bluuebunny/crossref_metadata_2025 huggingface dataset:
The recurring AGCT motif in S region DNA specifically recruits 14‐3‐3 adaptor proteins that are critical for the unfolding of CSR
Authors: Xu Zhenming , Fulop Zsolt , Zhang Jinsong , Al‐Qahtani Ahmed , Pone Egest , Pal Zsuzsanna , Park Seok‐Rae , Zan Hong , Fu Haian , Yates John , Casali Paolo
Published: June 2021

Somatic hypermutation (SHM) and class switch DNA recombination (CSR) are central to the maturation of the antibody response and occur mainly in germinal centers (GCs). To identify critical switch (S) region cis‐elements, we analyzed S region DNA sequences and identified AGCT motifs occurring at a high frequency in Sμ and all other downstream S regions in human, mouse and Xenopus laevis. 14‐3‐3 adaptor proteins were the major AGCT‐binding proteins and preferentially localized to S regions in switching B cells. Expression of 14‐3‐3 was induced in GC B cells in vivo and B cells stimulated to undergo CSR in vitro. The differential induction of the seven 14‐3‐3 isoforms in these B cells suggests an isoform‐specific role of 14‐3‐3 in CSR. This was confirmed by different degrees of CSR inhibition in B cells that were null in 14‐3‐3γ or expressed a semi‐dominant 14‐3‐3σ mutant or reduced levels of 14‐3‐3τ or 14‐3‐3ε. Further, 14‐3‐3 interacted with critical CSR factors, such as AID and Ku80/Ku86. Finally, difopein, a pan‐14‐3‐3 isoform peptide inhibitor that disrupts interaction between 14‐3‐3 and its protein ligands, reduced the level of resected double strand DNA breaks (DSBs), the obligatory intermediates in CSR. Thus, our findings demonstrate that the recurring AGCT motif in S regions specifically recruit 14‐3‐3 adaptor proteins that are critical for the unfolding of CSR.

Supported by NIH grants AI 45011, AI 60573 and AR 40908.
Gene regulatory interactions mediating neural crest formation
Authors: Bronner‐Fraser Marianne
Published: June 2021

The neural crest is a population of multipotent, migratory cells that forms at the neural/nonneural ectoderm border in vertebrate embryos, migrate extensively and differentiate into diverse cells. Data compiled from various vertebrates suggest that a network of interacting transcriptional regulators and downstream effector genes confer properties like multipotency and migratory capacity to nascent neural crest cells. These regulatory interactions can be divided into distinct phases. First, inductive signals (e.g. Wnt, BMP, FGF) establish the neural plate border, by up‐regulation of border specifier genes like Msx1/2, Pax3/7, and Zic. These border genes in turn up‐regulate neural crest specifier genes like Slug/Snail, FoxD3 and SoxE, which in turn activate specific downstream targets that render the neural crest migratory and multipotent. We are testing linkages in this hypothetical neural crest gene regulatory network in chick and lamprey by systematically perturbing a subset of the transcription factors and examining the effect on likely downstream genes. In addition, we are isolating cis‐regulatory regions of neural crest specifier genes to identify neural crest enhancers and determine direct inputs. The results suggest that a series of gene regulatory circuits produce migratory neural crest cells and that many of these events are conserved to the base of vertebrates.
Altered innate immunity contributes to the development of colitis in PI3K p110d mutant mice
Authors: Uno Jennifer Kikue , Rao Kavitha , Matsuoka Katsuyoshi , Li Fengling , Sartor R. Balfour , Plevy Scott E
Published: June 2021

The phosphoinositide‐3 kinase (PI3K) pathway is an important negative regulator of innate immune signaling. Initial characterization of mice with a targeted mutation in the p110δ subunit of PI3K revealed chronic, focal colitis. However, immunomodulatory effects of PI3K p110δ in innate immunity remain unclear. We demonstrate that PI3K p110δ mutant macrophages (splenic, bone marrow derived & intestinal) are hyperresponsive to TLR signaling, producing increased levels of IL‐12 p40, IL‐12 p70, and IL‐23. Likewise, increased colonic IL‐12/23, IFN‐γ, and IL‐17 are detected, indicating that aberrant regulation of innate immune signaling could contribute to the development of Th1/Th17 mediated colitis. An innate mucosal inflammatory response is essential for clearance of enteric microorganisms that breach the epithelial barrier. However, excessive and prolonged activation of these inflammatory pathways is detrimental to the host and may contribute to the pathogenesis of IBD. We therefore sought to determine the role of PI3K p110δ signaling in killing of intracellular enteric bacteria by macrophages. P13K p110δ mutant macrophages display significantly decreased killing of intracellular K12 E. coli compared to wild type macrophages. In addition, decreased bacterial killing was associated with marked increases in IL‐12 p40 production in p110δ mutant macrophages as compared to wild type macrophages (mutant vs. wild type, p= 0.05, n=3). In conclusion, PI3K p110δ plays an important role in dampening innate immune responses necessary for maintaining gut homeostasis.
A dual function for RELMβ in promoting TH1 immune responses and infection‐induced intestinal inflammation
Authors: Nair Meera G , Guild Katherine J , Du Yurong , Stevens Sean , Karow Margaret , Artis David
Published: June 2021

