptotic TUNEL staining.

Results: 1) TCR‐I cells expand systemically and acquire effector function 2) Tumor specific CD8+T cell responses are aepitope‐I specific. 3) CD40 ligation enhanced TCR‐I cell proliferate. 4) In tumor‐bearing hosts TCR‐I cells proved capable of lysing 100% of target cells in vivo. 5) TCR‐I cells infiltrates salivary gland and tumor and enhanced with CD40 ligation. 6) CD40 ligation enhanced anti tumor activity detected by apoptosis using TUNEL.

Conclusion: T ag expressed as a tumor antigen in osteosarcomas mice is strongly immunogenic. CD40 ligation enhanced responses to T‐ag as measured by TCR‐I proliferation, activation, migration into tumors and antitumor cell cytotoxicity.
HLA‐B‐Associated Transcript 3 (BAT3), a ligand for Nkp30 is secreted from cells as Exosomes and enhances the activity of Natural Killer cells
Authors: Simhadri Venkateswara Rao , Reiners Katrin S , Hansen Hinrich P , Engert Andreas , von Strandmann Elke Pogge
Published: June 2021

Natural Killer (NK) cells, defined as innate lymphocytes also serve as regulators of adaptive immunity via cytokine secretion and cross‐talk with dendritic cells (DCs). Both killing and maturation of DCs by NK cells is dependent on NKp30, a Natural Cytotoxicity Receptor (NCR) expressed on NK cells. However, the NKp30 specific cellular ligands have remained elusive.

Recently BAT3 was identified as the tumor associated ligand for NKp30 (Immunity, 2007 in press). It is a nuclear protein characterized by a conserved BAG domain (BclBcl‐2 associated anthogene), that interacts with Hsp70.

This abstract describes that BAT3 is secreted into the extracellular environment in exosomal fractions upon exposure to stress signals. The exosomes from the tumor cells and iDCs possess BAT3 on the surface. These exosomes activate NK cells and stimulate the cytokine release TNFalpha and IFNgamma. This release was enhanced by exosomes from cells overexpressing BAT3 and was inhibited by depleting the overexpressed BAT3. Moreover, the exosomes’ stimulation was inhibited when the cells were targeted with siRNA of BAT3.

Thus we postulate that BAT3, a novel cellular ligand for NKp30 receptor has a role in the reciprocal activation of NK‐DC cross‐talk. We present evidence that BAT3 is secreted into exosomes, a novel group of immune regulators “Nucleokines”.

Research was supported by Deutsche Forschungsgemeinschaft (DFG) (STR 530/5‐1).
Systemic Flt3L and intratumoral HSV1‐TK treatment eradicates intracranial GBM in a syngeneic model
Authors: Muhammad A.K.M Ghulam , King Gwendalyn D. , Candolfi Marianela , Curtin James , Liu Chunyan , Kroeger Kurt , Lowenstein Pedro R. , Castro Maria G.
Published: June 2021

Adenoviral vector (Ad) expressing FMS‐like tyrosine kinase 3 ligand (Flt3L) was delivered systemically to make less invasive our recently developed combined gene therapy for GBM. Six days after intra‐striatal stereotactic implantation of 4,500 CNS‐1 cells in Lewis rats, intravenous pre‐dosing with an empty Ad (Ad0) was performed and within 4~6 hours, AdFlt3L was delivered via the tail vein followed by intra‐tumoral herpes simplex type 1 thymidine kinase (AdTK) delivery. Controls (treated with Ad0 systemically and AdTK intra‐tumor) succumbed by day 22, whereas, 80% of the treated group survived long term (p<0.05). The survivors were rechallenged with GBM in the contralateral striatum and 70% survived long‐term without additional treatment. Brains of long‐term survivors displayed no residual tumor or local neurotoxicity; CD68+ macrophages, few CD8+ T cells, and MHC II immune positive cells were detected; no liver toxicity was encountered. Positive delayed type hypersensitivity reaction in the treatment group indicated the presence of memory T cells. In conclusion, gene therapy combining immune stimulation using systemic AdFlt3L and intra‐tumor cytotoxic/oncolytic treatment (AdTK) elicits tumor regression and immunological memory inhibiting tumor recurrence.
Activation of quiescent memory T lymphocytes from human tumors is enhanced by co‐cultivation with autologous tumor‐associated stromal fibroblasts
Authors: Barnas Jennifer Lynn , Bankert Richard B
Published: June 2021

