bluuebunny/crossref_metadata_2025 huggingface dataset:
Androgen Effects On Soluble Epoxide Hydrolase Expression and Cardioprotection
Authors: Batchu Sri Nagarjun , Pawa Jasmine , Zhang Yunfang , Myers Page , Clark James , Miller‐Degraff Laura , Zeldin Darryl C , Sinal Christopher J , Goralski Kerry , Seubert John M
Published: June 2021

Androgens can regulate hepatic and renal expression of soluble epoxide hydrolase (sEH or EPHX2) which metabolize epoxyeicosatrienoic acids (EETs) to less bioactive metabolites. To examine the role of EETs and testosterone in ischemia reperfusion injury, mice with targeted disruption of soluble epoxide hydrolase (sEH null) were utilized. Gonadectomies were performed on randomly selected mice either at post‐natal day1 (PN1) or 3 months of age (cast). Hearts were excised and perfused in a Langendorff apparatus, subjected to 20min of ischemia followed by 40min of reperfusion. PCR analysis of EPHX2, αMHC, βMHC and GAPDH expression was performed. Improved postischemic recovery of left ventricular function was observed in male sEH null mice compared to WT (LVDP: 63±3 and 22±2%), which was diminished in females (LVDP: 40±6 and 22±5%). Gonadectomy improved postischemic recovery of LVDP (WT: control 25±3; PN1 68±11; cast 67±12%; sEH null: control 64±3; cast 88±14%). Renal expression of EPHX2 decreased following gonadectomy and was restored following androgen treatment. In contrast, no differences in cardiac EPHX2 expression were observed between treatment groups. Increased βMHC:αMHC ratio gene expression in gonadectomized mice was observed. While gender differences play a role in postischemic functional recovery our data would suggest that androgens do not influence cardiac expression of sEH.
Characterization of the human sEH phosphatase by site directed mutagenesis and LC‐MS/MS analysis
Authors: Cronin Annette , Homburg Shirli , Dürk Heike , Richter Ingrid , Adamska Magdalena , Frere Frederic , Arand Michael
Published: June 2021

The bifunctional human soluble epoxide hydrolase (sEH) is implicated as a regulator of diverse physiological processes due to the brakedown of arachidonic acid derived signalling molecules like epoxyeicosatrienoic acids (EETs) by its epoxide hydrolase domain. Recently, we discovered that the sEH N‐terminal domain displays a novel phosphatase activity. sEH accepts the generic substrate 4‐NPP as well as lipid phosphates, but the physiological role remains uncertain.

The phosphatase domain contains three conserved sequence motifs, including the potential catalytic nucleophile Asp9, and several residues implicated in substrate turnover and/or Mg2+ binding. To enlighten the proposed catalytic mechanism we constructed active site mutants by site directed mutagenesis, which were recombinantly expressed as soluble proteins, purified and analysed for their kinetic properties. An exchange of Asp9, Lys160, Asp184 or Asn189 results in a complete loss of phosphatase activity, emphasising the requirement of these amino acids for catalysis, whereas a substitution of Asp11, Thr123, Asn124 and Asp185 leads to sEH mutant proteins with residual phosphatase activity. To confirm the role of Asp9 as catalytic nucleophile we presently analyze the presumed phosphoester intermediate by mass spectrometry. The dual phosphatase and epoxide hydrolase activities give new insights into the physiological role of human sEH.
Soluble epoxide hydrolase inhibitor improves cardiac ischemic tolerance in Ren‐2 transgenic rats
Authors: Neckar Jan , Cervenka Ludek , Hammock Bruce D , Hwang Sung H , Imig John D , Kolar Frantisek
Published: June 2021

Arachidonic acid metabolites of the cytochrome P450 epoxygenase pathway, the epoxyeicosatrienoic acids (EETs), have been shown to protect hearts against ischemia/reperfusion injury. Here, we examined effects of inhibition of the EETs‐degrading enzyme, soluble epoxide hydrolase (sEH), on cardiac ischemic tolerance in hypertensive transgenic rats harboring murine Ren‐2 gene (TGR). Anesthetized (pentobarbital) open‐chest rats were subjected to 20‐min LAD coronary artery occlusion and 3‐h reperfusion for infarct size determination (tetrazolium staining). The sEH inhibitor, cis‐4‐[4‐(Adamantan‐1‐yl‐ureido)‐cyclohexyloxy]‐benzoic acid (c‐AUCB), was given in drinking water (0.5 mg/day) starting 3 days prior ischemia. Untreated TGR exhibited smaller infarction (58.2 ± 3.0 % of the area at risk, P<0.05) than control Hannover SD rats (71.0 ± 3.5 %). c‐AUCB decreased mean arterial blood pressure (by 31 mm Hg, P<0.05), further reduced infarct size (43.4 ± 2.9 %, P<0.05) and tended to suppress ischemic ventricular arrhythmias in TGR but not in SD rats. These data indicate that the inhibition of EETs degradation is cardioprotective in Ren‐2 transgenic rats.
Neutrophil expression of the C5a receptor is increased in type 2 diabetes
Authors: La Bonte Laura R , Davis‐Gorman Grace , Stahl Gregory L , McDonagh Paul F
Published: June 2021