The secreted goblet cell‐derived protein Resistin‐like moleculeβ (RELMβ) has been implicated in divergent functions including immunity to intestinal infection and promotion of chemical‐induced colitis. However, the mechanisms through which RELMβ influences proinflammatory responses and adaptive immunity are undefined. To investigate the function of RELMβ in this context, we employed a model of chronic Trichuris infection characterized by RELMβ expression, IFNγ production and severe intestinal inflammation. In contrast to WT mice, RELMβ −/− mice failed to establish chronic infection, and parasite clearance was associated with decreased production of IFNγ and TNFα and reduced intestinal inflammation. Based on these findings, we hypothesized a role for RELMβ in promoting proinflammatory responses by influencing accessory cell function and/or through direct effects on CD4 T cells. While recombinant RELMβ did not directly affect CD4 T cells, rRELMβ treatment of macrophages induced MHC Class II expression, secretion of IL‐12 and TNFα , and augmented their ability to promote antigen‐specific TH1 cell differentiation. Taken together, these data demonstrate a previously unidentified role for an intestinal goblet cell‐derived factor in the modulation of proinflammatory responses and adaptive immunity through direct effects on macrophages.
PD‐1 negatively regulates CD8 T cell‐mediated mucosal autoimmunity
Authors: Vezys Vaiva , Masopust David , Sharpe Arlene , Freeman Gordon , Newell Kenneth , Ahmed Rafi
Published: June 2021

Loss of T cell tolerance to mucosal self‐antigens can lead to debilitating diseases, such as inflammatory bowel disease. We wished to identify the developmental cues that discriminate between T cell tolerance induction vs. autoreactivity within the intestine. Some evidence suggests that signaling through the programmed death (PD) pathway may augment intestinal T cell responses, whereas responses to non‐mucosal proteins are oppositely affected. We assessed the role of PD‐1 and PD‐L1 in CD8 T cell‐mediated intestinal autoimmunity. Upon transfer of naive OT‐I cells to mice producing OVA exclusively within small intestinal epithelial cells, T cells expand in lymph nodes and migrate to the intestine, but cause no pathology, despite large numbers of cells within the antigen‐bearing tissue. With the inclusion of a blocking antibody to PD‐L1, OT‐I cell numbers and function increased dramatically and led to fatal autoimmunity, similar to disease induced with the inclusion of inflammatory agents. Therefore, PD‐1 signaling can be a critical regulator of tolerogenic vs. pathological responses. These results highlight the importance of negative regulatory mechanisms in preventing mucosal autoimmunity.
Generation and homeostatic regulation of CD8αβ effector memory T cells by CD8αα and its ligand, the thymic leukemia (TL) antigen
Authors: Huang Yujun , Kronenberg Mitchell , Schoenberger Stephen , Cheroutre Hilde
Published: June 2021

Abundant intra‐epithelial lymphocytes (IEL) reside within the intestinal epithelium making the intestinal IELs the largest T cell population in the body. The majority of CD8αβ T cell subset of intestinal IELs displays an effector memory phenotype. These experienced T cells populate the mucosal surface and provide rapid and efficient immune protection at the largest entry surface for pathogens. The conditions that lead to mucosal effector memory differentiation are poorly understood. We found that CD8αβ effector memory IELs are derived preferentially from primary effector cells that transiently induce CD8ααduring the effector phase. CD8αα not only plays a key role in this memory differentiation process, but it is also required for long‐term survival of CD8αβ+ effector memory IELs. TL, as a ligand for CD8αα, is not required for the generation or survival of CD8αβ effector memory IELs. Surprisingly however, TL expressed by the intestinal epithelial cells causes death of activated CD8αβ T cells. On the other hand, when interacting with TL, CD8αα rescues activated CD8αβ IEL from cell death induced by TL. Here we provide the first evidence that a unique memory process exists that leads to the specific differentiation of mucosal effector memory T cells that gain the capacity to reside long term at the mucosal surface where they provide immediate and highly effective immune responses upon re‐challenge.
BG1, a candidate gene affecting the occurrence of herpesvirus–associated avian lymphomas
Authors: Wang Yujun , Goto Ronald M. , Miller Marcia M.
Published: June 2021

Oncogenic herpesvirus infections contribute to the occurrence of lymphomas in the human population. Genetic makeup may have a role in susceptibility. Through genetic mapping with recombinant haplotypes we have identified BG1, a member of the extended butyrophilin gene family, as a gene having a significant role in MHC‐associated resistance to the occurrence of T‐cell lymphomas that often follow infection with gallid herpesvirus 2 in the chicken animal model. Significant differences in tumor incidence occur among congenic birds differing only at the BG1 locus. Two alleles, BG1*R2 and BG1*R4, are respectively associated with tumor resistance and susceptibility. The alleles differ only in the 3′‐untranslated region (3′‐UTR) with the longer 3′‐UTR of the BG1*R4 allele containing unique sequence. In dual Firefly/Renilla luciferase reporter assays we found that the 3′‐UTR of the BG1*R4 suppresses PMA‐stimulated translation of the firefly reporter gene, while the 3′‐UTR of BG1*R2 has a far less inhibitory influence. With further constructs we have narrowed the effective region of the BG1*R4 3′‐UTR to a region relatively rich in CG dinucleotides.

Supported in part by NCI R21 CA105426.
Age‐associated changes and importance of Natural Killer cell functions during primary influenza infection in mice
Authors: Nogusa Shoko , Ritz Barry W , Gardner Elizabeth M
Published: June 2021