While T lymphocytes from the tumor microenvironment are hyporesponsive to a short term (one hour) T cell receptor (TCR) activation, chronic TCR stimulation results in T cell activation and the synthesis of interferon‐γ (IFN‐γ). IFN‐γ production by T cells in response to chronic stimulation is significantly enhanced when patient T cells are cultured with autologous tumor‐derived fibroblasts. Our goal is to define the cellular and molecular events contributing to this fibroblast‐dependent enhancement of the T cell response to activation. Because others have established that there are molecular interplays between immunoreceptors and integrins with TCR signaling, we are testing the hypothesis that fibroblast enhancement of T cells results from T cell integrin binding to fibroblast generated extracellular matrix (ECM). Function blocking antibodies to the collagen binding integrin VLA‐2 inhibits the fibroblast dependent enhancement of T cell activation. These preliminary data are consistent with the possibility that the enhanced T cell response is mediated in part by T cell outside‐in signaling via integrin binding to ECM produced by fibroblasts. Supported by U.S. Public Health Service Grants R01‐CA108970 and the John R. Oishei Foundation.
STAT3 and immune escape in head and neck squamous cell carcinoma
Authors: Leibowitz Michael Shinichi , Lopez‐Albaitero Andres , Ferris Robert L
Published: June 2021

One strategy of immune escape involves downregulation of the antigen processing machinery (APM), enabling tumor cell evasion by cytotoxic T lymphocytes (CTL), however the mechanism responsible for this has not been determined. Previous work in our and other laboratories has shown that cell lines and tissues derived from HNSCC downregulate APM components such as transporter associated with antigen processing (TAP)‐1 and TAP2. Treatment with IFN‐γ upregulates APM components, and restores the ability of tumor antigen‐specific CTL to recognize and lyse these tumor cell lines. HNSCC tumors overexpress signal transducer and activator of transcription (STAT)‐3, a transcription factor activated by interleukin‐6 (IL‐6). Pre‐treatment of HNSCC cell lines with IL‐6 abrogates IFN‐γ mediated STAT1 activation, suggesting that STAT3 negatively regulates STAT1 activation. Thus, we hypothesized that overexpression of STAT3 in HNSCC may contribute to downregulation of APM components and CTL recognition by decreasing STAT1 activity, thereby contributing to immune escape. Preliminary studies indicate that IL‐6 pre‐treatment can decrease IFN‐γ mediated upregulation of APM components at both the transcriptional and translational level. Investigation is underway to elucidate the interaction of STAT1 and STAT3, and their potential contribution to APM component mediated tumor immune escape from CTL recognition.
Lewis Lung Carcinoma (LLC) alters the phenotype of murine lung mast cells resulting in a phenotype consistent with myeloid‐derived suppressor cells (MDSCs)
Authors: Mailloux Adam William , Young M Rita I
Published: June 2021

MDSCs are among the most potent immune suppressive phenotypes found in the tumor microenvironment. Because they share so many characteristics with myeloid progenitors, MDSCs are thought to arise as a product of tumor‐associated hematopoiesis. However, a clear MDSC progenitor has yet to be identified. We used flow cytometry to demonstrate a significant increase in CD11b+ infiltrate in a murine model of LLC as compared to normal lung tissue. A large portion of this infiltrate also expresses the granulocyte/myeloid marker Gr‐1. Interestingly, CD11b+/Gr‐1+ cells in normal lung tissue also express the high affinity IgE receptor FcεR1α, which is indicative of mast cells. However, this FcεR1α expression is lost on these cells in LLC‐bearing animals in a tumor burden‐dependant manner. Additionally, conventional methylene blue stains show highly granulated mast cells in normal lung tissue while showing a complete lack of granulated cells in LLC‐bearing lung tissue. We also immunomagnetically isolated Gr‐1+ mast cells out of normal lung tissue and incubated them in‐vitro with LLC‐conditioned media while monitoring their FcεR1α expression, granularity, and morphology in an attempt to reproduce the phenotypic changes seen in‐vivo. Our data suggests that in LLC, MDSCs may originate from fully differentiated mast cells rather than from myeloid precursors.

Supported by the Dept of Veteran's Affairs & NIH
Tumors induce regulatory dendritic cells that suppress CD8+ T cell antitumor immunity
Authors: Norian Lyse A. , Rodriguez Paulo , O’Mara Leigh. A. , Zabaleta Jovanny , Ochoa Augusto , Cella Marina , Allen Paul M.
Published: June 2021