Ischemic heart disease is associated with excessive complement activation leading to increased plasma complement component C5a. Some effects of C5a are mediated through binding to the C5a receptor (CD88) on circulating PMNs. Previously we found that diabetic PMNs are chronically activated, as measured by increased CD11b expression. Complement activation plays an important role in the inflammatory response and since type 2 diabetes is associated with chronic inflammation, we tested the hypothesis that CD88 expression is increased on PMNs from type 2 diabetic blood, perhaps partly explaining the observed basal increase in PMN CD11b expression. Whole blood samples were taken from Zucker Diabetic Fatty (ZDF) and non‐diabetic Sprague‐Dawley (SD) rats. Blood samples were incubated with labeled anti‐CD45, a pan‐leukocyte marker, for the detection of PMNs. and then with labeled anti‐CD88. Flow cytometry was used to measure the total fluorescence intensity (TFI) of CD88 in the CD45‐positive PMN population. We found that CD88 expression was significantly increased on PMNs from diabetic blood compared to non‐diabetic blood (Non‐diabetic: 16.4±1.3 TFI, Diabetic: 34.4±5.4 TFI, p<0.05). These results suggest that the chronic inflammation that accompanies type 2 diabetes influences the expression of the PMN C5a receptor. These results may partly explain the hyper‐responsiveness of diabetic PMNs to acute stressors such as ischemia/reperfusion. Supported by NIH 58859 and AHA 0610018Z.
The frequency dependence and perfect adaptation of osmo‐sensing in budding yeast
Authors: van Oudenaarden Alexander
Published: June 2021

The propagation of information through signaling cascades spans a wide range of time‐scales, including the rapid ligand‐receptor interaction and the much slower response of downstream gene expression. To determine which dynamic range dominates a response, we use periodic stimuli to measure the frequency dependence of signal transduction in the osmo‐adaptation pathway of Saccharomyces cerevisiae. We apply system identification methods from control engineering to infer a concise predictive model. We find that the dynamics of osmo‐adaptation is dominated by a fast‐acting negative feedback through the kinase Hog1 that does not require protein synthesis. At elevated osmo‐shocks an additional, much slower, negative feedback through gene expression allows cells to adapt faster to future stimuli.
Comparison of placenta‐derived MSC (pMSC) with bone marrow MSC (bmMSC) expanded in GMP‐compatible medium
Authors: Pilz Gregor A , Braun Julian , Abele Harald , Schäfer Richard , Aicher Wilhelm K.
Published: June 2021

Objectives:

Human term placenta is a promising source of mesenchymal stromal cells (MSC). After expansion in GMP‐compatible medium containing human‐platelet‐rich plasma, these cells could be utilized in regenerative medicine. In our study we examined the properties of pMSC in comparison to bmMSC.

Methods:

PMSC were isolated from term placenta and expanded in FCS‐free, GMP‐compatible medium. Characteristic MSC antigen expression of plastic adherent cells was observed by flow cytometry. Differentiation to adipogenic, osteogenic and chondrogenic lineages was detected by cytochemistry and immunostaining. Marker gene expression was measured by quantitative RT‐PCR.

Results:

PMSC can be expanded efficiently in GMP‐compatible medium while retaining their typical antigen expression and showing differentiation potential comparable to that of BM MSC.

Conclusion:

Human term placenta can be used as a ethically unobjectionable source of MSC and represent a non‐invasive alternative to bmMSC. The cultivation of MSC in “GMP‐compatible medium” allows rapid expansion to provide the necessary cell numbers for tissue engineering
Alpha‐synucleinopathy is associated with spinocerebellar ataxia type 2 with Parkinsonism
Authors: Takao Masaki , Sakiyama Yoshio , Saito Yuko , Aoyama Masahiro , Shiroyama Harumi , Kurisaki Hiroshi , Ishikawa Kinya , Tsuchiya Kuniaki , Mihara Ban , Murayama Shigeo
Published: June 2021

Levodopa‐responsive Parkinsonism may be dominant phenotype of patients with SCA2. In general, they have a shorter abnormal expansion of CAG repeats (less than 39) in ATXN2 gene. The aim is to elucidate neuropathologic basis of this variant SCA2 from different Japanese families and give a new perspective on pathogenesis of SCA2 with Parkinsonism.

Case 1: He was diagnosed as having Parkinson's disease at age 50. At age 65, he showed cerebellar ataxia and slow eye movement. Case 2: He developed unstable gait at age 40. In addition to the ataxia, he had rigidity, bradykinesia, stooped posture and freezing gait. Genetic analyses of ATXN2 showed an expanded CAG repeat with 22/38 (case 1) and 22/37 (case 2).

Neuropathology: There is an atrophy of the pons and cerebellum. The most striking finding was the presence of α‐synuclein immunoreactive Lewy bodies (LBs) and neuritis (LNs) in the locus coeruleus, dorsal motor nucleus of vagus, basal nucleus of Meynert and amygdala. LBs and LNs were also seen in the substantia nigra. The distribution of Lewy pathology was consistent with stage 2 (case 1) and stage 3 (case 2) according to Braak's criteria. The 1C2 antibody depicted intranuclear immunopositive deposits in the various regions. In addition, α‐synuclein deposits were not seen in SCA2 cases without Parkinsonism.

We conclude that α‐synucleinopathy is vital pathologic alterations of SCA2 with Parkinsonism. JSPS 19500311
Function after repair of skeletal muscle defect with muscle‐derived extracellular matrix
Authors: Merritt Edward K , Walters Thomas J , Baer David G , Farrar Roger P
Published: June 2021