The role of Natural Killer (NK) cells during the immune response to primary influenza infection in young and aged mice were investigated. Young (6∼8 weeks) and aged (22 months) C57BL/6 mice were infected intranasally with influenza A virus (H1N1, PR8) and NK cell‐mediated cytotoxicity was determined in lung and spleen during the first 4 days of infection. Aged mice demonstrated both a decrease in influenza‐inducible NK cytotoxicity and a reduction in the percentage and number of NK cells relative to young mice. While young mice exhibited a peak over a baseline on day 2 post infection (p.i.) with a statistical significance, such an increase was not observed in aged mice. Young mice also exhibited 2‐fold increase in perforin and granzyme at day 2 p.i. coinciding with a peak of NK cytotoxicity activity. In order to further establish the importance of NK cells in controlling influenza infection, NK cells were depleted in young mice using anti‐NK1.1 antibody prior to infection. Young mice depleted of NK cells exhibited increased weight loss and lung virus titer during early course of infection compared to young or aged mice. These data indicate that NK cells function is impaired in response to primary influenza infection in aged mice. More importantly, these results underscore the essential role of NK cells in controlling virus titers in lung, at the site of infection, during the early course of influenza infection, regardless of age.
Development of a multivalent vaccine against respiratory syncytial virus
Authors: Subbarayan Praseetha , Pillai Shreekumar , Vig Komal , Dennis Vida , Singh Shree Ram
Published: June 2021

Human Respiratory Syncytial Virus (RSV) has been acknowledged as the most universal cause of critical respiratory tract infections in newborn babies causing high rate of morbidity and mortality. As the virus is ubiquitous in all parts of the world, avoidance of infection is not possible. After RSV infection the immunity is incomplete and secondary infection can occur throughout life. There is no vaccine available and research in this field is ongoing. RSV fusion (F), attachment (G) and matrix (M2) proteins are most sought after due to their potential to induce high immune response. In the present study, we designed a multivalent protein which contained antigenic regions from three proteins of RSV (F, M2 and G). These antigenic regions were cloned into pET‐32 vector and a vector pET‐FM2G was generated. The recombinant protein was expressed and purified using His‐bind columns. The purified protein was analyzed by SDS‐PAGE and western blot to characterize the purity of protein and presence of RSV proteins. Currently, mice are being immunized using the multivalent vaccine to study the immune response. Mice will be challenged with RSV to ultimately test the protection capability of the immunogen. Financial support for this work by National Institutes of Health Grants 2S06 GM008219‐200012 and NSF‐CREST (HRD#0734232) is acknowledged.
Gene expression patterns induced by HPV‐16 L1 VLP in leukocytes from vaccine recipients
Authors: Pinto Ligia A , Garcia‐Pineres Alfonso J , Hildesheim Allan , Dodd Lori , Kemp Troy J , Yang Jun , Fullmer Brandie , Harro Clayton , Lowy Douglas R , Schiller John T , Lempicki Richard A
Published: June 2021

To determine the cellular pathways induced by an HPV‐16 L1 VLP vaccine, we compared the gene expression signature in L1 VLP‐stimulated PBMC from 19 vaccine and 7 placebo recipients before vaccination, and 1 month after receiving the second immunization. Cells were incubated for 72 hours with or without HPV‐16 L1 VLP before total RNA extraction. Gene expression was analyzed using the Human Genome Focus Affymetrix Array.

Vaccination with HPV 16 L1 VLP was associated with a differential expression of genes in the immune/defense response, cytokine, cell signaling and cell cycle pathways in VLP‐stimulated PBMC. In addition, there was up‐regulation of genes associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing antibody titers were found for cyclin d2 (CCND2) and the IL‐1 receptor antagonist (IL1RN). Twenty‐two differentially expressed genes were selected for confirmation by RT‐PCR in an independent group of 9 vaccine recipients. Agreement with the microarray data was seen for most of these genes.

In conclusion, microarray technology was used to identify host gene expression profiles induced by HPV‐16 L1 VLP in leukocytes from vaccine recipients. Further studies are needed to define their potential utility in prediction of HPV vaccination outcomes in clinical trials. (Funded by N01‐CO‐12400)
Cathelicidins protect mice from Bacillus anthracis spore challenge through their lytic and immunomodulatory abilities
Authors: Lisanby Mark , Swiecki Melissa , Pflughoeft Kathryn , Koehler Theresa , Kearney John
Published: June 2021

Cathelicidins are a family of antimicrobial peptides that are expressed in mammals and possess lytic and immunomodulatory properties. In this study, we determined the minimum effective concentration of cathelicidins from three different species against B. anthracis bacilli using standard radial diffusion and microbroth dilution assays. The effects of each peptide on spore germination and vegetative outgrowth were determined with microscopic and flow cytometric analyses. While no peptide prevented germination, one cathelicidin arrested outgrowth by directly lysing the plasma membrane of the developing bacilli. Cathelicidin administration protected C57BL/6 mice during subcutaneous spore challenge but failed to protect A/J mice from pulmonary infection. Cellular recruitment and depletion studies showed that the protection in C57BL/6 mice resulted from the direct lysis of germinated spores as well as the increased recruitment of Mac‐1+/Gr‐1+ cells to the injection site. These findings suggest that cathelicidins might be utilized to augment the initial innate immune response to B. anthracis spore exposure and inhibit disease progression. This work was supported by grants from the National Institutes of Health: AI057699‐03 to J.F.K, and AI057156 and AI33537 to T.M.K. M.W.L was supported by T32AI55438 and K.J.P was supported by T32AI055449.
Isolation and Characterization of a novel staphylococcal enterotoxin from the Mu50 Vancomycin Resistant Strain of Staphylococcus aureus
Authors: Cottrell Christopher A , Domanski Tammy L , Schlessman Jamie L
Published: June 2021