Dendritic cells have a critical impact on the outcome of adaptive immune responses against growing tumors. Using a murine model of spontaneously arising mammary carcinoma, we found that phenotypically mature, MHC Cl II+/CD11b+/CD11chigh tumor infiltrating dendritic cells (TIDCs) act as regulatory DCs to suppress CD8+ T cell function, resulting in diminished T cell‐based antitumor immunity in vivo. Stimulation of naive T cells with TIDCs resulted in an altered cell fate program characterized by cell cycle arrest, impaired IFNg production, and anergy. Suppression by TIDCs overcame stimulatory signals provided by standard DCs and occurred independently of cognate interactions with T cells. TIDC suppression was mediated by arginase metabolism of the amino acid L‐arginine; blockade of arginase, but not iNOS, restored T cell proliferation. Thus, the tumor microenvironment can induce phenotypically mature MHC Cl II+ TIDCs to acquire regulatory functions and utilize arginine metabolism to suppress protective CD8+ T cell based antitumor immunity. Our results suggest that targeting these cells in vivo may enhance the efficacy of T cell based antitumor immunotherapies.
Anti‐tumor immune response induced by homeostatic proliferation and CD28 signaling
Authors: Suzuki Toshihiro , Kishimoto Hidehiro , Abe Ryo
Published: June 2021

Recent studies suggested that homeostatic proliferation (HP) could lead immune response against tumor‐associated antigen (TAA) resulting in rejection of tumor. Although there has been little evidence suggesting any requirement for co‐stimulatory signal and IL‐2 for the expansion and phenotypical changes of naive T cells in various models of lymphopenic host, we demonstrate that CD28 signaling is indispensable for the differentiation of donor CD8+ T cells into functional CTL during HP. Administration of IL‐2 on early time points i.e. day 0 to 5 of HP could not enhance the anti‐tumor effects, however, the tumor specific immune response induced by HP was significantly enhanced when IL‐2 was administrated systemically at 10 to 15 days after treatments. Surprisingly, systemic administration of IL‐2 for both late and early phase of HP did not recover the defect of anti‐tumor effect in the absence of CD28 signaling. Therefore, we concluded that IL‐2 production is not critical factor for the initiation of the anti‐tumor immune response induced by HP. Since our observation indicated that donor naive CD8+ T cells differentiated into TEM cells through TCM cells during HP, the mechanisms of the induction of TAA‐specific CTL in lymphopenic host is distinct from generation of effector T cells with foreign antigen and adjuvant; nevertheless CD28 is most important co‐stimulant molecule for the induction of fully functional CTLs.
Role of Endothelial Cells in a Novel Mechanism of Tumor‐Induced Immune Suppression
Authors: Mulligan Jennifer , Young M. Rita I.
Published: June 2021

Previous studies by our lab have shown that Lewis lung carcinoma (LLC)‐secreted products skew endothelial cells to suppress T cell, NK cell and macrophage functions. LLC‐derived VEGF has been identified as inducing endothelial cells to suppress T‐cell functions. In our current studies, we examined the role of endothelial cells in mediating suppression in human head and neck squamous cell carcinoma (HNSCC) and in the in vivo LLC tumor model. Antibody neutralization of HNSCC‐derived VEGF blocked tumor induced increases in endothelial cells production of VEGF and PGE2. T cells treated with HNSCC‐exposed endothelial cell conditioned media had decreased IFN‐γ production, compared to control treated T cells. Treatment of HNSCC‐conditioned media with VEGF neutralizing antibody prevented induction of suppressive endothelial cells toward T cell functions. We also examined the ability of tumors to induce suppressive endothelial cells in vivo. Conditioned media from endothelial cells isolated from the lungs of tumor‐bearing mice suppressed IFN‐γ production by CD8+T cells and IL‐2 by CD4+ T cells as compared to normal lung controls. Studies are ongoing to determine the role of VEGF in the induction of suppressive endothelial cells in vivo. Together these studies implicate induction of suppressive endothelial cells as a novel mechanism of tumor‐induced immune suppression. Supported by Dept of Veterans Affairs & NIH
Substance P modulation of anti‐tumor immunity in a mouse model: characterization of NK and T cells
Authors: Brand Lucas , Kastelic Amanda , Spampinato Christine , Manske Jill
Published: June 2021

Substance P (SP) is a neuropeptide that has been shown to have immunoregulatory properties including effects on mediators involved in anti‐tumor immunity. In previous studies we have shown that treatment of mice with SP provides protection against tumor growth. This protection requires both T cells and NK cells, and adoptive transfer cells from SP‐treated animals can transfer tumor protection. In this study we characterized NK and T cells from SP treated mice. Mice were implanted with osmotic pumps that delivered SP for 5, 9, or 14 days. Following treatment, we examined the expression of the CD25 as a marker of T cell activation, and CD69 expression on NK cells. NK cells from SP‐treated mice showed up regulation of CD69. No SP effect was observed on CD25 expression. To further characterize the T cells involved in this response, we examined the effect of SP on proinflammatory cytokines. Levels of intracellular Interleukin‐2 (IL2), gamma‐Interferon (IFNγ), and Tumor Necrosis Factor (TNFα) were examined. SP treated mice had increased levels of IFNγ after five days of SP treatment, even in the absence of additional in vitro stimulation. These results suggest that the role of T cells in SP mediated tumor protection may be via production and release of proinflammatory cytokines which may, in turn, affect activation of NK cells.