The loss of a significant portion of skeletal muscle poses a unique challenge for the normally robust regenerative capacity of muscle tissue. A partial transection injury with tissue loss, which causes a large gap to develop in the muscle, will not fully heal as the fibers are unable to span the gap to reconnect the disconnected ends. In order to determine a possible functional repair technique of a skeletal muscle defect, a 1 x 1 cm portion of the lateral gastrocnemius of Lewis rats was removed. A skeletal muscle derived extracellular matrix (ECM) was implanted in place of the lost tissue. Without ECM repair, the maximal isometric tetanic force of the defected lateral gastrocnemius was 59.6% ± 4.7, 72.9% ± 2.3, and 78.0% ± 1.5 of the nonoperated contralateral limb 7, 14, and 28 days post‐injury. With ECM repair, the maximal force of the defected lateral gastrocnemius was 80.7% ± 8.4, 77.4% ± 4.2, and 84.0% ± 3.6 of the nonoperated limb. These data suggest that after 28 days of recovery from a defect injury, the lateral gastrocnemius will not recover full functionality. Addition of an extracellular matrix to fill the defect results in more force 7, 14, and 28 days post‐defect, but full recovery still does not occur. This work funded in part by the U.S. Army Medical Research and Material Command grant DAMD W81XWH‐06‐1‐0540 to RPF
Yeast High Mobility Group Protein HMO2 preferentially binds to DNA ends
Authors: Ray Sreerupa , Grove Anne
Published: June 2021

High Mobility Group Proteins (HMG) are non‐histone chromosomal proteins which are common in eukaryotes. They bind to DNA in a non sequence specific manner. HMO2 from Saccharomyces cerevisiae is a HMGB protein that is a component of the DNA‐damage dependent chromatin remodeling complex INO80, which is involved in double strand break repair (DSBs) in yeast. The gene encoding HMO2 was amplified from yeast genomic DNA. It was expressed in E. coli Rosetta Blue cells and purified using nickel affinity chromatography. DNA binding assays indicate that HMO2 binds to supercoiled DNA with higher affinity than linear DNA. Like other HMGB proteins it has a preference for distorted DNA such as four‐way junctions () and DNA with loops, but it has no preference for nicks, gaps and overhangs. DNA end‐joining and exonuclease protection assays show that it binds preferentially to the DNA ends, both blunt and cohesive ends, indicating that it might play a role in DNA repair. Further, HMO2 is also able to supercoil relaxed DNA in presence of T4 DNA ligase. Consistent with the distinct functions of HMO2 and its closely related homolog HMO1, HMO2 does not rescue an hmo1 knockout phenotype.
Charge‐driven surface‐colloid interaction for detection biomolecules on microarrays.
Authors: Sun Ye , Golovlev Val
Published: June 2021

The aim of this study is to demonstrate a new approach for high‐multiplexed analysis of RNA, DNA, and proteins without chemical modification of target molecules for detection. The approach utilizes nanometer‐sized particles, which carry electric charges and are attracted by electrostatic force to target molecules captured on a solid surface. The nano‐particles bind to the surface proportionally to the number of target molecules. Location and surface density of the bound particles can be measured by a low‐cost optical detection system. In our study the charge‐driven particle‐surface interaction was investigated using Gouy‐Chapman‐Stern‐Graham model. The model predicts experimental conditions at which nano‐particles bind selectively to large target molecules and do not bind to small capture molecules present on the same surface. Important implication of this result is the possibility to detect DNA, RNA, and proteins immobilized on a surface with no need for chemical modification or labeling of the target molecules. The detection approach has been demonstrated for gene expression analysis and for protein detection using microarrays.

This study is supported by NIH grant 2R44GM074311.
Mutagenesis of Membrane Subunits of the Escherichia coli Complex I
Authors: Michel Jose , Vik Steven B
Published: June 2021

Complex I plays a major role in the electron transport chains of mitochondria and of many bacteria, but little is known about its mechanism. The bacterial version is considered a minimal version of the mammalian complex I. Oxidation of NADH, reduction of ubiquinone, and proton translocation are three essential functions of Complex I. To identify critical amino acids required for these functions we used site‐directed mutagenesis to create amino acid substitutions in subunits L, M and N. These subunits are the largest subunits in the membrane arm of the complex, and are thought to be involved in proton translocation. Substitutions were found that led to reduced activity, and in some cases to a total loss of activity. In addition, truncations and deletions of individual membrane subunits were constructed and analyzed. In general, function of the complex was lost, but truncation or deletion of subunit L was the least deleterious. In addition, western blots of membrane fractions from the deletion strains indicated that the membrane arm of the complex does not assemble when individual subunits are missing.

Supported by a grant from the Welch Foundation.
Expression and Characterization of a Functional Leishmania Variant of Cytochrome b5 Reductase
Authors: Barber Michael J , Roma Glenn William , Seyfang Andreas
Published: June 2021

Cytochrome b5 reductase (cb5r), a member of the FNR family of flavoprotein transhydrogenases, catalyzes the NADH‐dependent reduction of cytochrome b5 (cb5). The putative nucleotide sequence for the L. donovani enzyme was used to clone and generate a histidine‐tagged form of the soluble, catalytic domain of cb5r. The recombinant protein was purified to homogeneity and exhibited absorption and CD spectra comparable to the wild type rat cb5r diaphorase domain, suggesting similar protein folding and flavin incorporation. Initial‐rate kinetic studies showed a decreased turnover rate in the Leishmania diaphorase domain, (kcat of 283s‐1) for the NADH:FR as compared to the rat domain (800s‐1). Km for NADH was comparable to that of wild type rat cb5r. Thermal stability studies revealed decreased thermal stability (49oC) compared to that of the rat protein (55oC). Spectral binding studies indicated comparable affinity for and binding of H4NAD and NAD+ for both variants. Oxidation‐reduction potentials in both the absence (−275mV) and presence (−195mV) of NAD+ were comparable to values previously obtained for the corresponding rat domain (−274 mV and −190 mV). These results provide direct comparison of an enzyme from two divergent phylogenetic sources using identical expression systems and indicate a species‐based difference in the structural, spectroscopic, kinetic and thermodynamic properties of the enzyme.
Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine
Authors: Matsuo Yasuhiro , Fisher Edward , Patton‐Vogt Jana , Marcus Stevan
Published: June 2021