Staphylococcal enterotoxins are a class of superantigens that cause potentially lethal infections including pneumonia, endocarditis, necrotizing fasciitis, scarlet fever, and toxic shock syndrome. The first step in developing effective treatments to staphylococcal enterotoxin exposure is to understand fully the mechanism by which these toxins cause illness. To this end, a hypothetical enterotoxin was selected by examining the genomic sequence of the Mu50 strain of S. aureus in the NCBI database. An ORF with 31% amino acid sequence similarity to previously identified enterotoxins was amplified using PCR, cloned into a p‐MAL expression vector and transformed into competent E. coli host cells. Following confirmation of the gene sequence by bidirectional sequencing, the p‐MAL plasmid containing the toxin gene was transformed into competent E. coli BL21 DE3 host cells. Induction conditions were optimized to yield the toxin as a maltose binding protein fusion. The protein was purified from the cell extract using an amylose resin affinity column and the toxin was released from the affinity tag by Factor Xa protease cleavage. Purity of the toxin was confirmed using SDS‐PAGE and MALDI‐TOF/MS. A MTT colorimetric CD4+ T‐cell proliferation assay was used to determine the superantigenicity of the hypothetical enterotoxin.
Macrophages pulsed with intact Streptococcus pneumoniae play an active role as antigen‐presenting cells in eliciting an anti‐protein response upon adoptive transfer into naïve mice
Authors: Vasilevsky Sam , Colino Jesus , Canaday David H. , Snapper Clifford M
Published: June 2021

Macrophages, in contrast to dendritic cells (DC) are regarded as ineffective for priming naïve CD4+ T cells, but the relative role of these cells in promoting T cell‐dependent humoral immunity in a naïve host is largely unknown. We isolated bone marrow‐derived (BMM) and peritoneal (PerM) macrophages, as well as bone marrow‐derived DC (BMDC) and pulsed them with intact S. pneumoniae (Pn) followed by transfer into naive WT mice. Both Pn14‐pulsed BMM and PerM, elicited an IgM and/or IgG anti‐protein and anti‐polysaccharide (PS) response comparable to that of BMDC. Whereas the IgG anti‐protein response by Pn‐pulsed BMM exhibited an absolute requirement for T cells in the recipient, the induced anti‐PS response was largely T cell‐independent. Nevertheless, in vitro, Pn‐pulsed BMDC secreted significantly more pro‐ and anti‐inflammatory cytokines, underwent a greater degree of phenotypic maturation, and presented pneumococcal surface protein A (PspA) to a PspA‐specific T hybridoma more efficiently than either BMM or PerM. Pn‐pused BMM that were paraformaldehyde‐fixed prior to transfer or lacking expression of CD40 or MHC‐II failed to elicit an anti‐protein response, although the anti‐PS response was largely unaffected. These data are the first to demonstrate that macrophages can play an active role as APCs for eliciting a protein‐specific Ig response in vivo in a naïve host.

Supported by NIH grant 2R01 AI49192
CD8 T cell response to Mycobacterium tuberculosis
Authors: Einarsdottir Thorbjorg , Flynn JoAnne L
Published: June 2021

The immune response against Mycobacterium tuberculosis (Mtb) is generally sufficient to contain, but not clear, the infection. Memory forms following Mtb infection and BCG vaccination, but doesn't protect from reinfection with Mtb and has limited effect on bacterial load. Our research is aimed at better understanding CD8 T cell responses to Mtb and identify host factors that prevent clearing of the bacteria. We created recombinant Mtb expressing OT1 and OT2 epitopes, recognized by CD8 and CD4 T cells respectively, which allows us to follow Ag‐specific cells during murine infection. Using tetramers, ELISpot, in vivo cytotoxicity assay and intracellular cytokine staining, OT1 responses were compared to responses against an endogenous Mtb epitope (from Rv0125, designated GAP).

Our results indicate that memory cells reside in lung and lymph node after bacterial clearance and can respond faster than naïve cells. Despite this, the frequency of responding cells is not appreciably higher during memory response. This low frequency may contribute to inefficiency of the response. Additionally, most CD8 T cells are cytolytic (characterized by CD107 surface expression) or IFNg producers, but not both. Cytolysis and IFNg are both important in controlling Mtb, and the inabilityof CD8 T cells to perform both functions may contribute to persistence of bacteria in the host.

Funding: NIH/NIAID AI37859‐10
B cells are required for the host protection to the gastrointestinal nematode parasite Heligmosomoides polygyrus
Authors: Liu Qian , Gaydo Andrew , Lin Hongxia , Liu Zhugong , Urban Joseph F. , Gause William C.
Published: June 2021

Previous studies from our laboratory have shown an important role for B cells in the development of Ag‐specific Th2 cells during a polarized Th2‐type response. In this study we examined the role of B cell in host protection to a nematode parasite, Heligmosomoides polygyrus (Hp). Hp is characterized by chronic infection following primary inoculation, but following drug‐induced worm clearance, secondary inoculation triggers a host protective memory Th2‐type response resulting in worm clearance. B cell deficient JhD and control WT mice were orally inoculated with Hp, followed by anti‐helminthic drug treatment on day 14, and one month later challenged with Hp. Compared to WT mice, JhD mice were much more susceptible to Hp infection with high numbers of worms. B cell deficiency did not affect T cell activation, IL‐4 and IL‐13 production by CD4 T cells in mesenteric lymph nodes, and alternatively activated macrophage activation in granulomas surrounding developing larvae in the submucosa. However, immunofluorescent staining of granulomas showed that there were more Gr‐1+ neutrophils adjacent to the parasites, and less Gr‐1+ F4/80+ cells surrounding these Gr‐1+ cells in JhD mice compared to WT mice. These results suggest that B cells are required for the development of an effective memory response culminating in parasite expulsion.
Characterization of antibody response in women with naturally acquired HPV 16 infection using pseudovirion‐based neutralization assay
Authors: Unger Elizabeth R. , Panicker Gitika , Meadows Kristi S. , Lee Daisy R. , Blalock Emily L.
Published: June 2021