This work was funded in part by a Young Scholars grant from the University of St. Thomas
B7‐H1 induced Apoptosis on Epstein‐Barr Virus transformed B cells through Fas‐Fas Ligand expression
Authors: Park Ga‐Bin , Kim Yeong‐Seok , Song Hyunkeun , Lee Hyun‐Kyung , Lee Wang Jae , Hur Dae Young
Published: June 2021

B7‐H1 encompasses a recently discovered cell surface glycoprotein within the B7 family of T‐cell co‐regulatory molecules. Some human tumors acquire the ability to aberrantly express B7‐H1. Tumor‐associated B7‐H1, as well as B7‐H1 on activated lymphocytes, has been shown to impair antigen‐specific T‐cell function and survival in vitro. In contrast, the monoclonal antibody‐mediated stimulation of B7‐H1(reverse signaling) on cancer cells expressing B7‐H1 molecule itself has not been studied. Our group has recently reported that B7‐H1 is induced on EBV‐transformed B cells via immunohistochemical staining and flow cytometric analysis. In addition, We observed that B7‐H1 ligation with anti‐B7‐H1 mAb enhanced the apoptosis of EBV‐transformed B cells. And B7‐H1 ligation induced the expression of both FasL and Fas in theses cells. Furthermore, anti‐Fas blocking antibody, ZB4, blocked B7‐H1‐mediated apoptosis effectively and caspase inhibitors also effectively blocked B7‐H1‐induced cell death. Western blot analysis showed down‐regulated expression of procaspase‐8, 3 and PARP. The release of cytochrome c and translocation to nucleus of endoG and AIF were showed by confocal micoscopy. The findings suggested that Fas/FasL pathway was activated by B7‐H1 ligation on EBV‐transformed B cells.
Association between CD44 polymorphism and breast cancer
Authors: Zhou Juhua , Nagarkatti Prakash , Zhong Yin , Nagarkatti Mitzi
Published: June 2021

CD44 is a cell‐surface glycoprotein involved in many cellular functions including lymphocyte activation, recirculation and homing, hematopoiesis, and tumor metastasis. Thus, CD44 may play an important role in anti‐tumor immunity. Here we aimed to elucidate if CD44 polymorphisms are associated with breast cancer. Nucleotide sequencing analysis demonstrated that a unique polymorphism (designated as Ex2+13 A>G) in the CD44 exon2 upstream intron was identified in 80% of breast cancer patients, which had a significantly increased frequency than normal donors (Odds ratio, 5.16; p < 0.05). Besides, different polymorphic changes in the CD44 exon2 coding region were identified in ~ 40% of breast cancer patients, which were totally absent in normal donors. Mann‐Whitney U test showed that the patients containing the CD44 polymorphisms in CD44 exon2 coding sequence had breast cancer at earlier stage, 43±2 vs. 58±2 (p < 0.0005). Immunolocalization indicated that CD44 was down‐regulated on tumor‐infiltrating lymphocytes in breast tumor tissues. We propose that CD44 polymorphisms may be responsible for CD44 down‐regulation on tumor‐infiltrating lymphocytes, facilitating breast tumor cell growth. Our results suggested that CD44 polymorphisms may play an important role in breast cancer development (Supported in part by NIH grants R01AI053703, R01ES09098, R01 AI058300, R01DA016545, R01HL058641 and P01AT003961).
THE ROLE OF CM1 ON INCREASE OF PROSTAGLANDIN E2 PRODUCTION FROM HUMAN BURKITT'S LYMPHOMA
Authors: Bae Seyeon , Kang Jae Seung , Lee Seung Koo , Kim Jee Eun , Jung Da Jung , Lee Na Eun , Kong Joo Myoung , Lee Seona , Shin Dong Hoon , Hwang Young‐il , Lee Wang Jae
Published: June 2021