We describe the characterization of the pps1 gene encoding a predicted phosphatidylserine (PS) synthase in the fission yeast, Schizosaccharomyces pombe. pps1 Δ mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable PS in vivo and possess negligible in vitro PS synthase activity, indicating that pps1 encodes the major PS synthase activity in S. pombe. Supplementation of media with ethanolamine partially suppresses the growth defect of pps1 Δ cells, reflecting the likely importance of PS as a precursor for phosphatidylethanolamine in S. pombe. pps1 Δ mutants exhibit morphology, cytokinesis, cytoskeletal, and cell wall defective phenotypes. Overexpression of pps1 also leads to cell morphology and cytokinesis defects, implicating PS as a dosage‐dependent regulator of these processes. During log phase, GFP‐Pps 1p fusion proteins are concentrated at the cell and nuclear peripheries and presumptive ER membranes, while in stationary phase cells, they are redistributed to unusual cytoplasmic structures. Moreover, stationary phase pps1 Δ cultures retain poor viability relative to wild type S. pombe cells, even in medium containing ethanolamine, demonstrating a critical role for PS in stationary phase survival. Our findings demonstrate novel cellular functions for PS and the usefulness of S. pombe as a model organism for elucidating its molecular functions.
Apoptosis is the Primary Phenotype Evoked by p190RhoGAP Overexpression
Authors: Ludwig Kirsten , Parsons Sarah J.
Published: June 2021

p190RhoGAP (p190) is a RhoGTPase activator protein (GAP) that has been shown to regulate actin cytoskeletal dynamics via Rho‐dependent signaling pathways. By enhancing Rho‐mediated hydrolysis of RhoGTP to RhoGDP, p190 negatively regulates association of RhoGTP with its downstream effectors. The p190 gene is located on chromosome 19q13.3, which is mutated in several solid tumor types, suggesting its involvement in malignant transformation. Findings that p190 reduced the incidence of PDGF‐induced gliomas in mouse models further implicate it as a tumor suppressor.

To gain insight into its mechanism of action, an overexpression approach was taken. Previous studies in our lab revealed that overexpression of p190 in epithelial cells led to a multi‐nucleated phenotype, suggesting that it negatively regulates mitosis. Chromatin condensation, an apoptotic marker, was also frequently observed. In contrast, overexpression of p190 in fibroblasts led to dendrite formation or chromatin condensation. To further elucidate which cellular processes p190 influenced most, both epithelial cells and fibroblasts were examined for the frequency of the three phenotypes in cells overexpressing p190. The most prominent phenotype in both cell types was apoptosis, as determined by TUNEL staining and caspase 3 cleavage. Real‐time microscopy in epithelials revealed that multi‐nucleation resulted in apoptosis, as did dendrite formation in fibroblasts. In conclusion, we found that overexpression of p190RhoGAP primarily resulted in apoptosis of both cell types, and that multi‐nucleation and dendrite formation appeared to be intermediate phenotypes leading to apoptosis in epithelial and fibroblastic cells, respectively. These results suggest that p190 may utilize different pathways in different cell types to mediate its anti‐tumor effects.

National Institutes of Health CA39438 (SJP)
Autophagic cell death on reductive cardiac development in STZ‐induced diabetic rats
Authors: Lee Youngjeon Lee , Lee Sang‐kil , Won Jinyoung , Lee Narae , Hong Yonggeun
Published: June 2021

Diabetes is a major predictor of heart failure. However, little is known regarding mechanisms how it causes heart to pathological remodeling and even cardiac dysfunction. Here, we tested the role of autophagy in diabetic cardiac dysfunction. Here, we hypothesized that diabetes induces cardiac autophagy. When H9c2 cells were exposed to low‐glucose and/or glucose‐free media, we observed augmented LC3 expression, which serves as a marker protein for autophagosomes. To confirm whether it does reflection of in vitro evidence compared to in vivo diabetic animal model, diabetic mellitus was induced using by streptozotocin (60mg/kg, IP) at 4‐wks‐age in rats. Body weight and blood glucose level were measured throughout this experiment. The left ventricle (LV) including the interventricular septum was isolated and weighed for body weight and tibial bone length. Diabetic rats have shown the reduction of size of LV rather than hypertrophy according to growth retardation and higher level of LC3 protein expression, suggesting activated autophagic cell death. Taken together, these findings implicate that progression of pathological remodeling of heart development is associated with autophagy. Thereby we suggest further studies regarding effect of diabetic cardiac autophagy on heart dysfunction including heart failure. [Supported by KRF–2006–E00344).
Evaluation of iTunes University Podcasts on Undergraduate Biology Education
Authors: Smith Christopher J , Beliz Tania , Martin Theresa , Gibbs David , Mach Jean
Published: June 2021

The recent emergence of podcast use in education has enabled faculty to offer students access to digitally recorded lectures and course material. These approaches to education present the student with supplements and alternatives to study, lecture review and learning. This study was undertaken to evaluate these efforts to support student learning in undergraduate‐level science courses. Unedited podcasts of recorded lectures of biology classes were posted on College of San Mateo's iTunes University website provided by Apple Corporation. Surveys were administered to students at the course mid‐point to evaluate the use, effectiveness and outcome of the recorded lectures. Survey results were grouped according to level of use, subjective student impression of use, and the outcome of use in order to explore the effect on actual performance outcome. The results showed that in second level classes the majority of students accessed the recorded lectures. In contrast, the majority of students did not access the recorded lectures in introductory level biology classes. Students that used the podcasts the most felt that doing so improved their performance in the class. It was also indicated that the group of second level students most in need of review used the podcasts the most. Further studies are underway to evaluate the effectiveness of podcast use on performance in order to more effectively use the podcasts to support both in‐ and out‐of‐class learning and contribute to student success.
Evolutionarily conserved cysteines in the stalk region of CD3‐epsilon are critical for T cell development and function
Authors: Wang Yibing , Becker Dean , Vass Tibor , White Janice , Marrack Philippa , Kappler John W.
Published: June 2021