Antibody response to HPV16 infection has mainly been studied using Virus‐like Particle (VLP)‐based ELISA assays of serum. Local antibody response at the cervix may be a better indicator of protection. In this study, the pseudovirion–based neutralization assay was adapted to measure antibody titers in response to naturally acquired HPV 16 infection in both serum and cervical mucous samples. Samples were collected from women (n=230) attending colposcopy clinics and matched for age, race and HPV16 DNA status. Results showed that 54.1% of sera and 17.2% of mucous samples had positive antibody titers. Correlation of titers between the matched sera and mucosal samples was poor (ρ= 0.54), with sera being more likely to be positive for an antibody response (κ= 0.25). Univariate analysis of the data showed HPV 16 DNA status was significantly associated with both serum and mucosal antibody titer, but not with HPV 16 viral load or cervical disease. Comparison of the neutralization assay to the standard HPV 16 VLP‐based IgG ELISA also showed a fair correlation (ρ= 0.74) between titers and absorbance. In naturally acquired infections, serum antibodies are a more sensitive measure of exposure than mucosal antibodies. This study was supported in part by the National Cancer Institute's Early Detection Research Network Interagency Agreement Y1‐CN‐0101‐01.
IL‐17 is Required for CD4‐Mediated Graft‐Versus‐Host Disease
Authors: Willis‐Kappel Lucy , Goldberg Gabrielle , Ivanov Ivaylo , Suh David , King Christopher , Ligh Cassandra , Smith Odette , Iwakura Yoichiro , Littman Dan R. , Brink Marcel R.M. van den
Published: June 2021

A new CD4+ T cell subset, characterized by IL‐17 secretion (Th17 cells), has been identified. Th17 cells produce high levels of proinflammatory cytokines, including IL‐17, IL‐21 and IL‐22, and have been implicated in allograft rejection of solid organs and many autoimmune diseases. Thus far no studies have addressed the functional role of Th17 cells in acute graft‐versus‐host disease (GVHD). We performed DNA microarray analyses of the small intestine from GVHD mice and found increased expression of Th17 related genes. Using FACS we detected donor Th17 cells at early time points (day 10–14) after transplant in the spleen, lymph nodes (LN) and lamina propria in mice with GVHD, but not in untreated mice or transplanted mice without GVHD. We confirmed these findings in two MHC‐disparate models, as well as ELISA of the supernatant of in vitro stimulated cells from the spleen and LN of GVHD mice. Additionally, transplantation of IL‐17−/− allogeneic T cells induced significantly less GVHD morbidity and mortality as compared to WT transplant recipients. Finally, transplantation of RORγ (a transcription factor necessary for the development of Th17 cells) deficient donor T cells resulted in a significant survival benefit with respect to WT donor T cells. These data suggest that CD4+ Th17 cells contribute to the development of acute GVHD
CXCL9 promotes development of IFN‐γ‐producing alloreactive CD8 T cells but is not a dominant factor in directing graft infiltration following heterotopic cardiac allografting
Authors: Rosenblum Joshua , Fairchild Robert L.
Published: June 2021

Factors influencing T cell polarization during priming to cardiac allografts are poorly understood. We investigated the role of CXCL9 (MIG) in priming CD8 T cells to allografts and whether MIG recruits primed CD8 T cells to the graft. To measure MIG in the priming site, DCs and B cells were purified from B6 recipients of MHC‐mismatched A/J grafts on d 7 post‐transplant (pTx), and qRT‐PCR was performed. In allograft recipients, DCs produced 50x greater MIG mRNA than B cells. To monitor effector development in the absence of MIG, wt or MIG−/− allografts were performed and purified CD8 T cells were tested by IFN‐γ ELISPOT on d 7 pTx. For MIG−/− donors, the frequency of IFN‐γ‐producing CD8 T cells was 40% lower than wt allografts. With MIG−/− recipients, the frequency of IFN‐γ‐producing T cells approximates naïve levels. To test the role of MIG in graft infiltration, Thy1.1 donor‐primed CD8 T cells were transferred to 1) B6 Thy1.2 allograft recipients or 2) MIG−/− B6 Thy1.2 recipients of MIG−/− allografts. Grafts were harvested on d 2 post‐transfer and analyzed using FACS. Primed CD8 T cells infiltrate allografts equally regardless of MIG in the graft or recipient. Collectively, these data indicate that APCs produce MIG in the priming site and that MIG promotes development of IFN‐γ‐producing effectors. It appears that MIG is not required to recruit primed T cells to the allograft. Work was supported by RO1 A1 51620 and NIH T32 GM07250.
Underweight and overweight cluster equally with child stunting in Indonesia
Authors: Doak Colleen , Baron Ruth , Ponce Maiza Campos
Published: June 2021

Background: Previous published results showed a dual burden of underweight and overweight within households in Indonesia. Aim: This study documents the dual burden in stunted pre‐school children from Indonesia in 2000.

Methods: Definitions for stunting, underweight, wasting and overweight/obesity are based on the WHO international growth standards. Maternal weight status is assessed based on BMI definitions for underweight, overweight, or normal weight.

Results: Stunted children were predominantly normal weight (86.7%) whereas the prevalence of overweight (6.7%) and wasting (6.6%) were nearly equal. Furthermore, mothers of stunted children were also predominantly normal weight (69.1%) with only a slightly higher prevalence of overweight (17.2%) than underweight (13.7%). Comparing maternal under and overweight, stunted children two and under more frequently clustered together with underweight mothers. In contrast, the older children more frequently had overweight mothers.