CM1 (centrocyte/‐blast marker 1) is originally defined as a germinal center (GC) B cell marker and it plays a critical role on B cell development in GC. In addition, CM1 is expressed on Burkitt's lymphoma cell lines. It suggests that CM1 plays a role in the pathogenesis of tumor cells. Cyclooxygenase‐2 (COX‐2) and its product, prostaglandin E2 (PGE2) are well‐known molecules, which are closely related with the pathogenesis of several kinds of tumor. Therefore, we investigated the role of CM1 on the regulation of COX‐2 expression and PGE2 production from Burkitt's lymphoma cell lines, such as Raji and Ramos. When CM1 was ligated with anti CM1 mAb, PGE2 production was increased and CM1‐induced PGE2 production was inhibited by the pre‐treatment of NS‐398, COX‐2 specific inhibitor and kamebakaurin, specific inhibitor of NF‐kB. Finally, the effect of increased PGE2 production by CM1 ligation on activation induced cytidine deaminase (AID) was evaluated. The expression of AID was increased by CM1 ligation and it was suppressed by the pre‐treatment of NS‐398 and kamebakaurin. Taken together, CM1‐induced PGE2 production increases the expression of AID via the activation of NF‐kB and increase of enzymatic activity of COX‐2.
The cancer‐germline antigen TRAG‐3 stimulates Th1‐type, Toll‐like receptor 8‐negative antigen‐specific CD4+ regulatory T cells
Authors: Kudela Pavol , Janjic Bratislav , Fourcade Julien , Kirkwood John M. , Maillere Bernard , Zarour Hassane M.
Published: June 2021

TRAG‐3 is a cancer‐germline antigen expressed by tumors of different histological types including melanoma, breast, and lung cancers. We have previously demonstrated that TRAG‐3 stimulates spontaneous Th1‐type CD4+ T cell response against a single immunodominant and promiscuous MHC class II epitope in patients with stage IV melanoma. Here, we have shown that some of the TRAG‐3‐specific CD4+ T cell clones isolated from the PBLs of melanoma patients and normal donors inhibited the proliferation of allogenic naïve CD4+ T cells in response to anti‐CD3 antibody stimulation and acted in a cell‐to‐cell contact‐dependent fashion. The capability of these TRAG‐3‐specific CD4+ T cell clones to suppress IL‐2 production by responder cells was depended upon their activation by the cognate antigen. The TRAG‐3‐specific regulatory T cell clones did not express Toll‐like receptor 8 and no correlation was found between the level of Foxp3 expression and regulatory functions. Collectively, our findings demonstrate the capability of the same cancer‐germline‐derived epitope to stimulate both antigen‐specific helper and regulatory T cells and emphasize the need for novel therapeutic interventions to preferentially stimulate antigen‐specific helper T cells in vivo. This work was supported by NIH/NCI Grants CA90360 and CA112198.
Role of TNF in colon carcinogenesis
Authors: Gyulai Zsofia , Salcedo Rosalba , Dai Ren‐Ming , Anderson Kimberley , Lyakh Lyudmila , Mazzoni Alessandra , Young Matthew , Colburn Nancy , Oshima Akira , Grivennikov Sergei , Nedospasov Sergei
Published: June 2021

The degree of inflammation and the type of inflammatory/immune response are responsible for tilting the balance between tumor progression and regression. TNF‐α has conflicting roles in cancer, as being both a necrotic and promoting/growth factor. There is much clinical interest in blocking TNF in anti‐cancer therapy but relatively little is known about the exact role of TNF in the development of cancer. We focused on defining the contribution of tissue specific TNF towards chronic colitis promotion and cancer using a chemical carcinogenesis model. TNF and conditional TNF deficient mice were treated with a single dose of the mutagenic agent azoxymethane followed by 3 or 4 cycles of 2% dextran sodium sulfate (AOM/DSS) administered in the drinking water. Mice were monitored for weight loss and intestinal bleeding. At the end of the treatment, mice were sacrificed for macroscopic and histological analysis of the intestine. TNF −/− mice developed significantly less pathology when compared with floxed TNF control mice. Most lesions were localized to the distal portion of the colon. To elucidate the contribution of TNF produced by different cell types, experiments with conditional TNF deficient mice are in progress. Our data indicate that the lack of TNF plays a protective role in the AOM/DSS carcinogenesis model.
Characterization of The Bovine Mammary Serotonergic System
Authors: Hernandez Laura Lorraine , Limesand Sean W. , Baumgard Lance H , Horseman Nelson D , Collier Robert J
Published: June 2021