The T cell receptor (TCR) is a multimeric complex. The basic elements of an alpha‐beta TCR include TCR‐alpha, TCR‐beta, CD3‐epsilon, CD3‐delta, CD3‐gamma, and zeta chain. The CD3 proteins have an extracellular stalk region which has a highly conserved CXXC motif. The two cysteines can be found in every CD3 molecule that exists today. Previous studies have shown that the two cysteines are important for TCR assembly. However, the conclusion has been controversial. We have generated transgenic mice that carry only either wild type‐like or cysteine‐mutated CD3‐epsilon. The transgenic mice with mutated CD3‐epsilon have revealed distinct features different from normal mice. Two key findings have been made: 1) replacing those two cysteines with serines in CD3‐epsilon does not block assembly of TCR complex but does cause a series of T cell‐related physiologic defects in mice; 2) the mutation in CD3‐epsilon severely impairs the ability of T cell signaling initiated from the TCR. We have concluded that the two conserved cysteines in the stalk region of CD3‐epsilon are not crucial for TCR assembly. Instead, they play a critical role for T cell development and function by controlling TCR signaling. This work has been supported by National Institutes of Health Grant AI‐17134 and AI‐18785.
Age‐related extrinsic factors affect naive CD4 T cell responses
Authors: Maue Alexander , Eaton Sheri , Lanthier Paula , Haynes Laura
Published: June 2021

Declines in immune function with age contribute to the reduced efficacy of vaccines in the elderly. Age‐related intrinsic defects in CD4 T cell function lead to reduced B cell helper activity. New studies have found extrinsic age‐related changes that influence the responses of naïve CD4 T cells. Aged mice displayed a significant increase in the number of CD4 T cells that express molecules associated with immune regulation as compared to young, including IL‐10, CD25, GITR and CTLA‐4. IL‐10 production resided almost exclusively within the CD62Llo subset of CD4 T cells. In vivo, naïve young transgenic (Tg) CD4 cells transferred into young or aged hosts exhibited decreased antigen‐specific expansion in aged hosts. This effect was attributable to host CD4 T cells, as in vivo CD4 depletion prior to adoptive transfer alleviated the impediment to proliferation. To determine this mechanism, an in vitro proliferation model was established in which young naïve Tg CD4 cells were primed in the presence of CD44hi or CD44lo polyclonal cells from young or aged mice. CD44hi cells impeded dilution of CFSE within naïve cells regardless of donor age. Furthermore, IL‐10R blockade within CD44hi‐containing cultures abrogated the decreased proliferation of transgenic CD4 cells. Our results demonstrate that external factors in aged mice may act to inhibit naïve CD4 T cell responses.
TIM‐1 signaling in T cell activation
Authors: Lin Jean Yean‐Jin , Kane Lawrence P.
Published: June 2021

T cell immunoglobulin and mucin 1 (TIM‐1) is capable of acting as a co‐stimulatory molecule to enhance T cell activation and to modulate CD4+ T cell responses. Currently, relatively little is known about the signal transduction pathways downstream of TIM‐1 that are responsible for controlling these immune responses. Previous work from this laboratory has demonstrated that src‐kinase dependent tyrosine phosphorylation of the cytoplasmic tail of TIM‐1 is important in transmitting the TIM‐1 response in the T cell. The purpose of this study is to explore additional tyrosine phosphorylated proteins that may be downstream of TIM‐1 signaling. Whole cell lysates from TIM‐1 stimulated D10 cells were probed for tyrosine phosphorylation. This work has suggested a previously unrecognized phosphorylated substrate that can be induced by agonistic anti‐TIM‐1 antibodies. We are currently working to identify this protein as well as to better define the conditions under which it is phosphorylated.

Supported by NIAID and NCI.
Role of Foxo1 in T cell Activation and Survival
Authors: Tinoco Roberto , Beisner Daniel R , DeJean Anne , Kerdiles Yann , Hedrick Stephen M
Published: June 2021

FOXO1 is a transcription factor, a member of a class of evolutionary conserved forkhead genes that also includes Foxo3a, Foxo4, and Foxo6. FOXOs regulate the expression of apoptotic, cell cycle progression, and detoxification genes. In response to growth factor signaling, FOXOs are phosphorylated and excluded from the nucleus to prevent their transcriptional activity. FOXOs have been extensively studied and shown to be important in longevity and cancer. FOXO1 is highly expressed in humans and mice; however, its function in T cells has not been examined. Foxo1 was conditionally deleted in T cells, Foxo1 KO mice displayed a higher proportion of activated T cells. In addition, Foxo1 KO mice had an increased number of CD4+ but not CD8+ T cells in lymph node and spleen. Upon activation, FOXO1 KO T cells, proliferated, yet failed to accumulate due to impaired survival. This accumulation defect after activation was also observed in FOXO1 KO TCR transgenic T cells. When we examined the role of FOXO1 in survival and cytokine rescue in activated CD4+ T cells, we observed that FOXO1 KO CD4+ T cells were less sensitive to growth factor withdrawal induced apoptosis. This FOXO1‐mediated survival effect can be partially attributed to decreased levels of Bim expression. These findings demonstrate that FOXO1 plays a very important role in regulating T cell activation and survival.
TGF‐beta REGULATES ANTI‐VIRAL IMMUNITY
Authors: Garidou Lucile , Truong Phi , McGavern Dorian
Published: June 2021