Conclusions: In 2000, stunted Indonesian children and their mothers were predominantly normal weight. Furthermore, by the age of 3, stunted children have more overweight than underweight mothers. More research is needed to understand the role of food quality and/or infectious disease to the dual burden of child under‐nutrition and overweight/obesity.
A study on health‐related lifestyle, nutrition intake and diet quality of Korean male workers according to cigarettes smoking (pack/year)
Authors: Chang Ria , Sohn Hee‐Sook
Published: June 2021

This study was performed to compare the lifestyles, dietary behaviors, nutrient intakes, and diet quality of male adult workers aged 20–50 years in Korea according to smoking history. Subjects were recruited and divided into three groups according to the smoking history (pack‐year), which included non‐smoker (n=40), light‐smoker (n=26; pack‐year ≤10), and heavy‐smoker (n=24; pack‐year > 10). The heavy‐smokers more often ate irregular meals compared with the other two groups (p< 0.001). Intakes of carbohydrate (p< 0.05), Vitamin C (p< 0.05), Plant calcium (p<0.05), Iron (p< 0.01), animal iron (p<0.05), Zinc (p<0.05) of light‐smokers and heavy‐smokers were lower than non‐smoker. INQ (Index of Nutritional Quality) of these nutrients in the two smoker groups were lower than for non‐smokers. Male smokers have low intake status of Vitamin C, Calcium, Iron, Zinc, and partly low diet quality. This research suggested that increased nutrition education and revised guidelines for smokers are needed.
Dissociation Between Acute Alcohol (EtOH) Intoxication‐Induced Increases in Atrogin‐1 and MuRF1 mRNA and Rates of Proteolysis in Skeletal Muscle
Authors: Lang Charles H. , Frost Robert A. , Vary Thomas C.
Published: June 2021

EtOH abuse causes skeletal muscle wasting resulting, in part, from a reduction in protein synthesis. However, whether alcohol enhances muscle proteolysis remains controversial. The current study determined whether acute EtOH stimulated muscle protein breakdown by measuring the mRNA content for the muscle‐specific ubiquitin E3 ligases atrogin‐1 and MuRF1 (aka atrogenes) and assessing the in vitro rate of proteolysis. IP injection of EtOH in rats increased atrogene mRNA in a time‐dependent manner, peaking at 8 h (4‐fold) and returning to baseline by 24 h. The EtOH‐induced rise in atrogenes was dose‐dependent, with increases observed in rats with blood alcohol levels >150 mg/dL. In contrast, there was no EtOH‐induced change in atrogene mRNA in heart or soleus under these conditions. Elevated atrogene expression appears mediated indirectly because neither perfusion of an EtOH‐containing buffer into the isolated hindlimb of control rats nor incubation of C2C12 myocytes with EtOH increased atrogene mRNA. Finally, IP injection of a maximally effective dose of EtOH showed no change in proteolysis determined in the isolated epitrochlearis muscle. These data demonstrate EtOH selectively increases atrogene mRNA in fast‐twitch skeletal muscle, most likely via production of a secondary mediator, but this change was not associated with a concomitant stimulation of muscle protein breakdown. (AA11290 and AA12814).
The effect of dietary lysine and genetics on lysine metabolism and growth in rainbow trout
Authors: Pomeroy Stephanie Katherine , Silverstein Jeffrey T. , Barrows Frederic T , Cleveland Beth M , Blemings Kenneth P
Published: June 2021

Since feed cost represents about 70% of production cost, inexpensive protein alternatives are desirable replacements for fish meal. One drawback to the replacement of fish meal with soybean meal is that the latter protein source is first limiting in lysine. To investigate if different genetic lines of fish would respond differently to a lysine‐deficient (LD) diet, five families with high feed efficiency and 5 with low feed efficiency were studied. These 10 families were fed a LD, soybean meal containing, diet. The highest and lowest feed efficiency families also received a lysine‐adequate (LA) diet as a positive control. For each diet x family treatment combination, 5 replicates were used. Feed efficiency and thermal growth coefficient were greater (P<0.05) while hepatic somatic index (HSI) and ammonia excretion were lower (P<0.05) for fish fed the LA diet. There was a family effect on feed efficiency (P<0.0001) and thermal growth coefficient (P<0.0001), but not ammonia excretion, HSI, or indices of lysine catabolism. A single family had a numerically more favorable feed efficiency and thermal growth coefficient, when fed the LD diet, indicating that it may possess an enhanced genetic potential for performance when consuming lysine‐deficient, soybean meal containing diets.

Support: USDA/CSREES, WV‐AFMPD
Recent insights into the role of IRF6 in craniofacial and skin development
Authors: schutte brian c , dunnwald martine , kinoshita akira
Published: June 2021

Genetic variation in Interferon Regulatory Factor 6 (IRF6) causes, and contributes risk for, orofacial clefting disorders. In one of these disorders, in addition to orofacial clefts, individuals have skin webbing of the lower limbs. Thus, IRF6 is a key component of palatal and skin development in humans. In mice, Irf6 is expressed in the medial edge epithelium (MEE) that is dependent on Tgfb signaling. And, Irf6 is expressed in the spinous layer of the epidermis. Moreover, mice deficient for Irf6 have abnormal skin, limb and craniofacial development, including cleft palate. In the epidermis, in vivo and in vitro analyses demonstrate that Irf6 is necessary for keratinocyte differentiation. Furthermore, keratinocytes from Irf6–/– epidermis fail to close a scratch wound and display abnormal clonal growth, suggesting abnormal cellular proliferation and migration. In the palate, growth of the shelves is not defective, but they fail to elevate above the tongue, may be due to oral adhesions. The palatal epithelium is highly adherent, but the MEE fails to undergo its normal pattern of dissolution. Mice that are compound heterozygotes for the null and hypomorphic alleles (Irf6–/neo) display less severe abnormalities than the Irf6 deficient mice. In the oral cavity, Irf6–/neo mice have less severe oral adhesions. We are assessing the Irf6–/neo mice for defects in palatal development and epithelial differentiation.
Bone mineral density of overweight sedentary women chronically consuming low calcium diets
Authors: Thomas David Travis , Lovelady Cheryl , Wideman Laurie
Published: June 2021