Previous research indicates serotonin (5‐HT) as a feedback inhibitor of lactation in the bovine. We utilized quantitative real‐time PCR (qRT‐PCR) and in situ hybridization of tissue cryosections to identify 5‐HT receptors in bovine brain (BB), mammary tissue (BMT) and primary bovine mammary epithelial cells (BMEC). Additionally, we utilized immunohistochemistry in BB and BMT cryosections to determine presence of tryptophan hydroxlase 1 (TPH‐1), aromatic amino acid decarboxylase (AADC), and the serotonin reuptake transporter (5HTT). Serotonin receptors 1b, 2a, 2b, 4, and 7 were detected in the BB, BMT and BMEC by PCR and qRT‐PCR. In BMT, fold expression of receptors relative to BB, (highest to lowest) was: 5‐HT 1b (2,477; P < 0.0001), 5‐HT 4 (389), 5‐HT 2b (89), 5‐HT 7 (18), and 5‐HT 2a (3.77). In BMEC, the fold expression of receptors relative to BB (highest to lowest) was: 5‐HT 4 (230, P < 0.0001), 5‐HT 2b (44), 5‐HT 7 (37), 5‐HT 1b (30) and 2a (1.64). Serotonin receptors 1a, 1d, 1f, and 2c were only detected in the BB by PCR. Immunostains for TPH‐1, AADC, and 5HTT were positive in both BB and BMT. We conclude that BMT and BMT enriched with BMEC contain the 5‐HT 1b, 2a, 2b, 4 and 7 receptors. The enrichment of tissue preparation with BMEC had the highest expression of the 5‐HT 4 receptors relative to BMT, while BMT had the highest expression of 5‐HT 1b receptors relative to BMEC. Manipulation of 5‐HT receptors with specific antagonists present in BMEC could lead to increased milk protein synthesis.

*Funded by USDA‐NRI#2007‐35206‐17898
Breastfeeding as a predictor of body mass index in 2‐year‐old children at risk for overweight
Authors: Street Erin E , Lovelady Cheryl , Ostbye Truls , Pendzich Margaret , Fuemmeler Bernard
Published: June 2021

The purpose of this study was to determine if breastfeeding was associated with BMI of children at 2 yrs who were at risk for obesity. Overweight/obese mothers (BMI = 32.9 ± 7.3 kg/m2) and their children were studied from 7 wks until 2 yrs postpartum (PP). Baseline data included race, education, income, age, marital status, gestational wt gain, and birth wt. At 1 yr PP mothers reported infant feeding. Women were assigned 0 if formula feeding only, 1 if combining formula and breastfeeding, and 2 if breastfeeding only for each month during the first year PP. A lactation score was calculated by totaling the values for each month. Scores were used to define two groups: 1) low intensity/duration (score=0–11, N=129) and 2) high intensity/duration (score=12–24, N=74). Race was 51% white, 46% African American and 3% other. At 2 yrs PP, 20% of children were overweight and 24% were obese using WHO growth standards. Multiple regression analysis was used to determine predictors of child BMI at 2 yrs. Variables were baseline data, gender, mothers BMI and breastfeeding group. The model accounted for 16% (p<0.001) of the variation in child BMI. Birth wt (p=0.01), mother BMI (p=0.03), and gestational wt gain (p=0.06) predicted an increase, while high intensity and long duration of breastfeeding (p=0.07) predicted a decrease in child BMI at 2 yrs. These results suggest breastfeeding may reduce obesity in children born to overweight/obese women.

Supported by R01‐DK064986
Vitamin B12 intake above current recommendations may be needed to ensure optimal vitamin B12 status in sub‐groups of healthy adults
Authors: von Castel‐Roberts Kristina , Kauwell Gail P.A. , Maneval David , Young Linda J , Nexo Ebba , Bailey Lynn B
Published: June 2021

The study objectives were to assess vitamin B12 (B12) intake and status of non‐supplement consuming healthy young (18–49 y) adults (n=299) and to characterize dietary patterns most likely associated with normal B12 status. Vitamin B12 status was assessed by plasma B12, serum MMA, and serum holo‐TC. Dietary intake was estimated using a modified Dietary History Questionnaire and reduced rank regression was used to identify dietary patterns predicting B12 status. Vitamin B12 status was abnormal for at least one biomarker (B12 < 148 pmol/L, MMA > 270 nmol/L, holo‐TC < 35 pmol/L) in 30% of individuals (23% meat consumers and 42% non‐meat consumers). Dietary patterns predicting variance in B12 biomarkers consisted primarily of fortified foods (eg RTE breakfast cereals and vegetarian meat substitutes), and fish, which were positively associated with vitamin B12 status. The median B12 intake associated with high vs normal MMA and low vs normal holo‐TC was 2.9 vs 4.7 g/d and 3.0 vs 4.8 g/d, respectively. The median B12 intake associated with the cut‐off for normal B12 status ranged from 3.2 to 4.1 μg/d for all biomarkers. Based on regression analysis, these data suggest that vitamin B12 requirement of healthy young adults may currently be underestimated. Individuals who wish to reduce meat consumption somewhat, may enhance B12 intake by including fish and fortified foods in their diet.
Hypoadiponectinemia is associated with obesity, and dyslipidemia in Korean patients with type 2 diabetes mellitus(DM)
Authors: Lee Eun‐hee , Ryu Han‐kyoung , Yu So‐young , Lee Hyun‐sook , Choi Young‐ju , Huh Kap‐bum , Kim Wha‐young
Published: June 2021