Because persistent infections burden the immune system in several ways, it is important to understand the factors that regulate immune responsiveness and therapies that achieve eradication of persistent pathogens. Administration of virus‐specific memory T cells (referred to as adoptive immunotherapy) is an efficacious strategy to purge persistent viruses from humans and mice. When memory T cells are administered to mice burdened with a persistent lymphocytic choriomeningitis virus (LCMV) infection from birth, the virus is purged from all tissues without inducing fatalities, suggesting that the transferred T cells are exquisitely regulated. Transforming growth factor beta (TGF‐β) is an immunosuppressive cytokine known to thwart the development of autoimmunity, but its role in the regulation of pathogen‐specific T cells is not entirely understood. TGF‐β is capable of preventing CD8 cytotoxicity by inhibiting perforin expression and can also inhibit IFN‐γ production in T cells. To evaluate the role of TGF‐β in the clearance of a persistent viral infection, we generated memory T cells in mice expressing a dominant negative TGF‐β receptor type II (dnTGF‐β RII) on T cells. Importantly, we revealed TGF‐β signaling is critical in the development of a LCMV‐specific primary T cell response and the formation of T cell memory. Moreover, when dnTGF‐β RII memory cells were administered to mice persistently infected with LCMV, they were unable to reduce viral loads, but caused severe immunopathology and increased mortality rates. These data demonstrate that TGF‐β regulates multiple stages of pathogen‐specific T cell development and function.
Bronchoalveolar Lavage (BAL) Matrix Metalloproteinases (MMPs) from Pulmonary Alveolar Proteinosis Patients (PAP) Processes BAL MCP‐3.
Authors: Bonfield Tracey Leigh , Koloze Mary , Ziady Assem , Thomassen Mary Jane
Published: June 2021

PAP is an anti‐GM‐CSF autoimmune lung disease. PAP BAL cells express increased levels of active MMP‐2 and MMP‐9 protein and mRNA relative to healthy controls (HC). Further, inhibitors of MMPs including TIMP‐1, TIMP‐2, TIMP‐3 or β2 macroglobulin were not elevated, suggesting lack of MMP deactivation. MMPs can proteolytically process chemokines such as MCP‐3, converting receptor agonists to antagonists. MCP‐3 is elevated in PAP BAL fluid (2.0±0.5 ng/ml, n=22, p<0.001) but there is a lack of robust recruitment. We hypothesize that PAP BAL MMPs contribute to processing of MCP‐3. PAP and HC BAL fluid were evaluated for chemotactic activity using the THP‐1 cells. PAP BAL fluid was chemotactic (1.3±0.2×104/ml, n=4) relative to HC (0.5±.04×104 n=4, p=0.03) which could be partially reversed by anti‐MCP‐3. PAP BAL fluid spiked with MCP‐3 (10ng/ml) had no additional chemotactic effect on the THP‐1 cells. MCP‐3‐spiked HC BAL had 4 fold greater chemotactic capacity (n=3, p=0.003). To determine if the MMPs in the PAP BAL fluid had the capacity to process MCP‐3 chemotaxis experiments were done in the presence of a MMP pan‐inhibitor. MCP‐3 spiked PAP BAL with the MMP inhibitor increased THP‐1 chemotaxis by 65% (n=4, p=0.04) relative to the PAP BAL fluid alone. PAP and control BAL fluid were evaluated using tandom mass spectrometry (MS/MS) spectra for a protein product consistent with the mass‐to‐charge predicted 780.7 MMP processed MCP‐3 peptide product. PAP BAL fluid contained a 780.7 peak consistent with the predicted size of truncated MCP‐3. These studies suggest that elevated MMPs in the lungs contribute to the processing of chemokines such as MCP‐3. This work was funded by NIH: AI:055840.
Accumulation of splenic plasma cells in lyn−/− mice is dependent on Bruton's tyrosine kinase but not IL‐6
Authors: Gutierrez Toni , Satterthwaite Anne
Published: June 2021

Deficiency of Lyn, a negative regulator of B and myeloid cell activation, results in B cell hyperresponsiveness, plasma cell accumulation, and production of autoantibodies in mice. Since accumulation of peripheral plasma cells is a common feature of both human and murine lupus, we have studied the mechanisms regulating this process in lyn−/− mice. Lyn‐deficient mice expressing reduced dosage of Btk (Btklo), a target of Lyn‐dependent inhibitory pathways, retain hypersensitivity to B cell receptor crosslinking but do not demonstrate increased plasma cell numbers or produce autoantibodies. Bone marrow‐derived macrophages from lyn−/− mice secrete more IL‐6, which promotes plasma cell differentiation and survival, than do wild type cells upon stimulation with LPS. Intracelluar staining for IL‐6 in splenic CD11b+ cells reveals that a greater frequency of lyn−/− myeloid cells produce IL‐6 upon LPS exposure than do wild type cells. Also, sera IL‐6 is significantly increased in aged lyn−/− mice. To determine if these effects are Btk‐mediated, we examined lyn−/−btklo mice and found that both production of IL‐6 in splenic CD11b+ cells and sera IL‐6 levels are normalized in lyn−/−btklo mice. However, lyn−/−IL‐6−/− mice continue to exhibit both plasma cell accumulation and production of anti‐DNA antibodies. Thus, plasma cell and autoimmune phenotypes in lyn−/− mice are mediated by Btk but not IL‐6.
Differential activation of dendritic cells from aged and young subjects by human DNA
Authors: Agrawal Anshu , Tay Jia , Ton Steven , Agrawal Sudhanshu , Gupta Sudhir
Published: June 2021