Previous reports suggest that being overweight helps preserve bone mineral density (BMD) in women. Exercise and ethnicity can also influence bone preservation. The objective of this study was to investigate the relationship between BMD and low calcium diets in overweight sedentary women. Measures of lumbar spine (LS), hip and total body BMD were obtained by DXA in 3 African American and 5 Caucasian females between the ages of 33–43. Two dietary recalls were obtained by a multi‐pass approach using Nutrition Data System software. Mean body mass index was 28.2 ± 0.6 kg/m2. LS BMD was 1.28 ± 0.04 g/cm2 with a T‐Score of 0.7 ± 0.3. Total body BMD was 1.22 ± 0.03 g/cm2 and total hip BMD was 1.04 ± 0.04 g/cm2. Mean BMDs were within normal limits. T‐Scores for total body BMD and total hip BMD were 1.2 ± 0.3 and 0.3 ± 0.3 respectively. Mean calcium intake was 533 ± 50 mg/d. Correlations were examined between calcium intake and LS BMD (r = −0.65; p = 0.08), total hip BMD (r = −0.34; p = 0.41), and total body BMD (r = −0.51; p = 0.20). Until we attain our larger, final sample size, these data suggest a trend toward a relationship between low calcium intake and low LS BMD in a diverse ethnic sample. While these findings are preliminary in nature, they suggest that being overweight may not protect LS BMD when calcium intake is low in sedentary women. Exercise and adequate calcium intake remain prudent strategies for osteoporosis prevention.
Effects of Obesity on Serum Insulin‐Like Growth Factor 1 (IGF‐1) Levels Using DMBA‐Induced Ovariectomized Zucker Rat Model.
Authors: Hakkak Reza , Shaaf Saeid , Macleod Stewart , Cockrell Gail , Korourian Soheila
Published: June 2021

Obesity is associated with increased risk for post‐menopausal women. Several studies have suggested that IGF‐1 plays an important role in promotion and progression of breast cancer. The main objective of this study was to use DMBA‐induced ovariectomized obese zucker rat model to investigate the effects of obesity on serum IGF‐1 levels. Lean and obese rats were sham operated or ovariectomized at age 40 days and were gavaged at age 50 days with 65 mg/kg DMBA. Rats were killed 135 days post‐DMBA treatment. Serum IGF1 was measured by ELISA. Thirty‐six (36%) of the obese ovariectomized rats (O/O) developed mammary tumors while lean ovariectomized (L/O) rats developed no mammary tumors (P<0.001). The obese sham (O/S) rats developed mammary tumors (59%) compared to 30% of the lean sham (L/S) rats (P<0.05). Ovariectomy caused a significant (P<0.001) increase in serum IGF‐1 for ovariectomized lean group but not in obese group. Also, obesity significantly increased (P<0.001) serum IGF‐1 in both sham and ovariectomized rats compared to lean rats. Our results suggest that obesity is a major risk for DMBA‐induced mammary tumor development and elevated IGF‐1 levels may be associated with the increased susceptibility to DMBA‐induced mammary tumors. Other mechanisms responsible for increased mammary tumor susceptibility in obese rats are under investigation. (Supported by Susan G. Komen Foundation and ABI to RH).
Evaluation of dietary factors in relation to the biomarkers of oxidative stress and inflammation in breast cancer patients
Authors: Yeon Jee‐Young , Suh Young‐Jin , Kim Sang‐Wook , Baik Hyun‐Wook , Kim Hyun‐Sook , Sung Mi‐Kyung , Sung Chung‐Ja
Published: June 2021

Recent epidemiological evidences have suggested a positive association between the intake of antioxidant nutrients and breast cancer (BC) risk. The purpose of this study was to compare the intake of antioxidant nutrients and oxidative stress and inflammation‐related biomarkers between BC patients (n = 153) and their healthy counterparts (n = 151). The mean caloric and fat intake of BC patients and controls was not significantly different from each other. The intake of folate and antioxidant nutrients including vitamin A, vitamin C and vitamin E were significantly lower in BC patients compared to the control subjects (p<0.001). Plasma concentrations of interleukin (IL)‐1β (p<0.001), IL‐6 (p<0.001) and prostaglandin E2 (p<0.05) were significantly higher in the BC patients. A significantly higher level of plasma concentrations of 8‐OHdG (8‐hydroxy‐2′‐deoxyguanosine, p<0.001) was observed in BC patients. Positive correlations were observed between inflammatory biomarkers, while antioxidant nutrient intakes were not related to the level of inflammatory markers. IL‐6 level was positively associated with the intake of total calorie, fat, or carbohydrate. Our results suggest that oxidative stress and inflammatory response may be related to the BC progress and dietary factors may play a role. This work was supported by the SRC/ERC program of MOST/KOSEF (RESEARCH CENTER FOR WOMEN's DISEASES)
In Vitro Fermentation Characteristics of Native Soluble Fiber Sources by Human Colonic Bacteria
Authors: Hussein Hussein S. , Yobi Abou , Sakuma Toshie , Bollinger Laurie M. , Wolf Bryan W. , Garleb Keith A.
Published: June 2021