Adiponectin has insulin‐sensitizing and anti‐inflammatory actions that suggests a protective role in diabetes development. The purpose of this study is to investigate the association between adiponectin level and anthropometric, and biochemical variables in Korean patients with type 2 DM. The subjects were recruited from a diabetes clinic in Seoul and classified into quartile according to serum adiponectin level. Waist circumference, BMI, and % body fat were significantly higher in lowest quartile than in highest quartile. Lowest quartile showed significantly higher serum concentrations of triglycerides, and insulin and lower HDL‐cholesterol than highest quartile. Also, higher prevalence of obesity, central obesity, and dyslipidemia was seen in lowest quartile. Nutrient intakes were not different among the groups. However, lowest quartile showed significantly higher intakes of high‐fat food than highest quartile. In conclusion, hypoadiponectinemia is a significant risk factor for the development of obesity, and dyslipidemia in Korean patients with type 2 DM. Consequently, type 2 DM patients with hypoadiponectinemia were more vulnerable to complications of diabetes. It is utmost importance to modify diet to keep proper level of serum adiponectin for type 2 DM patients management. (This work was supported by the second stage of BK21 Project in 2007 and by Seoul R&BD. Program, No. 10526)
The quantification of anti‐epileptic drugs with medication score and its use in assessing ketogenic therapy
Authors: Shah Sweny , Borum Peggy R.
Published: June 2021

Ketogenic Therapy (KT), a diet high in fat, low in carbohydrate, and adequate in protein, is used to treat intractable epilepsy in the pediatric population. The two main goals of the KT are to reduce seizures and the use of anti‐epileptic drugs (AEDs). The problem associated with assessing the reduction of AEDs is there has not been an effective way to quantify AEDs. Historically, the number (no.) of AEDs has been used, yet this may not give complete information as it does not take other factors into consideration, such as dosage and patient weight. We have introduced an AED Medication Score (MS), which is the ratio of the prescribed dosage to the patient specific minimum recommended dosage. Each MS is summed to give a total MS for each patient. To test for the applicability of MS, analysis was done on 26 patients using no. of AEDs and MS data from initiation and their last clinic visit. The linear correlation between the change in no. of AEDs and the change in MS is significant but weakly moderate (p = 0.03, R = 0.42). The significance is evidence for the reasoning that the change in no. of AEDs and MS measures the same variable but the weak linear correlation illustrates that MS provides different and more information than the change in number of AEDs. For example, after starting KT, 7 patients were on 2 fewer AEDs but their change in MS varied from −12 to 5. The analysis indicates that MS may be a more useful indicator of efficacy of KT.
Hepatic Lipid Molecules Synergize with Insulin to Induce GK Gene Expression
Authors: Gao Yifei , Lu Danhong , Newgard Christopher B , Chen Guoxun
Published: June 2021

Hepatic glucokinase (GK, Hexokinase IV) plays a key role in maintaining glucose homoestasis. A variety of hormonal and nutritional stimuli, including insulin, regulate its activity by controlling its gene transcription. Here, we report a simple method to extract lipid molecules from rat livers and measure their effects on GK gene expression in primary rat hepatocytes using real‐time polymerase chain reaction. To this end, we observed that a lipid extract (LE) prepared rat liver by saponification, induces the levels of GK transcripts and potentates insulin‐mediated induction of GK gene expression. In hepatocytes, LE or 1 nM insulin alone induced GK mRNA by 7.6 ± 5.9‐ and 19.3 ± 12.4 –fold, respectively. The combination of LE and 1 nM insulin induced the GK expression by 155 ± 72 –fold, much higher than the sum of the induction fold by them individually (7.6+19.3=26.9), suggesting a synergy between LE and insulin. The synergy exists for all of the insulin concentrations examined ranging from 0.1 to 100 nM. LE does not induce the levels of GK transcripts in pancreatic islets and INS‐1 insulinoma cells, demonstrating a specific effect on hepatic GK expression. The actions of LE start as early as 3 hours and last at least 12 hours. Moreover, it does not alter the stability of GK transcripts, suggesting a transcriptional mechanism. We conclude that endogenous lipid molecules from liver can modulate hepatocyte GK gene expression.
Digestibility and prebiotic potential of nondigestible carbohydrate fractions from novel maize‐based fibers in a dynamic in vitro model of the human intestine
Authors: Maathuis Annet , Hoffman Andrew , Evans Annette , Sanders Lisa , Venema Koen
Published: June 2021