Dendritic cells act as the interface between the innate and adaptive immunity to direct either an immunogenic or tolerogenic response. They are involved in maintaining peripheral self‐tolerance through clearance of apoptotic cells. Aging is associated with chronic inflammation and autoimmunity. There is impaired dendritic cell mediated phagocytosis. This can result in accumulation of late apoptotic cells that can lyse and release nuclear self antigens such as DNA. Our objective was to determine whether human dendritic cells react to self DNA and if there is a difference between the aged and young subjects. Our results show that‐ 1) intracellular human DNA can activate human dendritic cells by upregulating costimulatory molecules, CD80 and CD86 and inducing the secretion of IFN‐alpha and IL‐6. 2) dendritic cells from aged are more reactive to human DNA as compared to dendritic cells from young. 3) Co‐culture of dendritic cells from aged subjects (pre‐exposed to DNA) with T cells from young resulted in proliferation in contrast to dendritic cells from young individuals where we observed an inhibition of T cell proliferation. These data suggest that dendritic cells from aged individuals may be impaired in their capacity to maintain peripheral self tolerance. This may contribute to the chronic inflammation and autoimmunity associated with aging. Grant support‐ AG027512 from NIH and Ellison Medical Foundation.
Effect of Albuterol Isomers on TNF‐alpha; Induced ICAM‐1 and Eotaxin Expression in Human Parasympathetic Neurons
Authors: Nie Zhenying , Jacoby David B , Fryer Allison D
Published: June 2021

Airway parasympathetic neurons express ICAM‐1 and eotaxin which are required for eosinophil recruitment to the nerves. Major basic protein released by eosinophils blocks inhibitory neuronal M2 muscarinic receptors, increasing acetylcholine release and potentiating reflex bronchoconstriction. TNF‐α induces eotaxin and ICAM‐1 in parasympathetic neurons. Here we test whether albuterol, which is used to treat asthma, changes TNF‐α induced eotaxin and ICAM‐1 expression. Airway parasympathetic neurons were isolated from human trachea and grown in serum‐free medium for 1 week. Cells were incubated with 1uM of either R‐albuterol (the active isomer), S‐albuterol (the inactive isomer) or RS‐albuterol for 90 minutes before addition of 2ng/ml TNF‐α for another 4 hours. In the absence of TNF‐α baseline eotaxin and ICAM‐1 expression was not changed by any isomer of albuterol as measured by real time RT‐PCR. TNF‐α induced ICAM‐1 expression was inhibited by R‐albuterol but not by S or R,S‐albuterol. TNF‐α induced eotaxin expression was not inhibited by any isomer of albuterol. The suppressive effect of R‐albuterol on neural ICAM‐1 expression may decrease eosinophil recruitment to the airway nerves, thereby decreasing reflex bronchoconstriction.

Funded by a grant from Sepracor Inc. and grants from NIH: HL55543, HL54659, HL071795, ES014601
Diabetes awareness in the Hmong
Authors: Yang Sua
Published: June 2021

Over 200,000 Hmong live in the U.S., primarily in California, Minnesota, and Wisconsin. The central valley is home to the largest Hmong communities in California. The Hmong demonstrate one of the highest poverty rates. Like other Asian groups, the Hmong are at increased risks for diabetes. The research objective was to identify perspectives and beliefs surrounding topic areas of diabetes, overweight, physical activity, food choices, and effective delivery of health messages. Young adults from Northern California participated in quasi focus group discussions. This sample was targeted because of their understanding of cultural traditions, beliefs and concepts of chronic disease (unfamiliar to Hmong immigrants). Participation (n=19) included females and males, 18–23 years old. Although 42% of participants had a family member with diabetes, there was a lack of awareness and knowledge about diabetes prevention. The majority said Hmong were not concerned about diabetes, 36% said there is concern but minimal knowledge. Most were concerned about diabetes only if diagnosed. Too much soda, sugar, fats, and meat were seen as concerns for the family diet. There is substantial need for educational intervention for health promotion in the home. To effectively address health concerns, involvement of Hmong health care professionals, clan leaders, and the media is required.
Graham Snack Consumption in After‐School Snack Programs Based on Whole Wheat Flour Content
Authors: Sadeghi Leila , Grosser Maggie , Anderson Amy , Reicks Marla , Marquart Len
Published: June 2021

The 2005 Dietary Guidelines for Americans recommend that children consume at least 3 servings of whole grain foods per day. Data from a pilot after‐school snack program indicated that graham crackers, containing 5g whole wheat per serving, were consumed in the range of 73–88%, similar to the refined grain counterpart. This study examined graham snack consumption containing varying levels of whole wheat flour per serving by school‐aged children. Subjects included ~125 elementary school children (grades K‐6) participating in after‐school snack programs in 4 elementary schools. Over 8 weeks, children were offered graham snacks in a random order containing 5, 8, 12, and 16g of whole wheat flour per 30g serving. Plate waste was used to measure graham snack consumption, as children placed their unfinished snacks in a waste receptacle and subsequent waste was measured after each session. At week 9, a subgroup of children participated in focus groups to provide input about their perceptions for product quality and suggestions for improvement. Consumption for the 5, 8, 12, and 16g graham snacks was 78%, 76%, 85%, and 78%, respectively. This study suggests that these snacks can contribute a significant dietary source of whole grain when offered through an after‐ school snack program.