Fermentation of 20 soluble fibers (alginate [ALG], apple pectin [AP], arabinogalactan [AG], carrageenan [CG], carboxymethylcellulose [CMC], citrus pectin [CP], gellan gum [GLG], guar gum [GUG], gum Arabic [GA], gum ghatti [GGH], gum karaya [GK], hydrolyzed guar gum [HGG], konjac flour [KF], locust bean gum [LBG], methylcellulose [MC], oat‐β‐glucan [OBG], psyllium [PS], tomato pectin [TP], tragacanth gum [TG], and xanthan gum [XG]) was evaluated in vitro using three human fecal inocula. Serum bottles (n = 1,008; 125 mL each) were used in a completely randomized design experiment with treatments being arranged as a 20 (substrates) × 8 (incubation times; 1.5, 3, 4.5, 6, 9, 12, 18, and 24 h) factorial. At 24 h, in vitro dry matter disappearance varied (P < 0.05) as it was lowest (20.3%) for GK, moderate (40.1 to 70.6%) for AP, CP, GLG, GGH, and PS, and highest (≥ 91.0%) for the others. At 24 h, gas production also varied (P < 0.05) and was highest (≥ 74.0 mL) for GUG, HGG, KF, LBG, OBG, PS, TP, and TG. Of the remaining substrates, four (AP, AG, CG, and CP) had moderate amounts (30.5 to 58.7 mL), four (ALG, GA, GGH, and XG) had very small amounts (≤ 26 mL), and four (CMC, GLG, GK, and MC) had no gas produced. Several soluble fiber sources appear to have high potential in supporting human colon health. The concern with high gas production by some of these fiber sources, however, may limit their supplementation.
Investigating potential anti‐diabetic effects of specific soy isoflavones and metabolites in female obese Zucker rats
Authors: Banz William J , James H Bryce , Ajuwon Kolapo M
Published: June 2021

The purpose of this study was to investigate possible anti‐diabetic effects of the soy isoflavone, daidzein and its metabolite equol in an insulin resistant rodent model. Female obese Zucker rats were randomly assigned to four groups for 11 weeks: casein control (C), injected daidzein (CD), injected equol (CE), and high isoflavone soy protein (H). To bypass GI modification, daidzein and equol were injected and not orally administered. At week 9 animals were subjected to an oral glucose tolerance test (OGTT). At and subsequent to sacrifice, physiological, biochemical, and molecular parameters were examined. Animals on the H diet gained significantly more weight and exhibited a significant increase in food intake when compared to all other groups and also had significantly lower liver weight versus C, CD and CE groups. Additionally, the H animals demonstrated a significantly lower non‐fasted blood glucose level versus the CD and CE groups and the same trend (P<0.07) was observed versus the C group. However, the OGTT did not reveal a significant improvement in glucose tolerance between groups. There was a significant decrease in cholesterol and increase in triglyceride levels in the H group compared to all other groups. Insulin levels were not significantly different between groups. Overall, these results demonstrate differential responses to soy and specific injected soy components and metabolites.
Bitter melon extracts suppressed differentiation and triglyceride content of 3T3‐L1 adipocytes
Authors: Tam Ka Shing , Wu Jiajun , Tse Iris , Tam Tsun Ming , Wang Ming Fu , Li Edmund Tsze Shing
Published: June 2021

Bitter melon (BM; Momordica charantia) supplementation slows weight gain and tissue fat accumulation in fat diet‐induced obese rat. Liver and muscle carnitine palmitoyl transferase‐I expression was enhanced in BM rats, suggesting increased lipid oxidation. This study was designed to determine if extracts from BM juice can suppress triglyceride (TG) content in the 3T3‐L1 cell line. 3T3‐L1 pre‐adipocytes were induced to differentiate in the presence or absence of an ethanol‐water extract of BM juice. The latter was obtained by stepwise gradient elution of an organic extract of BM juice. Two fractions (eluted by 10% & 30% ethanol respectively and then freeze‐dried; F2 at 350μg/mL & F3 at 50μg/mL) were found to reduce TG content on day 10 (p<0.01, F3) & day 12 (p<0.01, both); and GPDH activity on day 10 & 12 (p<0.01, both) of a 12‐day study. Two‐way ANOVA indicated reduced mRNA expression of fatty acid synthase (p<0.001), acetyl CoA carboxylase 1 (p<0.01) and Glut‐4 (p<0.01) in BM‐treated adipocytes. With F3, these effects primarily occurred between days 8–10. Downregulation of PPARγ (p<0.001) and SREBP‐1c (p<0.001) mRNA expression was observed in F3‐treated cells on day 12. The data suggest that bioactive ingredient(s) in BM may suppress TG synthesis in 3T3‐L1 cells through suppressing the expression of certain lipogenic genes and transcription factors. (Supported by the Faculty Development Fund, HKU)
Green Leafy Vegetable Extracts Prevent Cu2+‐Induced Oxidation of Human Low Density Lipoprotein
Authors: SIM HYUN JUNG , SON CHAN WOK , LIU XI WEN , KIM MEE REE
Published: June 2021

Objective: Oxidation of low density lipoprotein(LDL) is regarded to play an important role in the development of atherosclerosis. Green leafy vegetables contain mixed antioxidants are responsible for their health benefits, and their advantage over single antioxidant may be due to a combination of additive and/or synergistic effects. In this respect, we evaluated that the total phenol content and antioxidant activity against LDL oxidation of green leafy vegetables.

Methods: Freeze‐dried green leafy vegetables were extracted with methanol. we selected the vegetables showing strong antioxiant activity based on the DPPH radical scavenging activity. The selected vegetable extracts were evaluated antioxidant action of Cu2+‐induced oxidation of human LDL. In addition, total phenol content was determined colorimetrically using Folin‐Ciocalteu reagent.

Results: The extracts of vegetables expressing a strong inhibition of LDL oxidation with EC50 values of less than 100 μg/mL were angelica, dandelion, mustard leaf and water spinach, which contain high total phenolic content. Highly positive correlations between inhibition of LDL oxidation and total phenol content were observed (p<0.001).

Conclusion: Based on these results, it is suggested that salad vegetables, especially angelica, dandelion, mustard leaf, could be employed as easily accessible sources of natural antioxidants, especially associated with anti‐atherosclerosis purpose.