A well balanced intestinal microbiota can offer numerous potential health benefits to the host. Some nondigestible dietary fibers act as prebiotics by stimulating the growth of beneficial – or minimizing the growth of undesirable – microbes. In this study, the prebiotic potential of novel fibers were evaluated using an in vitro model of the human intestine (TIM). Resistant starch (RS), resistant maltodextrin (RM), soluble corn fiber (SCF), soluble fiber dextrin (SFD) and biogum (BG) were pre‐digested in the model of the stomach and small intestine (TIM‐1) and monosaccharides separated by chromatography. The remaining fraction was presented to the model of the large intestine (TIM‐2; inoculated with microbiota of American origin) at a rate of 10 g / 24 h for a 72 h period. Samples were obtained from the lumen of the model every 24 h and short chain fatty acids (SCFA), branched chain fatty acids (BCFA), lactate and ammonia measured. DNA from luminal samples were hybridized to DNA arrays printed with probes to detect group level and individual species of microbes. Compared to a low fiber carbohydrate mixture, all novel fibers stimulated the growth of certain Bifidobacteria species at least 2 fold. SCF, SFD and BG treatments decreased several Bacteroides species by a factor of 2 or more. Butyrate generation was greatest and ammonia production was least in the BG and SCF treatments as compared to cellulose, a non‐fermentable control. Lactate accumulation was lowest in the RS and RM treatments. Overall, these novel fibers show different yet favorable fermentation profiles and prebiotic potential in this dynamic in vitro system of the human intestine. Research Support: Tate & Lyle
Effects of dietary high protein on renal health in the pig model
Authors: Jia Yong , House Jim D , Ogborn Malcolm R , Weiler Hope A , Aukema Harold M
Published: June 2021

The long‐term impact of a high protein (HP) diet at the upper limit of the acceptable macronutrient distribution range (AMDR) on kidney health is unknown. Therefore, 16 adult female Cotswold pigs were randomized to receive either normal (NP, 15% energy from protein) or HP (35% energy from protein) diets for 16 weeks. Each diet contained whole proteins from animal and plant sources and were balanced for energy, fat, vitamins and minerals. Body composition was measured by dual‐energy X‐ray absorptiometry, renal hypertrophy was assessed by kidney volume and glomerular hypertrophy was analyzed by measuring glomeruli on fixed sections. The HP diet increased kidney volume (p=0.003) and weight (p= 0.0276). Consistant with this, HP compared to NP pigs had larger glomeruli (p=0.078). However, diet had no significant effect on renal function as measured by glomerular filtration rate and proteinuria. HP consumption decreased body weight (p=0.0232) and body fat percentage (p<0.05) and increased percent bone mineral content (p=0.036) and lean mass (p<0.05). These findings suggest that despite the potential benefit of the HP diet on body composition, protein intakes at the upper limit of the AMDR may alter renal hemodynamics by increasing kidney and glomerular volume. The long term effects on glomerular injury remain to be elucidated (Supported by Canadian Institutes of Health Research).
Raisins and increased walking affect lipoprotein metabolism, reduce inflammation and alter satiety hormones
Authors: Puglisi Michael J , Mutungi Gisella , Vaishnav Ushma , Volek Jeff S , Fernandez Maria Luz
Published: June 2021

Raisins are a significant source of dietary fiber and polyphenols, which may reduce cardiovascular disease (CVD) risk by affecting lipoprotein metabolism and inflammation. Walking represents a low intensity exercise intervention that may also reduce CVD risk. The purpose of this study was to determine the effects of consuming raisins, increasing steps walked, or a combination of these interventions on plasma apolipoproteins and inflammatory cytokines. Thirty‐four men and postmenopausal women were matched for weight and gender and randomly assigned to consume 1 cup raisins/d (RAISIN), increase the amount of steps walked/d (WALK) or a combination of both interventions (RAISIN + WALK). The subjects completed a 2 wk run‐in period, followed by a 6 wk intervention. Plasma apolipoproteins (apo), inflammation markers and appetite hormones were measured at baseline and post‐intervention. Plasma apo CIII was decreased for WALK (P < 0.05), which most likely contributed to the reduction in plasma triglycerides (TG) concentrations from 1.49 mmol/L to 1.2 mmol/L for WALK (P < 0.05 for group effect). Plasma TNF‐α was decreased from 3.5 ng/L to 2.1 ng/L for RAISIN (P < 0.025 for time and group X time effect). All subjects had a reduction in plasma sICAM‐1 (P < 0.01). Plasma ghrelin was increased for both the RAISIN and the RAISIN + WALK groups (P < 0.05), possibly related to an increase in leptin due to raisin intake (P< 0.01). This research shows that simple lifestyle modifications such as adding raisins to the diet or increasing steps walked can alter lipoprotein metabolism and decrease inflammation, resulting in distinct beneficial effects on CVD risk [Supported by the California Raisin Marketing Board]