Kraft Foods, Inc.
Dynamic Shifts in Chinese Eating Behaviors
Authors: Wang Zhihong , Zhai Fengying , Zhang Bing , Du Shufa , Popkin Barry M
Published: June 2021

Research on the nutrition transition has ignored methods of cooking and snacking. This study explores dynamics of these behaviors and their associations with socioeconomic status (SES). Two waves of data from the China Health and Nutrition Survey from 1991 (11780 subjects aged 2 and above) and 2004 (11169 subjects) are used. Major increases in the frying of food and snacking behavior are the focus of further analysis. Logistic regressions of pooled data were performed to evaluate how SES was associated with two eating behaviors: snacking and consuming excessive fried foods. Simulation techniques were used to clarify the effects of the results that included significant interaction terms. Snacking behavior is beginning to emerge and there are important shifts away from steaming and boiling of food to less healthy frying of food. These changes are occurring across all age groups. An analysis of the SES factors linked with these shifts shows income is positively related to consumption of both snacks and excessive fried food. Urban residents are also more likely to snack and to consume excessive fried foods than rural residents. These trends, particularly the frying of food, are seen as less healthy options that will be difficult to change. Future health promotion programs targeting the higher‐SES population will exert far‐reaching effects on the improvement of health status.
Nutritional status in Mexican rural children: Probabilistic surveys from 12 states results.
Authors: Galindo‐Gómez Carlos , Ávila Abelardo , Ávila Marco , Quiroz Marco , Juárez Ma. Jesús Liliana , Rodríguez Gerardo
Published: June 2021

The objetive was to establish the nutritional status in Mexican rural preschool children and its association with socioeconomic variables and feeding practices. Data was obtained from probabilistic surveys from 12 states (2004–2006) in rural areas of three Mexican regions (North, Center and South). Anthropometric, socioeconomic level (SES) and feeding practices of children < 5 y was collected. Prevalence of malnutrition was estimated. Logistic regression models were calculated to establish the relationship between nutritional status and select variables. All analyses were adjusted by surveys design. In North region the prevalence of malnutrition was: underweight (WA <‐2z) 3.4%; wasting (WH <‐2z) 4.4%; stunting (HA <‐2z) 11.3% and obesity (HWH >2z) 5%. In Center region was: WA 8%; WH 6.4%; HA 20.8% and HWH 7.7%. In the South region was: WA 10.4%; 3 WH.6%; HA 22.9% and HWH 5.7%. Migration was associated with underweight in the North region (OR 0.11, p=0.01) and with obesity in Center region (OR 1.8, p=0.045). Indigenous was associated with underweight in the Center region (OR 1.47, p=0.04) and with stunting in South region (OR 1.35, p=0.02). Low SES (OR 1.75, p<0.01) and middle SES (OR 1.60, p = 0.001) were associated with stunting in South region. Malnutrition is a problem in Mexican rural areas.
Food Insecurity Associated with Maternal Psychological Distress, but not Child Overweight
Authors: McGarve November , Harrison Gail , Whaley Shannon , Jenks Eloise
Published: June 2021

The study objective is to assess potential relationships between food insecurity, maternal psychological distress, child feeding strategies, and child overweight. Data come from a case‐control study (n=552) of low‐income, primarily Latina recipients of a Los Angeles based WIC Program and their pre‐school age children. Statistical methods include bivariate chi‐square analyses and logistic regression. Food insecurity was significantly associated with maternal psychological distress (p<0.001), but unlike some findings from the research literature, neither food insecurity nor maternal psychological distress were significantly associated with child overweight. Ten child feeding strategies were examined; three were significantly associated at p<0.05 with child overweight (child takes food from refrigerator between meals, mother restricts child from play/television until s/he eats, and mother offers alternative foods) and one with maternal psychological distress (mother scolds child for not eating well). Findings suggest social policy should address food security and mental health issues in this population. Potential protective factors insulating children from overweight consequences of food insecurity and maternal psychological distress need exploration.
Iron supplementation and changes in iron absorption and iron status during pregnancy and lactation
Authors: Woodhouse Leslie R , Abrams Steven A , Chen Zhensheng , Griffin Ian J , King Janet C , Mendoza Concepcion , Mukherjea Ratna , Viteri Fernando
Published: June 2021

Iron supplementation (FeS) is often prescribed for pregnancy. Thus, we assessed the effect of FeS (65mg) on Fe absorption (FeAb) and Fe status during pregnancy and lactation. 3 groups of women were studied: FeS at breakfast (AM; n=13); FeS at bedtime (no food) (PM; n=11); and an unsupplemented group (N; n=10). FeAb was measured at 12–15 (T1) and 32–34 (T3) wks of gestation and at 7–9 wks postpartum (LC). FeAb was measured from the diet and from the supplement (AM group only) using a dual or triple stable isotope method (57Fe labeled bread was given at each meal on the study day, the 65mg supplement (AM group) was labeled with 54Fe, and 58Fe was given IV directly after the breakfast meal). FeAb from the breakfast and the supplement was measured from the relative increases in 57Fe, 54Fe and 58Fe erythrocyte enrichment after 14d. High resolution ICP‐MS with a Ni correction was used to determine Fe isotope ratios after an acid digestion of the erythrocytes. FeAb, (%; p<0.0001), from food for T1, T3 and LC in AM was 6.6a, 12.3b & 6.0a; in PM was 3.3a, 12.1b & 2.5a; and in N was 5.2a, 23.8b & 3.9a. FeAb was significantly higher at each time point in N vs. PM (p=0.012). FeAb (%; p<0.02) from the supplement in AM was 2.9ab, 4.4b & 1.6a. [Ferritin], (ln, ug/L, p<0.01) for T1, T3 & LC for AM (47b, 26a, 85c), PM (40b, 22a, 78c) & N (47b, 11a, 46b)shows that FeS, continuing into lactation, improves Fe status during pregnancy and lactation. [NIH # R01 HD38805 & WHNRC]